Fighting Spam: Moving Email to the Cloud

Science.gov (United States)

Schrader, Andrew

2011-01-01

On average, more than 90% of email is unwanted junk mail--spam. Many spam messages include annoying promotions for irrelevant products, but others include viruses and pornographic content, which can pose a severe legal issue for schools. Spam puts districts in a difficult situation as schools try to enhance learning in the classroom with advanced…

Spam:One Puzzled Problem for E-Commerce

Institute of Scientific and Technical Information of China (English)

FU Qiansheng; YANG Jingmin

2004-01-01

E-commerce has advantages of low bargaining cost, full field service, and convenient. Its development forms and promotes the key factor of the global economic growth, and the security problem of e-commerce becomes more and more important thereupon. It is the subject of a great meaning to structure the safe, convenient e-commerce running environment. The task of anti-spam is a concrete content among them. This paper discusses the source and danger of spam, and the precautionary and radical cure measures of spam are put forwarded, and the technologies of the anti-spam are discussed especially.

AN EFFECTIVE SPAM FILTERING FOR DYNAMIC MAIL MANAGEMENT SYSTEM

Directory of Open Access Journals (Sweden)

S. Arun Mozhi Selvi

2012-04-01

Full Text Available Spam is commonly defined as unsolicited email messages and the goal of spam categorization is to distinguish between spam and legitimate email messages. The economics of spam details that the spammer has to target several recipients with identical and similar email messages. As a result a dynamic knowledge sharing effective defense against a substantial fraction of spam has to be designed which can alternate the burdens of frequent training stand alone spam filter. A weighted email attribute based classification is proposed to mainly focus to encounter the issues in normal email system. These type of classification helps to formulate an effective utilization of our email system by combining the concepts of Bayesian Spam Filtering Algorithm, Iterative Dichotmiser 3(ID3 Algorithm and Bloom Filter. The details captured by the system are processed to track the original sender causing disturbances and prefer them to block further mails from them. We have tested the effectiveness of our scheme by collecting offline data from Yahoo mail & Gmail dumps. This proposal is implemented using .net and sample user-Id for knowledge base.

Take control of spam with Apple Mail

CERN Document Server

Kissell, Joe

2009-01-01

Questions answered in this book include: How does Mail's Junk Mail filtering really work?How does the spam filter use my Previous Recipients list?How could a spammer's email addresses get on my Previous Recipients list?What can I do to not get so much spam in the first place?What's the deal with email that pretends to come from my bank?What features should I look for in a spam-fighting utility or service?Should I try server-based filtering?

Detecting spam comments on Indonesia’s Instagram posts

Science.gov (United States)

Septiandri, Ali Akbar; Wibisono, Okiriza

2017-01-01

In this paper we experimented with several feature sets for detecting spam comments in social media contents authored by Indonesian public figures. We define spam comments as comments which have promotional purposes (e.g. referring other users to products and services) and thus not related to the content to which the comments are posted. Three sets of features are evaluated for detecting spams: (1) hand-engineered features such as comment length, number of capital letters, and number of emojis, (2) keyword features such as whether the comment contains advertising words or product-related words, and (3) text features, namely, bag-of-words, TF-IDF, and fastText embeddings, each combined with latent semantic analysis. With 24,000 manually-annotated comments scraped from Instagram posts authored by more than 100 Indonesian public figures, we compared the performance of these feature sets and their combinations using 3 popular classification algorithms: Na¨ıve Bayes, SVM, and XGBoost. We find that using all three feature sets (with fastText embedding for the text features) gave the best F 1-score of 0.9601 on a holdout dataset. More interestingly, fastText embedding combined with hand-engineered features (i.e. without keyword features) yield similar F 1-score of 0.9523, and McNemar’s test failed to reject the hypothesis that the two results are not significantly different. This result is important as keyword features are largely dependent on the dataset and may not be as generalisable as the other feature sets when applied to new data. For future work, we hope to collect bigger and more diverse dataset of Indonesian spam comments, improve our model’s performance and generalisability, and publish a programming package for others to reliably detect spam comments.

SPAM CLASSIFICATION BASED ON SUPERVISED LEARNING USING MACHINE LEARNING TECHNIQUES

Directory of Open Access Journals (Sweden)

T. Hamsapriya

2011-12-01

Full Text Available E-mail is one of the most popular and frequently used ways of communication due to its worldwide accessibility, relatively fast message transfer, and low sending cost. The flaws in the e-mail protocols and the increasing amount of electronic business and financial transactions directly contribute to the increase in e-mail-based threats. Email spam is one of the major problems of the today’s Internet, bringing financial damage to companies and annoying individual users. Spam emails are invading users without their consent and filling their mail boxes. They consume more network capacity as well as time in checking and deleting spam mails. The vast majority of Internet users are outspoken in their disdain for spam, although enough of them respond to commercial offers that spam remains a viable source of income to spammers. While most of the users want to do right think to avoid and get rid of spam, they need clear and simple guidelines on how to behave. In spite of all the measures taken to eliminate spam, they are not yet eradicated. Also when the counter measures are over sensitive, even legitimate emails will be eliminated. Among the approaches developed to stop spam, filtering is the one of the most important technique. Many researches in spam filtering have been centered on the more sophisticated classifier-related issues. In recent days, Machine learning for spam classification is an important research issue. The effectiveness of the proposed work is explores and identifies the use of different learning algorithms for classifying spam messages from e-mail. A comparative analysis among the algorithms has also been presented.

PENGIRIMAN E-MAIL SPAM SEBAGAI KEJAHATAN CYBER DI INDONESIA

Directory of Open Access Journals (Sweden)

Eka Nugraha Putra

2018-01-01

Full Text Available The Internet is part of the development of technology, where the internet provides many impacts, both positive and negative. Currently, privacy issues on the internet have also become a complicated legal issue, this is due to quite a number of privacy-related issues, but not all countries in the world manage privacy issues on the internet. As a means of communicating the Internet has introduced e-mail that provides convenience and practicality. But in its development e-mail has an adverse impact on its users in the form of e-mail spam. In terms of its actions, sending spam e-mail is quite a disadvantage, even violate the privacy. Some countries have also set it to one type of cybercrime (cybercrime. This research will discuss e-mail spam in Indonesia, how the legislation in Indonesia see the action of this spam e-mail, is there any possibility of spam e-mail is criminalized as a cybercrime. The research will also look at how spam e-mails violate privacy and review and analyze internet privacy settings in Indonesia in relation to the criminalization of spam email.

A new Spam fighting rule: Reverse SMTP Connect

CERN Multimedia

CERN Mail Service Team

2004-01-01

The CERN Mail Service is constantly improving Spam Fighting rules to minimize the number of Spams (unsolicited mails) delivered to the Inbox. On Wednesday 6th October, a new rule will be activated, the "Reverse SMTP Connect": if we cannot connect to the sender's mail server, we will not accept the mail (see http://cern.ch/mmms/Help/?kbid=171030 for details). According to intensive tests, we expect to reject 25% of mails that we currently accept, without generating false positives. Currently, around 90% of the mails received by CERN Mail gateways are Spams, 85% of these Spams are automatically rejected, using basic rules. As usual, statistics on Spam fighting, Mail traffic and Help pages can be found on the Mail Service site: http://cern.ch/mmm, and don't hesitate to report any problem that could be related to this new rule to Helpdesk@cern.ch (phone 78888). CERN Mail Service Team

Detection of Spam Email by Combining Harmony Search Algorithm and Decision Tree

Directory of Open Access Journals (Sweden)

M. Z. Gashti

2017-06-01

Full Text Available Spam emails is probable the main problem faced by most e-mail users. There are many features in spam email detection and some of these features have little effect on detection and cause skew detection and classification of spam email. Thus, Feature Selection (FS is one of the key topics in spam email detection systems. With choosing the important and effective features in classification, its performance can be optimized. Selector features has the task of finding a subset of features to improve the accuracy of its predictions. In this paper, a hybrid of Harmony Search Algorithm (HSA and decision tree is used for selecting the best features and classification. The obtained results on Spam-base dataset show that the rate of recognition accuracy in the proposed model is 95.25% which is high in comparison with models such as SVM, NB, J48 and MLP. Also, the accuracy of the proposed model on the datasets of Ling-spam and PU1 is high in comparison with models such as NB, SVM and LR.

SPONGY (SPam ONtoloGY): email classification using two-level dynamic ontology.

Science.gov (United States)

Youn, Seongwook

2014-01-01

Email is one of common communication methods between people on the Internet. However, the increase of email misuse/abuse has resulted in an increasing volume of spam emails over recent years. An experimental system has been designed and implemented with the hypothesis that this method would outperform existing techniques, and the experimental results showed that indeed the proposed ontology-based approach improves spam filtering accuracy significantly. In this paper, two levels of ontology spam filters were implemented: a first level global ontology filter and a second level user-customized ontology filter. The use of the global ontology filter showed about 91% of spam filtered, which is comparable with other methods. The user-customized ontology filter was created based on the specific user's background as well as the filtering mechanism used in the global ontology filter creation. The main contributions of the paper are (1) to introduce an ontology-based multilevel filtering technique that uses both a global ontology and an individual filter for each user to increase spam filtering accuracy and (2) to create a spam filter in the form of ontology, which is user-customized, scalable, and modularized, so that it can be embedded to many other systems for better performance.

SPONGY (SPam ONtoloGY: Email Classification Using Two-Level Dynamic Ontology

Directory of Open Access Journals (Sweden)

Seongwook Youn

2014-01-01

Full Text Available Email is one of common communication methods between people on the Internet. However, the increase of email misuse/abuse has resulted in an increasing volume of spam emails over recent years. An experimental system has been designed and implemented with the hypothesis that this method would outperform existing techniques, and the experimental results showed that indeed the proposed ontology-based approach improves spam filtering accuracy significantly. In this paper, two levels of ontology spam filters were implemented: a first level global ontology filter and a second level user-customized ontology filter. The use of the global ontology filter showed about 91% of spam filtered, which is comparable with other methods. The user-customized ontology filter was created based on the specific user’s background as well as the filtering mechanism used in the global ontology filter creation. The main contributions of the paper are (1 to introduce an ontology-based multilevel filtering technique that uses both a global ontology and an individual filter for each user to increase spam filtering accuracy and (2 to create a spam filter in the form of ontology, which is user-customized, scalable, and modularized, so that it can be embedded to many other systems for better performance.

SPONGY (SPam ONtoloGY): Email Classification Using Two-Level Dynamic Ontology

Science.gov (United States)

2014-01-01

Email is one of common communication methods between people on the Internet. However, the increase of email misuse/abuse has resulted in an increasing volume of spam emails over recent years. An experimental system has been designed and implemented with the hypothesis that this method would outperform existing techniques, and the experimental results showed that indeed the proposed ontology-based approach improves spam filtering accuracy significantly. In this paper, two levels of ontology spam filters were implemented: a first level global ontology filter and a second level user-customized ontology filter. The use of the global ontology filter showed about 91% of spam filtered, which is comparable with other methods. The user-customized ontology filter was created based on the specific user's background as well as the filtering mechanism used in the global ontology filter creation. The main contributions of the paper are (1) to introduce an ontology-based multilevel filtering technique that uses both a global ontology and an individual filter for each user to increase spam filtering accuracy and (2) to create a spam filter in the form of ontology, which is user-customized, scalable, and modularized, so that it can be embedded to many other systems for better performance. PMID:25254240

Fighting Spam on the Sender Side: A Lightweight Approach

NARCIS (Netherlands)

de Vries, Wouter Willem; Moreira Moura, Giovane; Pras, Aiko; Aagesen, Finn Arve; Knapskog, Svein Johan

2010-01-01

Spam comprises approximately 90 to 95 percent of all e-mail traffic on the Internet nowadays, and it is a problem far from being solved. The losses caused by spam are estimated to reach up to \\$87 billion yearly. When fighting spam, most of the proposals focus on the receiver-side and are usually

SPAM- SPECTRAL ANALYSIS MANAGER (DEC VAX/VMS VERSION)

Science.gov (United States)

Solomon, J. E.

1994-01-01

The Spectral Analysis Manager (SPAM) was developed to allow easy qualitative analysis of multi-dimensional imaging spectrometer data. Imaging spectrometers provide sufficient spectral sampling to define unique spectral signatures on a per pixel basis. Thus direct material identification becomes possible for geologic studies. SPAM provides a variety of capabilities for carrying out interactive analysis of the massive and complex datasets associated with multispectral remote sensing observations. In addition to normal image processing functions, SPAM provides multiple levels of on-line help, a flexible command interpretation, graceful error recovery, and a program structure which can be implemented in a variety of environments. SPAM was designed to be visually oriented and user friendly with the liberal employment of graphics for rapid and efficient exploratory analysis of imaging spectrometry data. SPAM provides functions to enable arithmetic manipulations of the data, such as normalization, linear mixing, band ratio discrimination, and low-pass filtering. SPAM can be used to examine the spectra of an individual pixel or the average spectra over a number of pixels. SPAM also supports image segmentation, fast spectral signature matching, spectral library usage, mixture analysis, and feature extraction. High speed spectral signature matching is performed by using a binary spectral encoding algorithm to separate and identify mineral components present in the scene. The same binary encoding allows automatic spectral clustering. Spectral data may be entered from a digitizing tablet, stored in a user library, compared to the master library containing mineral standards, and then displayed as a timesequence spectral movie. The output plots, histograms, and stretched histograms produced by SPAM can be sent to a lineprinter, stored as separate RGB disk files, or sent to a Quick Color Recorder. SPAM is written in C for interactive execution and is available for two different

A framework for unsupervised spam detection in social networking sites

NARCIS (Netherlands)

Bosma, M.; Meij, E.; Weerkamp, W.

2012-01-01

Social networking sites offer users the option to submit user spam reports for a given message, indicating this message is inappropriate. In this paper we present a framework that uses these user spam reports for spam detection. The framework is based on the HITS web link analysis framework and is

Clustering Spam Domains and Destination Websites: Digital Forensics with Data Mining

Directory of Open Access Journals (Sweden)

Chun Wei

2010-03-01

Full Text Available Spam related cyber crimes have become a serious threat to society. Current spam research mainly aims to detect spam more effectively. We believe the prosecution of spammers is a more effective way of stopping spam emails than filtering, therefore more research is needed to help forensic investigators to collect useful evidence. This research proposes an algorithm for clustering spam domains extracted from spam emails based on the hosting IP addresses and tracing the domains over a period of time. The results reveal several facts that merit law enforcement attention: many seemingly unrelated spam campaigns are actually related; spammers have a sophisticated mechanism for combating URL blacklisting by registering many new domain names every day and flushing out old domains; the domains are hosted at different IP addresses across several networks, mostly in China where legislation is not as tight as in US; old IP addresses are replaced by new ones from time to time, but still show strong correlation among them. These facts lead to the conclusion that spam-related cyber crimes are operated by well-organized criminal syndicates that have sufficient manpower to distribute a huge volume of spam through bots, purchase a large number of domain names and hosting servers and maintain websites to sell counterfeit products online. Traditional law enforcements technology has not scaled well in cases involving millions of data elements. This paper demonstrates an effective use of data mining to respond to this challenge.

Is Email Business Dying?: A Study on Evolution of Email Spam Over Fifteen Years

OpenAIRE

De Wang; Danesh Irani; Calton Pu

2014-01-01

With the increasing dedication and sophistication of spammers, email spam is a persistent problem even today. Popular social network sites such as Facebook, Twitter, and Google+ are not exempt from email spam as they all interface with email systems. While some report predicts that email spam business is dying due to the decreasing volume of email spam. Whether email spam business is really dying is an interesting question. In this paper, we analyze email spam trends on Spam Archive dataset, ...

An efficient incremental learning mechanism for tracking concept drift in spam filtering.

Directory of Open Access Journals (Sweden)

Jyh-Jian Sheu

Full Text Available This research manages in-depth analysis on the knowledge about spams and expects to propose an efficient spam filtering method with the ability of adapting to the dynamic environment. We focus on the analysis of email's header and apply decision tree data mining technique to look for the association rules about spams. Then, we propose an efficient systematic filtering method based on these association rules. Our systematic method has the following major advantages: (1 Checking only the header sections of emails, which is different from those spam filtering methods at present that have to analyze fully the email's content. Meanwhile, the email filtering accuracy is expected to be enhanced. (2 Regarding the solution to the problem of concept drift, we propose a window-based technique to estimate for the condition of concept drift for each unknown email, which will help our filtering method in recognizing the occurrence of spam. (3 We propose an incremental learning mechanism for our filtering method to strengthen the ability of adapting to the dynamic environment.

Is Email Business Dying?: A Study on Evolution of Email Spam Over Fifteen Years

Directory of Open Access Journals (Sweden)

De Wang

2014-05-01

Full Text Available With the increasing dedication and sophistication of spammers, email spam is a persistent problem even today. Popular social network sites such as Facebook, Twitter, and Google+ are not exempt from email spam as they all interface with email systems. While some report predicts that email spam business is dying due to the decreasing volume of email spam. Whether email spam business is really dying is an interesting question. In this paper, we analyze email spam trends on Spam Archive dataset, which contains 5.5 million spam emails over 15 years (1998 – 2013. We statistically analyze emails contents including header information (e.g. content type and embedded items (e.g. URL links. Also, we investigate topic drift using topic modeling technique. Moreover, we perform network analysis on sender-to-receiver IP routing networks. Our study shows the dynamic nature of email spam over one and a half decades and demonstrate that the email spam business is not dying but more capricious.

Identifying spam e-mail messages using an intelligence algorithm

Directory of Open Access Journals (Sweden)

Parichehr Ghaedi

2014-06-01

Full Text Available During the past few years, there have been growing interests in using email for delivering various types of messages such as social, financial, etc. There are also people who use email messages to promote products and services or even to do criminal activities called Spam email. These unwanted messages are sent to different target population for different purposes and there is a growing interest to develop methods to filter such email messages. This paper presents a method to filter Spam email messages based on the keyword pattern. In this article, a multi-agent filter trade based on the Bayes rule, which has benefit of using the users’ interest, keywords and investigation the message content according to its topic, has been used. Then Nested Neural Network has been used to detect the spam messages. To check the authenticity of this proposed method, we test it for a couple of email messages, so that it could determine spams and hams from each other, effectively. The result shows the superiority of this method over the previous ones including filters with Multi-Layer Perceptron that detect spams.

Hot Zone Identification: Analyzing Effects of Data Sampling on Spam Clustering

Directory of Open Access Journals (Sweden)

Rasib Khan

2014-03-01

Full Text Available Email is the most common and comparatively the most efficient means of exchanging information in today's world. However, given the widespread use of emails in all sectors, they have been the target of spammers since the beginning. Filtering spam emails has now led to critical actions such as forensic activities based on mining spam email. The data mine for spam emails at the University of Alabama at Birmingham is considered to be one of the most prominent resources for mining and identifying spam sources. It is a widely researched repository used by researchers from different global organizations. The usual process of mining the spam data involves going through every email in the data mine and clustering them based on their different attributes. However, given the size of the data mine, it takes an exceptionally long time to execute the clustering mechanism each time. In this paper, we have illustrated sampling as an efficient tool for data reduction, while preserving the information within the clusters, which would thus allow the spam forensic experts to quickly and effectively identify the ‘hot zone’ from the spam campaigns. We have provided detailed comparative analysis of the quality of the clusters after sampling, the overall distribution of clusters on the spam data, and timing measurements for our sampling approach. Additionally, we present different strategies which allowed us to optimize the sampling process using data-preprocessing and using the database engine's computational resources, and thus improving the performance of the clustering process.

Analysis of Web Spam for Non-English Content: Toward More Effective Language-Based Classifiers.

Directory of Open Access Journals (Sweden)

Mansour Alsaleh

Full Text Available Web spammers aim to obtain higher ranks for their web pages by including spam contents that deceive search engines in order to include their pages in search results even when they are not related to the search terms. Search engines continue to develop new web spam detection mechanisms, but spammers also aim to improve their tools to evade detection. In this study, we first explore the effect of the page language on spam detection features and we demonstrate how the best set of detection features varies according to the page language. We also study the performance of Google Penguin, a newly developed anti-web spamming technique for their search engine. Using spam pages in Arabic as a case study, we show that unlike similar English pages, Google anti-spamming techniques are ineffective against a high proportion of Arabic spam pages. We then explore multiple detection features for spam pages to identify an appropriate set of features that yields a high detection accuracy compared with the integrated Google Penguin technique. In order to build and evaluate our classifier, as well as to help researchers to conduct consistent measurement studies, we collected and manually labeled a corpus of Arabic web pages, including both benign and spam pages. Furthermore, we developed a browser plug-in that utilizes our classifier to warn users about spam pages after clicking on a URL and by filtering out search engine results. Using Google Penguin as a benchmark, we provide an illustrative example to show that language-based web spam classifiers are more effective for capturing spam contents.

Identification and Characterization of a Novel Issatchenkia orientalis GPI-Anchored Protein, IoGas1, Required for Resistance to Low pH and Salt Stress.

Directory of Open Access Journals (Sweden)

Akinori Matsushika

Full Text Available The use of yeasts tolerant to acid (low pH and salt stress is of industrial importance for several bioproduction processes. To identify new candidate genes having potential roles in low-pH tolerance, we screened an expression genomic DNA library of a multiple-stress-tolerant yeast, Issatchenkia orientalis (Pichia kudriavzevii, for clones that allowed Saccharomyces cerevisiae cells to grow under highly acidic conditions (pH 2.0. A genomic DNA clone containing two putative open reading frames was obtained, of which the putative protein-coding gene comprising 1629 bp was retransformed into the host. This transformant grew significantly at pH 2.0, and at pH 2.5 in the presence of 7.5% Na2SO4. The predicted amino acid sequence of this new gene, named I. orientalis GAS1 (IoGAS1, was 60% identical to the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity, and 58-59% identical to Candida albicans Phr1 and Phr2, pH-responsive proteins implicated in cell wall assembly and virulence. Northern hybridization analyses indicated that, as for the C. albicans homologs, IoGAS1 expression was pH-dependent, with expression increasing with decreasing pH (from 4.0 to 2.0 of the medium. These results suggest that IoGAS1 represents a novel pH-regulated system required for the adaptation of I. orientalis to environments of diverse pH. Heterologous expression of IoGAS1 complemented the growth and morphological defects of a S. cerevisiae gas1Δ mutant, demonstrating that IoGAS1 and the corresponding S. cerevisiae gene play similar roles in cell wall biosynthesis. Site-directed mutagenesis experiments revealed that two conserved glutamate residues (E161 and E262 in the IoGas1 protein play a crucial role in yeast morphogenesis and tolerance to low pH and salt stress. Furthermore, overexpression of IoGAS1 in S. cerevisiae remarkably improved the ethanol fermentation ability at pH 2.5, and at pH 2.0 in the

SPAM-assisted partial volume correction algorithm for PET

Energy Technology Data Exchange (ETDEWEB)

Cho, Sung Il; Kang, Keon Wook; Lee, Jae Sung; Lee, Dong Soo; Chung, June Key; Soh, Kwang Sup; Lee, Myung Chul [College of Medicine, Seoul National Univ., Seoul (Korea, Republic of)

2000-07-01

A probabilistic atlas of the human brain (Statistical Probability Anatomical Maps: SPAM) was developed by the International Consortium for Brain Mapping (ICBM). It will be a good frame for calculating volume of interest (VOI) according to statistical variability of human brain in many fields of brain images. We show that we can get more exact quantification of the counts in VOI by using SPAM in the correlation of partial volume effect for simulated PET image. The MRI of a patient with dementia was segmented into gray matter and white matter, and then they were smoothed to PET resolution. Simulated PET image was made by adding one third of the smoothed white matter to the smoothed gray matter. Spillover effect and partial volume effect were corrected for this simulated PET image with the aid of the segmented and smoothed MR images. The images were spatially normalized to the average brain MRI atlas of ICBM, and were multiplied by the probablities of 98 VOIs of SPAM images of Montreal Neurological Institute. After the correction of partial volume effect, the counts of frontal, partietal, temporal, and occipital lobes were increased by 38{+-}6%, while those of hippocampus and amygdala by 4{+-}3%. By calculating the counts in VOI using the product of probability of SPAM images and counts in the simulated PET image, the counts increase and become closer to the true values. SPAM-assisted partial volume correction is useful for quantification of VOIs in PET images.

SPAM-assisted partial volume correction algorithm for PET

International Nuclear Information System (INIS)

Cho, Sung Il; Kang, Keon Wook; Lee, Jae Sung; Lee, Dong Soo; Chung, June Key; Soh, Kwang Sup; Lee, Myung Chul

2000-01-01

A probabilistic atlas of the human brain (Statistical Probability Anatomical Maps: SPAM) was developed by the International Consortium for Brain Mapping (ICBM). It will be a good frame for calculating volume of interest (VOI) according to statistical variability of human brain in many fields of brain images. We show that we can get more exact quantification of the counts in VOI by using SPAM in the correlation of partial volume effect for simulated PET image. The MRI of a patient with dementia was segmented into gray matter and white matter, and then they were smoothed to PET resolution. Simulated PET image was made by adding one third of the smoothed white matter to the smoothed gray matter. Spillover effect and partial volume effect were corrected for this simulated PET image with the aid of the segmented and smoothed MR images. The images were spatially normalized to the average brain MRI atlas of ICBM, and were multiplied by the probablities of 98 VOIs of SPAM images of Montreal Neurological Institute. After the correction of partial volume effect, the counts of frontal, partietal, temporal, and occipital lobes were increased by 38±6%, while those of hippocampus and amygdala by 4±3%. By calculating the counts in VOI using the product of probability of SPAM images and counts in the simulated PET image, the counts increase and become closer to the true values. SPAM-assisted partial volume correction is useful for quantification of VOIs in PET images

An Empirical Study of Spam and Prevention Mechanisms in Online Video Chat Services

OpenAIRE

Xing, Xinyu; Ahn, Junho; Lee, Wenke; Han, Richard; Mishra, Shivakant

2012-01-01

Recently, online video chat services are becoming increasingly popular. While experiencing tremendous growth, online video chat services have also become yet another spamming target. Unlike spam propagated via traditional medium like emails and social networks, we find that spam propagated via online video chat services is able to draw much larger attention from the users. We have conducted several experiments to investigate spam propagation on Chatroulette - the largest online video chat web...

Effect of chitosan on the heat stability of whey protein solution as a function of pH.

Science.gov (United States)

Zhao, Zhengtao; Xiao, Qian

2017-03-01

Chitosan was reported to interact with proteins through electrostatic interactions. Their interaction was influenced by pH, which was not fully characterized. Further research on the interactions between protein and chitosan at different pH and their influence on the thermal denaturation of proteins is necessary. In this research, the effect of chitosan on the heat stability of whey protein solution at pH 4.0-6.0 was studied. At pH 4.0, a small amount chitosan was able to prevent the heat-induced denaturation and aggregation of whey protein molecules. At higher pH values (5.5 and 6.0), whey proteins complexed with chitosan through electrostatic attraction. The formation of chitosan-whey protein complexes at pH 5.5 improved the heat stability of dispersions and no precipitation could be detected up to 20 days. The dispersion with a medium amount of chitosan (chitosan:whey protein 1:5) produced the most stable particles, which had an average radius of 135 ± 14 nm and a zeta potential value of 36 ± 1 mV. In contrast, at pH 6.0 only the dispersion with a high amount of chitosan (chitosan:whey protein 1:2) showed good shelf stability up to 20 days. It was possible to produce heat-stable whey protein beverages by regulating the interaction between chitosan and whey protein molecules. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Awareness, training and trust in interaction with adaptive spam filters

NARCIS (Netherlands)

Cramer, H.S.M.; Evers, V.; van Someren, M.W.; Wielinga, B.J.; Greenberg, S.; Hudson, S.E.; Hinckley, K.; Morris, M.R.; Olsen Jr., D.R.

2009-01-01

Even though adaptive (trainable) spam filters are a common example of systems that make (semi-)autonomous decisions on behalf of the user, trust in these filters has been underexplored. This paper reports a study of usage of spam filters in the daily workplace and user behaviour in training these

Evaluation of Spam Impact on Arabic Websites Popularity

Directory of Open Access Journals (Sweden)

Mohammed N. Al-Kabi

2015-04-01

Full Text Available The expansion of the Web and its information in all aspects of life raises the concern of how to trust information published on the Web especially in cases where publisher may not be known. Websites strive to be more popular and make themselves visible to search engines and eventually to users. Website popularity can be measured using several metrics such as the Web traffic (e.g. Website: visitors’ number and visited page number. A link or page popularity refers to the total number of hyperlinks referring to a certain Web page. In this study, several top ranked Arabic Websites are selected for evaluating possible Web spam behavior. Websites use spam techniques to boost their ranks within Search Engine Results Page (SERP. Results of this study showed that some of these popular Websites are using techniques that are considered spam techniques according to Search Engine Optimization guidelines.

Manipulating E-Mail Server Feedback for Spam Prevention

Directory of Open Access Journals (Sweden)

O. A. Okunade

2017-08-01

Full Text Available The cyber criminals who infect machines with bots are not the same as the spammers who rent botnets to distribute their messages. The activities of these spammers account for the majority of spam emails traffic on the internet. Once their botnets and campaigns are identified, it is not enough to keep on filtering the spam emails, it is necessary to deploy techniques that will carry the fight to their end. It is observed that spammers also take into account server feedback (for example to detect and remove non-existent recipients from email address lists. We can take advantage of this observation by returning fake information, thereby poisoning the server feedback on which the spammers rely. The results of this paper show that by sending misleading information to a spammer, it is possible to prevent recipients from receiving subsequent spam emails from that same spammer.

Catching the Drift : Using Feature-Free Case-Based Reasoning for Spam Filtering.

OpenAIRE

Delany, Sarah Jane; Bridge, Derek

2007-01-01

n this paper, we compare case-based spam filters, focusing on their resilience to concept drift. In particular, we evaluate how to track concept drift using a case-based spam filter that uses a feature-free distance measure based on text compression. In our experiments, we compare two ways to normalise such a distance measure, finding that the one proposed in [1] performs better. We show that a policy as simple as retaining misclassified examples has a hugely beneficial effect on handling con...

Analysis of Techniques for Protection Against Spam over Internet Telephony

NARCIS (Netherlands)

Quinten, Vincent M.; van de Meent, R.; Pras, A.; Pras, Aiko; van Sinderen, M.J.; van Sinderen, Marten J.

2007-01-01

Spam in Internet telephony (SPIT) networks is likely to become a large problem in the future, as more and more people and companies switch from traditional telephone networks to Voice over IP (VoIP) networks, and as it is easy to spam VoIP users. The goal of this survey paper is to identify

Aplikasi Manajemen Risiko pada Pembangunan Sistem Penyediaan Air Minum (SPAM Regional Jawa Tengah (Studi Kasus pada Pembangunan Jaringan Transmisi SPAM Regional Bregas

Directory of Open Access Journals (Sweden)

Hernoni Septiani

2016-06-01

Full Text Available The increasing need for drinking water and undistributed raw water source needs solution with SPAM, Central Java Region. One of it is SPAM Bregas Region which the service locations are in Brebes Districts, Tegal City, and Tegal District. In the process of the transmission network development, there come some risks that hamper so that it needs for further research. This research aims to identify risks, analyze the amount of risk probability and give policy recommendations. The responden of study are government agencies as providers the transmission pipelines development projects of Bregas Regional SPAM, also the supervision consultants and contractors as a service providers. The method used is to spread the questionnaire to each stakeholder for structuring the risk of using RBS (Risk Breakdown Structure and multiplying the value of the impact and frequency to obtain the value of the level of risk for each risk factor. The results showed the risks that happened in transmission network development in Bregas Regional SPAM are different from each stakeholder. Risks from perception of the supervising consultant 21.95% is accepted, 18.29% mitigated, and 59.76% avoided. Risk from owner perseption 32.93% is accepted, 23.17% mitigated, and 43.90% avoided. Rizks from contractor perseption 23.17% is accepted, 29.27 mitigated, and 47.56% avoided. Overall stakeholder’s biggest risk is land acquisition.

Characterization of PhPRP1, a histidine domain arabinogalactan protein from Petunia hybrida pistils.

Science.gov (United States)

Twomey, Megan C; Brooks, Jenna K; Corey, Jillaine M; Singh-Cundy, Anu

2013-10-15

An arabinogalactan protein, PhPRP1, was purified from Petunia hybrida pistils and shown to be orthologous to TTS-1 and TTS-2 from Nicotiana tabacum and NaTTS from Nicotiana alata. Sequence comparisons among these proteins, and CaPRP1 from Capsicum annuum, reveal a conserved histidine-rich domain and two hypervariable domains. Immunoblots show that TTS-1 and PhPRP1 are also expressed in vegetative tissues of tobacco and petunia respectively. In contrast to the molecular mass heterogeneity displayed by the pistil proteins, the different isoforms found in seedlings, roots, and leaves each has a discrete size (37, 80, 160, and 200 kDa) on SDS-PAGE gels. On the basis of their chemistry, distinctive domain architecture, and the unique pattern of expression, we have named this group of proteins HD-AGPs (histidine domain-arabinogalactan proteins). Copyright © 2013 Elsevier GmbH. All rights reserved.

Analysis of Naïve Bayes Algorithm for Email Spam Filtering across Multiple Datasets

Science.gov (United States)

Fitriah Rusland, Nurul; Wahid, Norfaradilla; Kasim, Shahreen; Hafit, Hanayanti

2017-08-01

E-mail spam continues to become a problem on the Internet. Spammed e-mail may contain many copies of the same message, commercial advertisement or other irrelevant posts like pornographic content. In previous research, different filtering techniques are used to detect these e-mails such as using Random Forest, Naïve Bayesian, Support Vector Machine (SVM) and Neutral Network. In this research, we test Naïve Bayes algorithm for e-mail spam filtering on two datasets and test its performance, i.e., Spam Data and SPAMBASE datasets [8]. The performance of the datasets is evaluated based on their accuracy, recall, precision and F-measure. Our research use WEKA tool for the evaluation of Naïve Bayes algorithm for e-mail spam filtering on both datasets. The result shows that the type of email and the number of instances of the dataset has an influence towards the performance of Naïve Bayes.

"I Am Sorry to Send You SPAM": Cross-Cultural Differences in Use of Apologies in Email Advertising in Korea and the U.S.

Science.gov (United States)

Park, Hee Sun; Lee, Hye Eun; Song, Jeong An

2005-01-01

A series of studies investigating cultural differences in apology usage in unsolicited email advertising messages (i.e., SPAM) are reported. Study 1 documented that in comparison to American SPAM, a greater percentage of Korean SPAM included apologies. The next five studies ("Ns" = 516, 3132, 662, 524, 536) tested various explanations…

Interactions between whey proteins and salivary proteins as related to astringency of whey protein beverages at low pH.

Science.gov (United States)

Ye, A; Streicher, C; Singh, H

2011-12-01

Whey protein beverages have been shown to be astringent at low pH. In the present study, the interactions between model whey proteins (β-lactoglobulin and lactoferrin) and human saliva in the pH range from 7 to 2 were investigated using particle size, turbidity, and ζ-potential measurements and sodium dodecyl sulfate-PAGE. The correlation between the sensory results of astringency and the physicochemical data was discussed. Strong interactions between β-lactoglobulin and salivary proteins led to an increase in the particle size and turbidity of mixtures of both unheated and heated β-lactoglobulin and human saliva at pH ∼3.4. However, the large particle size and high turbidity that occurred at pH 2.0 were the result of aggregation of human salivary proteins. The intense astringency in whey protein beverages may result from these increases in particle size and turbidity at these pH values and from the aggregation and precipitation of human salivary proteins alone at pH salivary proteins in the interaction is a key factor in the perception of astringency in whey protein beverages. At any pH, the increases in particle size and turbidity were much smaller in mixtures of lactoferrin and saliva, which suggests that aggregation and precipitation may not be the only mechanism linked to the perception of astringency in whey protein. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A systematic framework to discover pattern for web spam classification

OpenAIRE

Jelodar, Hamed; Wang, Yongli; Yuan, Chi; Jiang, Xiaohui

2017-01-01

Web spam is a big problem for search engine users in World Wide Web. They use deceptive techniques to achieve high rankings. Although many researchers have presented the different approach for classification and web spam detection still it is an open issue in computer science. Analyzing and evaluating these websites can be an effective step for discovering and categorizing the features of these websites. There are several methods and algorithms for detecting those websites, such as decision t...

REVEALING NON-ALPHABETICAL GUISES OF SPAM-TRIGGER VOCABLES

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SERGIO A. ROJAS-GALEANO

2013-01-01

Full Text Available El 75% del correo electrónico que se transmite hoy en día, corresponde a mensajes masivos no solicitados (comúnmente denominados spam, lo que evidencia la necesidad de continuar fortaleciendo los mecanismos de protección contra su propagación. Uno de los tretas más ingeniosas utilizadas últimamente por los spammers para sobrepasar los filtros basados en comparación de texto, es el enmascaramiento de las palabras vedadas mediante substituciones con símbolos no alfabéticos, de manera que aún visualmente logren transmitir la semántica del término original (por ejemplo, enmascarando viagra como v1@gr@ o como v-i-a-g-r-a. En este artículo se discute una técnica simple pero efectiva para contrarrestar esta sutil trampa, que consiste en la adaptación de un algoritmo reconocido de apareamiento de textos para revelar efectivamente la similitud existente entre vocablos desencadenadores de filtros spam y variantes alfanuméricas enmascaradas.

CoSpa: A Co-training Approach for Spam Review Identification with Support Vector Machine

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Wen Zhang

2016-03-01

Full Text Available Spam reviews are increasingly appearing on the Internet to promote sales or defame competitors by misleading consumers with deceptive opinions. This paper proposes a co-training approach called CoSpa (Co-training for Spam review identification to identify spam reviews by two views: one is the lexical terms derived from the textual content of the reviews and the other is the PCFG (Probabilistic Context-Free Grammars rules derived from a deep syntax analysis of the reviews. Using SVM (Support Vector Machine as the base classifier, we develop two strategies, CoSpa-C and CoSpa-U, embedded within the CoSpa approach. The CoSpa-C strategy selects unlabeled reviews classified with the largest confidence to augment the training dataset to retrain the classifier. The CoSpa-U strategy randomly selects unlabeled reviews with a uniform distribution of confidence. Experiments on the spam dataset and the deception dataset demonstrate that both the proposed CoSpa algorithms outperform the traditional SVM with lexical terms and PCFG rules in spam review identification. Moreover, the CoSpa-U strategy outperforms the CoSpa-C strategy when we use the absolute value of decision function of SVM as the confidence.

Series Pneumatic Artificial Muscles (sPAMs) and Application to a Soft Continuum Robot.

Science.gov (United States)

Greer, Joseph D; Morimoto, Tania K; Okamura, Allison M; Hawkes, Elliot W

2017-01-01

We describe a new series pneumatic artificial muscle (sPAM) and its application as an actuator for a soft continuum robot. The robot consists of three sPAMs arranged radially round a tubular pneumatic backbone. Analogous to tendons, the sPAMs exert a tension force on the robot's pneumatic backbone, causing bending that is approximately constant curvature. Unlike a traditional tendon driven continuum robot, the robot is entirely soft and contains no hard components, making it safer for human interaction. Models of both the sPAM and soft continuum robot kinematics are presented and experimentally verified. We found a mean position accuracy of 5.5 cm for predicting the end-effector position of a 42 cm long robot with the kinematic model. Finally, closed-loop control is demonstrated using an eye-in-hand visual servo control law which provides a simple interface for operation by a human. The soft continuum robot with closed-loop control was found to have a step-response rise time and settling time of less than two seconds.

Ligand binding turns moth pheromone-binding protein into a pH sensor: effect on the Antheraea polyphemus PBP1 conformation.

Science.gov (United States)

Katre, Uma V; Mazumder, Suman; Prusti, Rabi K; Mohanty, Smita

2009-11-13

In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of approximately 1:1. Mutation of His(70) and His(95) to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.

The phosducin-like protein PhLP1 impacts regulation of glycoside hydrolases and light response in Trichoderma reesei

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Tisch Doris

2011-12-01

Full Text Available Abstract Background In the biotechnological workhorse Trichoderma reesei (Hypocrea jecorina transcription of cellulase genes as well as efficiency of the secreted cellulase mixture are modulated by light. Components of the heterotrimeric G-protein pathway interact with light-dependent signals, rendering this pathway a key regulator of cellulase gene expression. Results As regulators of heterotrimeric G-protein signaling, class I phosducin-like proteins, are assumed to act as co-chaperones for G-protein beta-gamma folding and exert their function in response to light in higher eukaryotes. Our results revealed light responsive transcription of the T. reesei class I phosducin-like protein gene phlp1 and indicate a light dependent function of PhLP1 also in fungi. We showed the functions of PhLP1, GNB1 and GNG1 in the same pathway, with one major output being the regulation of transcription of glycoside hydrolase genes including cellulase genes in T. reesei. We found no direct correlation between the growth rate and global regulation of glycoside hydrolases, which suggests that regulation of growth does not occur only at the level of substrate degradation efficiency. Additionally, PhLP1, GNB1 and GNG1 are all important for proper regulation of light responsiveness during long term exposure. In their absence, the amount of light regulated genes increased from 2.7% in wild type to 14% in Δphlp1. Besides from the regulation of degradative enzymes, PhLP1 was also found to impact on the transcription of genes involved in sexual development, which was in accordance with decreased efficiency of fruiting body formation in Δphlp1. The lack of GNB1 drastically diminished ascospore discharge in T. reesei. Conclusions The heterotrimeric G-protein pathway is crucial for the interconnection of nutrient signaling and light response of T. reesei, with the class I phosducin-like protein PhLP1, GNB1 and GNG1 acting as important nodes, which influence light

CoFea: A Novel Approach to Spam Review Identification Based on Entropy and Co-Training

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Wen Zhang

2016-11-01

Full Text Available With the rapid development of electronic commerce, spam reviews are rapidly growing on the Internet to manipulate online customers’ opinions on goods being sold. This paper proposes a novel approach, called CoFea (Co-training by Features, to identify spam reviews, based on entropy and the co-training algorithm. After sorting all lexical terms of reviews by entropy, we produce two views on the reviews by dividing the lexical terms into two subsets. One subset contains odd-numbered terms and the other contains even-numbered terms. Using SVM (support vector machine as the base classifier, we further propose two strategies, CoFea-T and CoFea-S, embedded with the CoFea approach. The CoFea-T strategy uses all terms in the subsets for spam review identification by SVM. The CoFea-S strategy uses a predefined number of terms with small entropy for spam review identification by SVM. The experiment results show that the CoFea-T strategy produces better accuracy than the CoFea-S strategy, while the CoFea-S strategy saves more computing time than the CoFea-T strategy with acceptable accuracy in spam review identification.

Side-effects of improving CERN's spam filter

CERN Multimedia

2004-01-01

To further improve the fight against spam (unsolicited email), CERN implemented in June a mechanism that returns electronic mail messages to the sender if the originating server is wrongly configured. This "reverse DNS lookup" is an increasingly widespread technique to identify whether the sending e-mail server is legitimate and has a valid host name. This change has nearly doubled the spam rejection efficiency at CERN. However, it has a known side-effect. A small number of correspondents are sending legitimate electronic mail to CERN from mail servers that are incorrectly configured (in technical terms, they do not have a reverse DNS registration). These persons will have their messages returned to them with an error message, provided their sender address is valid. The typical error message that the correspondent receives in such cases is "Relaying denied. No reverse DNS configured for IP". If a correspondent complains to you that emails they are sending you at CERN are being returned with...

Spam nation the inside story of organized cybercrime-from global epidemic to your front door

CERN Document Server

Krebs, Brian

2014-01-01

In Spam Nation, investigative journalist and cybersecurity expert Brian Krebs unmasks the criminal masterminds driving some of the biggest spam and hacker operations targeting Americans and their bank accounts. Tracing the rise, fall, and alarming resurrection of the digital mafia behind the two largest spam pharmacies-and countless viruses, phishing, and spyware attacks-he delivers the first definitive narrative of the global spam problem and its threat to consumers everywhere. Blending cutting-edge research, investigative reporting, and firsthand interviews, this terrifying true story reveals how we unwittingly invite these digital thieves into our lives every day. From unassuming computer programmers right next door to digital mobsters like "Cosma"-who unleashed a massive malware attack that has stolen thousands of Americans' logins and passwords-Krebs uncovers the shocking lengths to which these people will go to profit from our data and our wallets. Not only are hundreds of thousands of Americans expos...

Digestion products of the PH20 hyaluronidase inhibit remyelination.

Science.gov (United States)

Preston, Marnie; Gong, Xi; Su, Weiping; Matsumoto, Steven G; Banine, Fatima; Winkler, Clayton; Foster, Scott; Xing, Rubing; Struve, Jaime; Dean, Justin; Baggenstoss, Bruce; Weigel, Paul H; Montine, Thomas J; Back, Stephen A; Sherman, Larry S

2013-02-01

Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation. Mouse OPCs grown in vitro were analyzed for hyaluronidase expression and activity. Gain of function studies were used to define the hyaluronidases that blocked OPC maturation. Mouse and human demyelinating lesions were assessed for hyaluronidase expression. Digestion products from different hyaluronidases and a hyaluronidase inhibitor were tested for their effects on OPC maturation and functional remyelination in vivo. OPCs demonstrated hyaluronidase activity in vitro and expressed multiple hyaluronidases, including HYAL1, HYAL2, and PH20. HA digestion by PH20 but not other hyaluronidases inhibited OPC maturation into OLs. In contrast, inhibiting HA synthesis did not influence OPC maturation. PH20 expression was elevated in OPCs and reactive astrocytes in both rodent and human demyelinating lesions. HA digestion products generated by the PH20 hyaluronidase but not another hyaluronidase inhibited remyelination following lysolecithin-induced demyelination. Inhibition of hyaluronidase activity lead to increased OPC maturation and promoted increased conduction velocities through lesions. We determined that PH20 is elevated in demyelinating lesions and that increased PH20 expression is sufficient to inhibit OPC maturation and remyelination. Pharmacological inhibition of PH20 may therefore be an effective way to promote remyelination in multiple sclerosis and related conditions. Copyright © 2012 American Neurological Association.

Interactions involved in pH protection of the alphavirus fusion protein

Energy Technology Data Exchange (ETDEWEB)

Fields, Whitney; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

2015-12-15

The alphavirus membrane protein E1 mediates low pH-triggered fusion of the viral and endosome membranes during virus entry. During virus biogenesis E1 associates as a heterodimer with the transmembrane protein p62. Late in the secretory pathway, cellular furin cleaves p62 to the mature E2 protein and a peripheral protein E3. E3 remains bound to E2 at low pH, stabilizing the heterodimer and thus protecting E1 from the acidic pH of the secretory pathway. Release of E3 at neutral pH then primes the virus for fusion during entry. Here we used site-directed mutagenesis and revertant analysis to define residues important for the interactions at the E3–E2 interface. Our data identified a key residue, E2 W235, which was required for E1 pH protection and alphavirus production. Our data also suggest additional residues on E3 and E2 that affect their interacting surfaces and thus influence the pH protection of E1 during alphavirus exit.

pH modulates the binding of early growth response protein 1 transcription factor to DNA.

Science.gov (United States)

Mikles, David C; Bhat, Vikas; Schuchardt, Brett J; Deegan, Brian J; Seldeen, Kenneth L; McDonald, Caleb B; Farooq, Amjad

2013-08-01

The transcription factor early growth response protein (EGR)1 orchestrates a plethora of signaling cascades involved in cellular homeostasis, and its downregulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with an increase in pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as His382 by virtue of the fact that its replacement by nonionizable residues abolishes the pH dependence of the binding of EGR1 to DNA. Notably, His382 inserts into the major groove of DNA, and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, His382 is mainly conserved across other members of the EGR family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating the protein-DNA interactions that are central to this family of transcription factors. Collectively, our findings reveal an unexpected but a key step in the molecular recognition of the EGR family of transcription factors, and suggest that they may act as sensors of pH within the intracellular environment. © 2013 FEBS.

Measures for Controlling Spam Messages%垃圾短信控制措施

Institute of Scientific and Technical Information of China (English)

乐炯; 陈信刚; 洪亮

2016-01-01

With the rapid development of Short Message Service(SMS)value-added services,spam messages repre-sented by advertising messages and fraud messages are increasing,and therefore the mobile phone user's legitimate rights and interests,as the right to tranquility in life,right to communication freedom and right to privacy,etc. are infringed. In order to effectively control the number of spam messages,the paper comprehensively analyzes spam messages types and their transmitting channels. Some control measures of spam messages,as the function of over-clocking intercept,keyword intercept,black list,white list,differentiated quality of services and SMS signature, etc. are deployed in six important network elements of the spam messages transmitting channels,such as SMS cen-ter,SMS gateway,device for mass messages,Service Provider(SP) platform,SP management platform and industry SMS platform. Through the implementation of the measures for controlling spam messages,complaints on spam messages are relatively reduced,and therefore as mobile phone user's experience improves significantly.%随着短信增值业务的快速发展，以广告短信、欺诈短信为代表的垃圾短信日益增加，手机用户的生活安宁权、通信自由权、隐私权等合法权益受到侵害。为有效控制垃圾短信数量，本文对垃圾短信种类及其发送通道进行了全面梳理分析，在短信中心、短信网关、短信群发器、SP平台、SP管理平台、行业短信平台六个垃圾短信发送通道中的重要网元上部署了超频拦截功能、关键字拦截功能、黑名单功能、白名单功能、差异化服务质量保障功能、短信签名功能等垃圾短信控制措施。通过垃圾短信控制措施的实施，垃圾短信投诉量得到较好的控制，手机用户的感知明显提升。

Filtering SPAM in P2PSIP Communities with Web of Trust

Science.gov (United States)

Heikkilä, Juho; Gurtov, Andrei

Spam is a dominant problem on email systems today. One of the reasons is the lack of infrastructure for security and trust. As Voice over IP (VoIP) communication becomes increasingly popular, proliferation of spam calls is only a matter of time. As SIP identity scheme is practically similar to email, those share the same threats. We utilized Host Identity Protocol (HIP) to provide basic security, such as end-to-end encryption. To provide call filtering, however, other tools are needed. In this paper, we suggest applying trust paths familiar from the PGP web of trust to prevent unwanted communication in P2PSIP communities.

A Comparative Study with RapidMiner and WEKA Tools over some Classification Techniques for SMS Spam

Science.gov (United States)

Foozy, Cik Feresa Mohd; Ahmad, Rabiah; Faizal Abdollah, M. A.; Chai Wen, Chuah

2017-08-01

SMS Spamming is a serious attack that can manipulate the use of the SMS by spreading the advertisement in bulk. By sending the unwanted SMS that contain advertisement can make the users feeling disturb and this against the privacy of the mobile users. To overcome these issues, many studies have proposed to detect SMS Spam by using data mining tools. This paper will do a comparative study using five machine learning techniques such as Naïve Bayes, K-NN (K-Nearest Neighbour Algorithm), Decision Tree, Random Forest and Decision Stumps to observe the accuracy result between RapidMiner and WEKA for dataset SMS Spam UCI Machine Learning repository.

Proposal and Implementation of An IDS for Potential SMS Spam Signaling Messages on SS7

OpenAIRE

Yeboah, Paul Ntim

2016-01-01

Problem Description Reports on spam activities reaching telecom operators reveal flaws in routing for SMS on the Signaling System 7 network. This thesis consists of the following tasks: A comprehensive Study of vulnerabilities on the SS7 network. Proposal and implementation of an intrusion detection system to mitigate signaling for SMS spam on the SS7 network. Evaluation of the proposed detection method.

REPTREE CLASSIFIER FOR IDENTIFYING LINK SPAM IN WEB SEARCH ENGINES

Directory of Open Access Journals (Sweden)

S.K. Jayanthi

2013-01-01

Full Text Available Search Engines are used for retrieving the information from the web. Most of the times, the importance is laid on top 10 results sometimes it may shrink as top 5, because of the time constraint and reliability on the search engines. Users believe that top 10 or 5 of total results are more relevant. Here comes the problem of spamdexing. It is a method to deceive the search result quality. Falsified metrics such as inserting enormous amount of keywords or links in website may take that website to the top 10 or 5 positions. This paper proposes a classifier based on the Reptree (Regression tree representative. As an initial step Link-based features such as neighbors, pagerank, truncated pagerank, trustrank and assortativity related attributes are inferred. Based on this features, tree is constructed. The tree uses the feature inference to differentiate spam sites from legitimate sites. WEBSPAM-UK-2007 dataset is taken as a base. It is preprocessed and converted into five datasets FEATA, FEATB, FEATC, FEATD and FEATE. Only link based features are taken for experiments. This paper focus on link spam alone. Finally a representative tree is created which will more precisely classify the web spam entries. Results are given. Regression tree classification seems to perform well as shown through experiments.

Human Neuronal Calcium Sensor-1 Protein Avoids Histidine Residues To Decrease pH Sensitivity.

Science.gov (United States)

Gong, Yehong; Zhu, Yuzhen; Zou, Yu; Ma, Buyong; Nussinov, Ruth; Zhang, Qingwen

2017-01-26

pH is highly regulated in mammalian central nervous systems. Neuronal calcium sensor-1 (NCS-1) can interact with numerous target proteins. Compared to that in the NCS-1 protein of Caenorhabditis elegans, evolution has avoided the placement of histidine residues at positions 102 and 83 in the NCS-1 protein of humans and Xenopus laevis, possibly to decrease the conformational sensitivity to pH gradients in synaptic processes. We used all-atom molecular dynamics simulations to investigate the effects of amino acid substitutions between species on human NCS-1 by substituting Arg102 and Ser83 for histidine at neutral (R102H and S83H) and acidic pHs (R102H p and S83H p ). Our cumulative 5 μs simulations revealed that the R102H mutation slightly increases the structural flexibility of loop L2 and the R102H p mutation decreases protein stability. Community network analysis illustrates that the R102H and S83H mutations weaken the interdomain and strengthen the intradomain communications. Secondary structure contents in the S83H and S83H p mutants are similar to those in the wild type, whereas the global structural stabilities and salt-bridge probabilities decrease. This study highlights the conformational dynamics effects of the R102H and S83H mutations on the local structural flexibility and global stability of NCS-1, whereas protonated histidine decreases the stability of NCS-1. Thus, histidines at positions 102 and 83 may not be compatible with the function of NCS-1 whether in the neutral or protonated state.

Assessing the Factors Affecting the Amount of E-Mail Spam Delivery in a Public School District

Science.gov (United States)

Yasenchock, David A.

2013-01-01

Eighty to ninety percent of e-mail is considered unsolicited commercial communication, or spam. Not only does spam violate the privacy of users, but it also incurs societal costs associated with time-related losses, serves as a vehicle for cyber-crime, and threatens the success of e-commerce by lowering consumer confidence. This quantitative…

Utilizing Multi-Field Text Features for Efficient Email Spam Filtering

Directory of Open Access Journals (Sweden)

Wuying Liu

2012-06-01

Full Text Available Large-scale spam emails cause a serious waste of time and resources. This paper investigates the text features of email documents and the feature noises among multi-field texts, resulting in an observation of a power law distribution of feature strings within each text field. According to the observation, we propose an efficient filtering approach including a compound weight method and a lightweight field text classification algorithm. The compound weight method considers both the historical classifying ability of each field classifier and the classifying contribution of each text field in the current classified email. The lightweight field text classification algorithm straightforwardly calculates the arithmetical average of multiple conditional probabilities predicted from feature strings according to a string-frequency index for labeled emails storing. The string-frequency index structure has a random-sampling-based compressible property owing to the power law distribution and can largely reduce the storage space. The experimental results in the TREC spam track show that the proposed approach can complete the filtering task in low space cost and high speed, whose overall performance 1-ROCA exceeds the best one among the participators at the trec07p evaluation.

Evaluating Third-Party Bad Neighborhood Blacklists for Spam Detection

NARCIS (Netherlands)

Moreira Moura, Giovane; Sperotto, Anna; Sadre, R.; Pras, Aiko; Seon Hong, C.; Diao, Y.; De Turk, F.

The distribution of malicious hosts over the IP address space is far from being uniform. In fact, malicious hosts tend to be concentrate in certain portions of the IP address space, forming the so-called Bad Neighborhoods. This phenomenon has been previously exploited to filter Spam by means of Bad

Designing a Web Spam Classifier Based on Feature Fusion in the Layered Multi-Population Genetic Programming Framework

Directory of Open Access Journals (Sweden)

Amir Hosein KEYHANIPOUR

2013-11-01

Full Text Available Nowadays, Web spam pages are a critical challenge for Web retrieval systems which have drastic influence on the performance of such systems. Although these systems try to combat the impact of spam pages on their final results list, spammers increasingly use more sophisticated techniques to increase the number of views for their intended pages in order to have more commercial success. This paper employs the recently proposed Layered Multi-population Genetic Programming model for Web spam detection task as well application of correlation coefficient analysis for feature space reduction. Based on our tentative results, the designed classifier, which is based on a combination of easy to compute features, has a very reasonable performance in comparison with similar methods.

A pH Switch Regulates the Inverse Relationship between Membranolytic and Chaperone-like Activities of HSP-1/2, a Major Protein of Horse Seminal Plasma.

Science.gov (United States)

Kumar, C Sudheer; Swamy, Musti J

2016-07-05

HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch.

Functional characterization of PhGR and PhGRL1 during flower senescence in the petunia.

Science.gov (United States)

Yang, Weiyuan; Liu, Juanxu; Tan, Yinyan; Zhong, Shan; Tang, Na; Chen, Guoju; Yu, Yixun

2015-09-01

Petunia PhGRL1 suppression accelerated flower senescence and increased the expression of the genes downstream of ethylene signaling, whereas PhGR suppression did not. Ethylene plays an important role in flowers senescence. Homologous proteins Green-Ripe and Reversion to Ethylene sensitivity1 are positive regulators of ethylene responses in tomato and Arabidopsis, respectively. The petunia flower has served as a model for the study of ethylene response during senescence. In this study, petunia PhGR and PhGRL1 expression was analyzed in different organs, throughout floral senescence, and after exogenous ethylene treatment; and the roles of PhGR and PhGRL1 during petunia flower senescence were investigated. PhGRL1 suppression mediated by virus-induced gene silencing accelerated flower senescence and increased ethylene production; however, the suppression of PhGR did not. Taken together, these data suggest that PhGRL1 is involved in negative regulation of flower senescence, possibly via ethylene production inhibition and consequently reduced ethylene signaling activation.

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

Science.gov (United States)

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

Individual whey protein components influence lipid oxidation dependent on pH

DEFF Research Database (Denmark)

Horn, Anna Frisenfeldt; Nielsen, Nina Skall; Jacobsen, Charlotte

In emulsions, lipid oxidation is expected to be initiated at the oil-water interface. The properties of the emulsifier used and the composition at the interface is therefore expected to be of great importance for the resulting oxidation. Previous studies have shown that individual whey protein...... by affecting the preferential adsorption of whey protein components at the interface. The aim of the study was to compare lipid oxidation in 10% fish oil-in-water emulsions prepared with 1% whey protein having either a high concentration of α-lactalbumin, a high concentration of β-lactoglobulin or equal...... amounts of the two. Emulsions were prepared at pH4 and pH7. Emulsions were characterized by their droplet sizes, viscosities, and contents of proteins in the water phase. Lipid oxidation was assessed by PV and secondary volatile oxidation products. Results showed that pH greatly influenced the oxidative...

Novel functions of CCM1 delimit the relationship of PTB/PH domains.

Science.gov (United States)

Zhang, Jun; Dubey, Pallavi; Padarti, Akhil; Zhang, Aileen; Patel, Rinkal; Patel, Vipulkumar; Cistola, David; Badr, Ahmed

2017-10-01

Three NPXY motifs and one FERM domain in CCM1 makes it a versatile scaffold protein for tethering the signaling components together within the CCM signaling complex (CSC). The cellular role of CCM1 protein remains inadequately expounded. Both phosphotyrosine binding (PTB) and pleckstrin homology (PH) domains were recognized as structurally related but functionally distinct domains. By utilizing molecular cloning, protein binding assays and RT-qPCR to identify novel cellular partners of CCM1 and its cellular expression patterns; by screening candidate PTB/PH proteins and subsequently structurally simulation in combining with current X-ray crystallography and NMR data to defined the essential structure of PTB/PH domain for NPXY-binding and the relationship among PTB, PH and FERM domain(s). We identified a group of 28 novel cellular partners of CCM1, all of which contain either PTB or PH domain(s), and developed a novel classification system for these PTB/PH proteins based on their relationship with different NPXY motifs of CCM1. Our results demonstrated that CCM1 has a wide spectrum of binding to different PTB/PH proteins and perpetuates their specificity to interact with certain PTB/PH domains through selective combination of three NPXY motifs. We also demonstrated that CCM1 can be assembled into oligomers through intermolecular interaction between its F3 lobe in FERM domain and one of the three NPXY motifs. Despite being embedded in FERM domain as F3 lobe, F3 module acts as a fully functional PH domain to interact with NPXY motif. The most salient feature of the study was that both PTB and PH domains are structurally and functionally comparable, suggesting that PTB domain is likely evolved from PH domain with polymorphic structural additions at its N-terminus. A new β1A-strand of the PTB domain was discovered and new minimum structural requirement of PTB/PH domain for NPXY motif-binding was determined. Based on our data, a novel theory of structure, function and

WARCProcessor: An Integrative Tool for Building and Management of Web Spam Corpora

Directory of Open Access Journals (Sweden)

Miguel Callón

2017-12-01

Full Text Available In this work we present the design and implementation of WARCProcessor, a novel multiplatform integrative tool aimed to build scientific datasets to facilitate experimentation in web spam research. The developed application allows the user to specify multiple criteria that change the way in which new corpora are generated whilst reducing the number of repetitive and error prone tasks related with existing corpus maintenance. For this goal, WARCProcessor supports up to six commonly used data sources for web spam research, being able to store output corpus in standard WARC format together with complementary metadata files. Additionally, the application facilitates the automatic and concurrent download of web sites from Internet, giving the possibility of configuring the deep of the links to be followed as well as the behaviour when redirected URLs appear. WARCProcessor supports both an interactive GUI interface and a command line utility for being executed in background.

WARCProcessor: An Integrative Tool for Building and Management of Web Spam Corpora.

Science.gov (United States)

Callón, Miguel; Fdez-Glez, Jorge; Ruano-Ordás, David; Laza, Rosalía; Pavón, Reyes; Fdez-Riverola, Florentino; Méndez, Jose Ramón

2017-12-22

In this work we present the design and implementation of WARCProcessor, a novel multiplatform integrative tool aimed to build scientific datasets to facilitate experimentation in web spam research. The developed application allows the user to specify multiple criteria that change the way in which new corpora are generated whilst reducing the number of repetitive and error prone tasks related with existing corpus maintenance. For this goal, WARCProcessor supports up to six commonly used data sources for web spam research, being able to store output corpus in standard WARC format together with complementary metadata files. Additionally, the application facilitates the automatic and concurrent download of web sites from Internet, giving the possibility of configuring the deep of the links to be followed as well as the behaviour when redirected URLs appear. WARCProcessor supports both an interactive GUI interface and a command line utility for being executed in background.

pH and Protein Sensing with Functionalized Semiconducting Oxide Nanobelt FETs

Science.gov (United States)

Cheng, Yi; Yun, C. S.; Strouse, G. F.; Xiong, P.; Yang, R. S.; Wang, Z. L.

2008-03-01

We report solution pH sensing and selective protein detection with high-performance channel-limited field-effect transistors (FETs) based on single semiconducting oxide (ZnO and SnO2) nanobelts^1. The devices were integrated with PDMS microfluidic channels for analyte delivery and the source/drain contacts were passivated for in-solution sensing. pH sensing experiments were performed on FETs with functionalized and unmodified nanobelts. Functionalization of the nanobelts by APTES was found to greatly improve the pH sensitivity. The change in nanobelt conductance as functions of pH values at different gate voltages and ionic strengths showed high sensitivity and consistency. For the protein detection, we achieved highly selective biotinylation of the nanobelt channel with through APTES linkage. The specific binding of fluorescently-tagged streptavidin to the biotinylated nanobelt was verified by fluorescence microscopy; non-specific binding to the substrate was largely eliminated using PEG-silane passivation. The electrical responses of the biotinylated FETs to the streptavidin binding in PBS buffers of different pH values were systematically measured. The results will be presented and discussed. ^1Y. Cheng et al., Appl. Phys. Lett. 89, 093114 (2006). *Supported by NSF NIRT Grant ECS-0210332.

Anti-Spam Legislation in Consideration of Personal Data Protection and Other Legal Instruments

Czech Academy of Sciences Publication Activity Database

Matejka, Ján

2016-01-01

Roč. 6, č. 2 (2016), s. 90-114 ISSN 1805-8396 R&D Projects: GA ČR(CZ) GA16-26910S Institutional support: RVO:68378122 Keywords : spam * personal data protection * e- marketing Subject RIV: AG - Legal Sciences

Arrestin-related proteins mediate pH signaling in fungi

OpenAIRE

Herranz, Silvia; Rodríguez, José M.; Bussink, Henk-Jan; Sánchez-Ferrero, Juan C.; Arst, Herbert N.; Peñalva, Miguel A.; Vincent, Olivier

2005-01-01

Metazoan arrestins bind to seven-transmembrane (7TM) receptors to regulate function. Aspergillus nidulans PalF, a protein involved in the fungal ambient pH signaling pathway, contains arrestin N-terminal and C-terminal domains and binds strongly to two different regions within the C-terminal cytoplasmic tail of the 7TM, putative pH sensor PalH. Upon exposure to alkaline ambient pH, PalF is phosphorylated and, like mammalian β-arrestins, ubiquitinated in a signal-dependent and 7TM protein-depe...

Effect of initial protein concentration and pH on in vitro gastric digestion of heated whey proteins.

Science.gov (United States)

Zhang, Sha; Vardhanabhuti, Bongkosh

2014-02-15

The in vitro digestion of heated whey protein aggregates having different structure and physicochemical properties was evaluated under simulated gastric conditions. Aggregates were formed by heating whey protein isolates (WPI) at 3-9% w/w initial protein concentration and pH 3.0-7.0. Results showed that high protein concentration led to formation of larger WPI aggregates with fewer remaining monomers. Aggregates formed at high protein concentrations showed slower degradation rate compared to those formed at low protein concentration. The effect of initial protein concentration on peptide release pattern was not apparent. Heating pH was a significant factor affecting digestion pattern. At pH above the isoelectric point, the majority of the proteins involved in the aggregation, and aggregates formed at pH 6.0 were more susceptible to pepsin digestion than at pH 7.0. At acidic conditions, only small amount of proteins was involved in the aggregation and heated aggregates were easily digested by pepsin, while the remaining unaggregated proteins were very resistant to gastric digestion. The potential physiological implication of these results on satiety was discussed. Copyright © 2013 Elsevier Ltd. All rights reserved.

LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

Science.gov (United States)

Zhang, Chunxiao; Hoang, Nam; Leng, Feng; Saxena, Lovely; Lee, Logan; Alejo, Salvador; Qi, Dandan; Khal, Anthony; Sun, Hong; Lu, Fei; Zhang, Hui

2018-03-09

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences

DEFF Research Database (Denmark)

Huang, Honggang; Larsen, Martin Røssel; Karlsson, Anders H

2011-01-01

phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24¿h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1¿h, but lowest at 24¿h, whereas the slow pH decline group showed the reverse case. The same pattern was also...... observed in most individual bands in 1-DE. The protein phosphorylation levels of 12 bands were significantly affected by the synergy effects of pH and time (p......Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein...

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

International Nuclear Information System (INIS)

Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

2011-01-01

Highlights: ► APPL1 regulates the protein level of EGFR in response to EGF stimulation. ► Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. ► Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

Proof of concept of a "greener" protein purification/enrichment method based on carboxylate-terminated carbosilane dendrimer-protein interactions.

Science.gov (United States)

González-García, Estefanía; Maly, Marek; de la Mata, Francisco Javier; Gómez, Rafael; Marina, María Luisa; García, María Concepción

2016-11-01

Protein sample preparation is a critical and an unsustainable step since it involves the use of tedious methods that usually require high amount of solvents. The development of new materials offers additional opportunities in protein sample preparation. This work explores, for the first time, the potential application of carboxylate-terminated carbosilane dendrimers to the purification/enrichment of proteins. Studies on dendrimer binding to proteins, based on protein fluorescence intensity and emission wavelengths measurements, demonstrated the interaction between carboxylate-terminated carbosilane dendrimers and proteins at all tested pH levels. Interactions were greatly affected by the protein itself, pH, and dendrimer concentration and generation. Especially interesting was the interaction at acidic pH since it resulted in a significant protein precipitation. Dendrimer-protein interactions were modeled observing stable complexes for all proteins. Carboxylate-terminated carbosilane dendrimers at acidic pH were successfully used in the purification/enrichment of proteins extracted from a complex sample. Graphical Abstract Images showing the growing turbidity of solutions containing a mixture of proteins (lysozyme, myoglobin, and BSA) at different protein:dendrimer ratios (1:0, 1:1, 1:8, and 1:20) at acidic pH and SDS-PAGE profiles of the corresponsing supernatants. Comparison of SDS-PAGE profiles for the pellets obtained during the purification of proteins present in a complex sample using a conventional "no-clean" method based on acetone precipitation and the proposed "greener" method using carboxylate-terminated carbosilane dendrimer at a 1:20 protein:dendrimer ratio.

Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

Science.gov (United States)

Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

2015-08-04

Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging.

Science.gov (United States)

Poëa-Guyon, Sandrine; Pasquier, Hélène; Mérola, Fabienne; Morel, Nicolas; Erard, Marie

2013-05-01

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

Effect of the coexistence of sodium caseinate and Tween 20 as stabilizers of food emulsions at acidic pH.

Science.gov (United States)

Perugini, Luisa; Cinelli, Giuseppe; Cofelice, Martina; Ceglie, Andrea; Lopez, Francesco; Cuomo, Francesca

2018-02-05

In the present investigation the properties of edible nanoemulsions were studied. Sodium caseinate represents a good candidate for food emulsion preparations thanks to its surface-active properties and because it is perceived as a natural product by consumers. Nevertheless, it is very sensitive to acidic pH close to its isoelectric point and, if used as emulsion stabilizer, this aspect can negatively affect the emulsion stability. In order to prevent this drawback, sodium caseinate was used in combination with a non-ionic surfactant (Tween 20) as emulsifier of oil/water nanoemulsions. For these reasons, nanoemulsions stabilized by Tween 20, sodium caseinate and by a blend of the two emulsifiers were studied and compared according to their response to pH variations. Nanoemulsions were characterized for size of the dispersed phase with variation of time and temperature, for their rheological properties, for surface charge as a function of pH and for protein fluorescence. Noticeably, it was ascertained that, at pH close to caseinate isoelectric point, emulsions stabilized with the blend of caseinate and Tween 20 were more stable, compared with emulsions stabilized only with sodium caseinate. Such behavior was explained according to the composition of the emulsifiers at the oil/water interface where, at acidic pH, the presence of Tween 20 ensured the steric stabilization thus improving the role of sodium caseinate as emulsion stabilizer. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses.

Science.gov (United States)

Clark, Amelia M; Nogales, Aitor; Martinez-Sobrido, Luis; Topham, David J; DeDiego, Marta L

2017-09-01

In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up

β-1,3-1,4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi.

Science.gov (United States)

Xu, Ting; Zhu, Tianhui; Li, Shujiang

2016-02-01

Bacillus velezensis is a known antifungal bacteria. To understand the role of β-1, 3-1, 4-glucanase played on B. velezensis about the mechanism which exerts effect on fungi, we isolated and cloned the β-1, 3-1, 4-glucanase gene (Bglu1) from B. velezensis ZJ20. The Bglu1 open reading frame was 732 bp that encoded a protein with 243 amino acids and a calculated molecular weight of 27.3 kDa. The same gene without the signal peptide, termed Bglu2, was also cloned and expressed in E. coli BL21. Among the two variants, only Bglu2 protein was expressed. Purified Bglu2 could be eluted with imidazole solution at concentrations ranging from 100 to 500 mM although the highest expression was observed at 150 and 200 mM and the purest was at 500 mM. In addition, activity of the crude enzyme was 1527 U ml(-1) and the highest activity of the purified enzyme was 1706 U ml(-1). The purified β-1, 3-1, 4-glucanase had activity on a wide range of pH and temperatures and displayed optimal activity at pH 5.0 and 35 °C. More importantly, the mycelial morphology of three pathogenic fungi was destroyed by the purified β-1, 3-1, 4-glucanase. In conclusion, β-1, 3-1, 4-glucanase from B. velezensis ZJ20 can be highly expressed in E. coli BL21 and the recombinant protein is pathogenic to fungi.

[Ph-Sensor Properties of a Fluorescent Protein from Dendronephthya sp].

Science.gov (United States)

Pakhomov, A A; Chertkova, R V; Martynov, V I

2015-01-01

Genetically encoded biosensors based on fluorescent proteins are now widely applicable for monitoring pH changes in live cells. Here, we have shown that a fluorescent protein from Dendronephthya sp. (DendFP) exhibits a pronounced pH-sensitivity. Unlike most of known genetically encoded pH-sensors, fluorescence of the protein is not quenched upon medium acidification, but is shifting from the red to green spectral range. Therefore, quantitative measurements of intracellular pH are feasible by ratiometric comparison of emission intensities in the red and green spectral ranges, which makes DendFP advantageous compared with other genetically encoded pH-sensors.

Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein.

Science.gov (United States)

Fitzgerald, Kerry D; Semler, Bert L

2013-09-01

Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein

Science.gov (United States)

Fitzgerald, Kerry D.; Semler, Bert L.

2013-01-01

Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. PMID:23830997

Cation modulation of hemoglobin interaction with sodium n-dodecyl sulphate (SDS iv: magnesium modulation at pH 7.20

Directory of Open Access Journals (Sweden)

Ali Akbar Moosavi-Movahedi

2016-03-01

Full Text Available We investigate the interaction of Mg2+ (0–2.30 mM and sodium n-dodecyl sulfate (SDS with hemoglobins (Hbs A and S at pH 7.20. SDS was used to model both membranes (0.60 mM SDS and proteases (5.0 mM SDS. Via UV-visible spectroscopy, second derivative and difference second derivative spectroscopy, we interrogated for difference(s in the interaction of these ligands with the proteins that can account for the HbS resistance to malaria parasite while been prone to sickling. Our results show that Mg2+ interaction with the proteins lowered the HbS oxygen affinity in comparison with the HbA. Additionally, [SDS]-protein interactions resulted in oxoferryl heme species formation that was prominent for the HbA and highly diminished for the HbS. [Mg2+] introduction to the [SDS]-protein mixture, however decreased the concentration of denatured protein species. The [Mg2+]-[SDS]-protein interactions suggest that while ionic or coulomb interactions for the HbA, in the presence of the surfactants, are [Mg2+] dependent, those of the HbS are not. Furthermore, hydrophobicity is a crucial force for the HbS interaction at neutral pH and is little-masked by ionic, electrostatic or coulombic interactions. In conclusion, at physiological pH, the Mg-SDS interaction decreased the HbS denaturation in comparison to the HbA.

Non-sticky translocation of bio-molecules through Tween 20-coated solid-state nanopores in a wide pH range

Science.gov (United States)

Li, Xiaoqing; Hu, Rui; Li, Ji; Tong, Xin; Diao, J. J.; Yu, Dapeng; Zhao, Qing

2016-10-01

Nanopore-based sensing technology is considered high-throughput and low-cost for single molecule detection, but solid-state nanopores have suffered from pore clogging issues. A simple Tween 20 coating method is applied to ensure long-term (several hours) non-sticky translocation of various types of bio-molecules through SiN nanopores in a wide pH range (4.0-13.0). We also emphasize the importance of choosing appropriate concentration of Tween 20 coating buffer for desired effect. By coating nanopores with a Tween 20 layer, we are able to differentiate between single-stranded DNA and double-stranded DNA, to identify drift-dominated domain for single-stranded DNA, to estimate BSA volume and to observe the shape of individual nucleosome translocation event without non-specific adsorption. The wide pH endurance from 4.0 to 13.0 and the broad types of detection analytes including nucleic acids, proteins, and biological complexes highlight the great application potential of Tween 20-coated solid-state nanopores.

Behind the Spam: A ``Spectral Analysis'' of Predatory Publishers

Science.gov (United States)

Beall, Jeffrey

2016-10-01

Most researchers today are bombarded with spam email solicitations from questionable scholarly publishers. These emails solicit article manuscripts, editorial board service, and even ad hoc peer reviews. These ``predatory'' publishers exploit the scholarly publishing process, patterning themselves after legitimate scholarly publishers yet performing little or no peer review and quickly accepting submitted manuscripts and collecting fees from submitting authors. These counterfeit publishers and journals have published much junk science? especially in the field of cosmology? threatening the integrity of the academic record. This paper examines the current state of predatory publishing and advises researchers how to navigate scholarly publishing to best avoid predatory publishers and other scholarly publishing-related perils.

A Genetically Encoded pH Sensor for Tracking Surface Proteins through Endocytosis**

OpenAIRE

Grover, Anmol; Schmidt, Brigitte F.; Salter, Russell D.; Watkins, Simon C.; Waggoner, Alan S.; Bruchez, Marcel P.

2012-01-01

We have combined our fluorogen activating peptide[1] with a new tandem dye molecule to develop a biosensor that labels a cell-surface protein and displays an easily detectable pH dependent emission color change by efficient intramolecular Förster resonant energy transfer. This probe has demonstrated pH variations in β2-adrenergic receptor trafficking and revealed a process of surface to endosome inter-cellular transfer in dendritic cells with potential significance in antigen transfer.

Market-oriented Means to Control the Spam Messages%用市场化手段治理垃圾短信顽疾

Institute of Scientific and Technical Information of China (English)

秦晓飞; 张敏

2013-01-01

After nearly 10 years of continuous governance, spam messages still cannot be treated radical, which is still the major obstacle for telecom operators to improve customer satisfactions. In this paper, in order to control the spam messages, the reasons for repeated spam messages were analyzed and the market-oriented methods were proposed to break the industry chain and find a win-win governance pattern for each side to guide it to user-accepted mobile advertising. Therefore, the spam message could be controlled radically.%经过近10年的持续治理，垃圾短信仍然没有得到根治，依然是影响各大电信运营商客户满意度提升的顽疾。分析了垃圾短信屡禁不止的原因，并针对垃圾短信的治理困局，试图通过市场化的手段，打破垃圾短信的成熟产业链结构，找到一种各方共赢的垃圾短信治理方式，引导垃圾短信成为用户认可的手机广告，从而推动垃圾短信得到根治。

Exploration of gaps in Bitly's spam detection and relevant counter measures

OpenAIRE

Gupta, Neha; Kumaraguru, Ponnurangam

2014-01-01

Existence of spam URLs over emails and Online Social Media (OSM) has become a growing phenomenon. To counter the dissemination issues associated with long complex URLs in emails and character limit imposed on various OSM (like Twitter), the concept of URL shortening gained a lot of traction. URL shorteners take as input a long URL and give a short URL with the same landing page in return. With its immense popularity over time, it has become a prime target for the attackers giving them an adva...

PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila.

Directory of Open Access Journals (Sweden)

Donghoon M Lee

Full Text Available The recruitment of GDP/GTP exchange factors (GEFs to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.

Turnover-dependent inactivation of the nitrogenase MoFe-protein at high pH.

Science.gov (United States)

Yang, Kun-Yun; Haynes, Chad A; Spatzal, Thomas; Rees, Douglas C; Howard, James B

2014-01-21

Proton uptake accompanies the reduction of all known substrates by nitrogenase. As a consequence, a higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high-pH approach would appear to be problematic in view of the observation reported by Pham and Burgess [(1993) Biochemistry 32, 13725-13731] that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that the MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While a normal, albeit low, level of substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially, the enzyme is reversibly inhibited for substrate reduction at pH 9.5, yet in a second, slower process, the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to that of the native MoFe-protein, yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by formation of a tight complex with the Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanism-based transformation that may provide a new probe of nitrogenase catalysis.

pH and Heat Resistance of the Major Celery Allergen Api g 1.

Science.gov (United States)

Rib-Schmidt, Carina; Riedl, Philipp; Meisinger, Veronika; Schwaben, Luisa; Schulenborg, Thomas; Reuter, Andreas; Schiller, Dirk; Seutter von Loetzen, Christian; Rösch, Paul

2018-05-25

The major celery allergen Api g 1 is a member of the pathogenesis-related 10 class protein family. Here we aimed to investigate the impact of heat and pH on the native protein conformation required for Immunoglobulin E (IgE) recognition. Spectroscopic methods, MS and IgE binding analyses were used to study the effects of pH and thermal treatment on Api g 1.0101. Heat processing results in a loss of the native protein fold via denaturation, oligomerisation and precipitation along with a subsequent reduction of IgE recognition. The induced effects and timescales are strongly pH depended. While Api g 1 refolds partially into an IgE-binding conformation at physiological pH, acidic pH treatment leads to the formation of structurally heat resistant, IgE-reactive oligomers. Thermal processing in the presence of a celery matrix or at pH conditions close to the isoelectric point (pI = 4.63) of Api g 1.0101 results in almost instant precipitation. Our data demonstrate that Api g 1.0101 is not intrinsically susceptible to heat treatment in vitro. However, the pH and the celery matrix strongly influence the stability of Api g 1.0101 and might be the main reasons for the observed temperature lability of this important food allergen. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

An Inexpensive and Easy Ultrasound Phantom: A Novel Use for SPAM.

Science.gov (United States)

Nolting, Laura; Hunt, Patrick; Cook, Thomas; Douglas, Barton

2016-04-01

Ultrasound models, commonly referred to as "phantoms," are simulation tools for ultrasound education. Commercially produced phantoms are available, but there are "homemade" alternatives such as raw poultry and gelatin molds. Precooked, processed meat, better known as SPAM (Hormel Foods Corporation, Austin, MN), can be used as an ultrasound phantom to teach several ultrasound applications. It is a versatile, hygienic, and easily manipulated medium that does not require refrigeration or preparatory work and can be easily discarded at the end of use. © 2016 by the American Institute of Ultrasound in Medicine.

Imaging Intracellular pH in Live Cells with a Genetically-Encoded Red Fluorescent Protein Sensor

OpenAIRE

Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

2011-01-01

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically-encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at...

Influence of pH value on microstructure of oil-in-water emulsions stabilized by chickpea protein flour.

Science.gov (United States)

Felix, Manuel; Isurralde, Nadia; Romero, Alberto; Guerrero, Antonio

2018-01-01

Food industry is highly interested in the development of healthier formulations of oil-in-water emulsions, stabilized by plant proteins instead of egg or milk proteins. These emulsions would avoid allergic issues or animal fat. Among other plant proteins, legumes are a cost-competitive product. This work evaluates the influence of pH value (2.5, 5.0 and 7.5) on emulsions stabilized by chickpea-based emulsions at two different protein concentration (2.0 and 4.0 wt%). Microstructure of chickpea-based emulsions is assessed by means of backscattering, droplet size distributions and small amplitude oscillatory shear measurements. Visual appearances as well as confocal laser scanning microscopy images are obtained to provide useful information on the emulsions structure. Interestingly, results indicate that the pH value and protein concentration have a strong influence on emulsion microstructure and stability. Thus, the system which contains protein surfaces positively charged shows the highest viscoelastic properties, a good droplet size distribution profile and non-apparent destabilization phenomena. Interestingly, results also reveal the importance of rheological measurements in the prediction of protein interactions and emulsion stability since this technique is able to predict destabilization mechanisms sooner than other techniques such as backscattering or droplet size distribution measurements.

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

OpenAIRE

Kadamur, Ganesh; Ross, Elliott M.

2016-01-01

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in...

Evolution of pH buffers and water homeostasis in eukaryotes: homology between humans and Acanthamoeba proteins.

Science.gov (United States)

Baig, Abdul M; Zohaib, R; Tariq, S; Ahmad, H R

2018-02-01

This study intended to trace the evolution of acid-base buffers and water homeostasis in eukaryotes. Acanthamoeba castellanii  was selected as a model unicellular eukaryote for this purpose. Homologies of proteins involved in pH and water regulatory mechanisms at cellular levels were compared between humans and A. castellanii. Amino acid sequence homology, structural homology, 3D modeling and docking prediction were done to show the extent of similarities between carbonic anhydrase 1 (CA1), aquaporin (AQP), band-3 protein and H + pump. Experimental assays were done with acetazolamide (AZM), brinzolamide and mannitol to observe their effects on the trophozoites of  A. castellanii.  The human CA1, AQP, band-3 protein and H + -transport proteins revealed similar proteins in Acanthamoeba. Docking showed the binding of AZM on amoebal AQP-like proteins.  Acanthamoeba showed transient shape changes and encystation at differential doses of brinzolamide, mannitol and AZM.  Conclusion: Water and pH regulating adapter proteins in Acanthamoeba and humans show significant homology, these mechanisms evolved early in the primitive unicellular eukaryotes and have remained conserved in multicellular eukaryotes.

PhDAHP1 is required for floral volatile benzenoid/phenylpropanoid biosynthesis in Petunia × hybrida cv 'Mitchell Diploid'.

Science.gov (United States)

Langer, Kelly M; Jones, Correy R; Jaworski, Elizabeth A; Rushing, Gabrielle V; Kim, Joo Young; Clark, David G; Colquhoun, Thomas A

2014-07-01

Floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis consists of numerous enzymatic and regulatory processes. The initial enzymatic step bridging primary metabolism to secondary metabolism is the condensation of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) carried out via 3-DEOXY-D-ARABINO-HEPTULOSONATE-7-PHOSPHATE (DAHP) synthase. Here, identified, cloned, localized, and functionally characterized were two DAHP synthases from the model plant species Petunia × hybrida cv 'Mitchell Diploid' (MD). Full-length transcript sequences for PhDAHP1 and PhDAHP2 were identified and cloned using cDNA SMART libraries constructed from pooled MD corolla and leaf total RNA. Predicted amino acid sequence of PhDAHP1 and PhDAHP2 proteins were 76% and 80% identical to AtDAHP1 and AtDAHP2 from Arabidopsis, respectively. PhDAHP1 transcript accumulated to relatively highest levels in petal limb and tube tissues, while PhDAHP2 accumulated to highest levels in leaf and stem tissues. Through floral development, PhDAHP1 transcript accumulated to highest levels during open flower stages, and PhDAHP2 transcript remained constitutive throughout. Radiolabeled PhDAHP1 and PhDAHP2 proteins localized to plastids, however, PhDAHP2 localization appeared less efficient. PhDAHP1 RNAi knockdown petunia lines were reduced in total FVBP emission compared to MD, while PhDAHP2 RNAi lines emitted 'wildtype' FVBP levels. These results demonstrate that PhDAHP1 is the principal DAHP synthase protein responsible for the coupling of metabolites from primary metabolism to secondary metabolism, and the ultimate biosynthesis of FVBPs in the MD flower. Copyright © 2014 Elsevier Ltd. All rights reserved.

Model for optimising the execution of anti-spam filters

Directory of Open Access Journals (Sweden)

David Ruano-Ordás

2016-12-01

Full Text Available During last years, the combination of several filtering techniques for the development of anti-spam systems has gained a enormous popularity. However, although the accuracy achieved by these models has increased considerably, its use has entailed the emergence of new challenges such as the need to reduce the excessive use of computational resources, the increase of filtering speed and the adjustment of the weights used for the combination of several filtering techniques. In order to achieve this goal we have been refined several aspects including: (i the design and development of small technical improvements to increase the overall performance of the filter, (ii application of genetic algorithms to increase filtering accuracy and (iii the use of scheduling algorithms to improve filtering throughput.

Behind the Spam: A "Spectral Analysis" of Predatory Publishers

Science.gov (United States)

Beall, Jeffrey

2015-08-01

Most researchers today are bombarded with spam email solicitations from questionable scholarly publishers. These emails solicit article manuscripts, editorial board service, and even ad hoc peer reviews. These "predatory" publishers exploit the scholarly publishing process, patterning themselves after legitimate scholarly publishers yet performing little or no peer review and quickly accepting submitted manuscripts and collecting fees from submitting authors. These counterfeit publishers and journals have published much junk science — especially in the field of cosmology — threatening the integrity of the academic record. This presentation examines the current state of predatory publishing and related scams such as fake impact factors and advises researchers how to navigate scholarly publishing to best avoid predatory publishers and other scholarly publishing-related perils.

Functional and rheological properties of proteins in frozen turkey breast meat with different ultimate pH.

Science.gov (United States)

Chan, J T Y; Omana, D A; Betti, M

2011-05-01

Functional and rheological properties of proteins from frozen turkey breast meat with different ultimate pH at 24 h postmortem (pH(24)) have been studied. Sixteen breast fillets from Hybrid Tom turkeys were initially selected based on lightness (L*) values for each color group (pale, normal, and dark), with a total of 48 breast fillets. Further selection of 8 breast samples was made within each class of meat according to the pH(24). The average L* and pH values of the samples were within the following range: pale (L* >52; pH ≤5.7), normal (46 meat, respectively. Ultimate pH did not cause major changes in the emulsifying and foaming properties of the extracted sarcoplasmic and myofibrillar proteins. An SDS-PAGE profile of proteins from low and normal pH meat was similar, which revealed that the extent of protein denaturation was the same. Low pH meat had the lowest water-holding capacity compared with normal and high pH meat as shown by the increase in cooking loss, which can be explained by factors other than protein denaturation. Gel strength analysis and folding test revealed that gel-forming ability was better for high pH meat compared with low and normal pH meat.Dynamic viscoelastic behavior showed that myosin denaturation temperature was independent of pH(24). Normal and high pH meat had similar hardness, springiness, and chewiness values as revealed by texture profile analysis. The results from this study indicate that high pH meat had similar or better functional properties than normal pH meat. Therefore, high pH meat is suitable for further processed products, whereas low pH meat may need additional treatment or ingredient formulations to improve its functionality.

GRP1 PH Domain, Like AKT1 PH Domain, Possesses a Sentry Glutamate Residue Essential for Specific Targeting to Plasma Membrane PI(3,4,5)P3

Science.gov (United States)

Pilling, Carissa; Landgraf, Kyle E.; Falke, Joseph J.

2011-01-01

During the appearance of the signaling lipid PI(3,4,5)P3, an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P3-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P2 and bind the rare PI(3,4,5)P3 target lipid with sufficiently high affinity. Our previous study of the E17K mutant of protein kinase B (AKT1) PH domain, together with evidence from Carpten et al (1), revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P2, thereby playing an essential role in specific PI(3,4,5)P3 targeting (2). The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P3-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P2 affinity and constitutive plasma membrane targeting. To test this hypothesis the present study investigates the E345 residue, a putative sentry glutamate, of General Receptor for Phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into GRP1 PH domain enhances PI(4,5)P2 affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P2 releases E345K GRP1 PH domain into the cytoplasm and the efficiency of this release increases when target Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K (1, 3). Analysis of available PH domain structures suggests that a lone glutamate residue (or, in some cases an aspartate) is a common, perhaps ubiquitous, feature of PI(3,4,5)P3-specific binding

Degradation kinetics of fisetin and quercetin in solutions affected by medium pH, temperature and co-existed proteins

Directory of Open Access Journals (Sweden)

Wang Jing

2016-01-01

Full Text Available Impacts of medium pH, temperature and coexisted proteins on the degradation of two flavonoids fisetin and quercetin were assessed by spectroscopic method in the present study. Based on the measured degradation rate constants (k, fisetin was more stable than quercetin in all cases. Increasing medium pH from 6.0 to 7.5 at 37°C enhanced respective k values of fisetin and quercetin from 8.30x10−3 and 2.81x10−2 to 0.202 and 0.375 h-1 (P<0.05. In comparison with their degradation at 37°C, fisetin and quercetin showed larger k values at higher temperature (0.124 and 0.245 h−1 at 50°C, or 0.490 and 1.42 h−1 at 65°C. Four protein products in medium could stabilize the two flavonoids (P<0.05, as these proteins at 0.10 g L-1 decreased respective k values of fisetin and quercetin to 2.28x10−2-2.98x10−2 and 4.37´10−2-5.97x10−2 h−1. Hydrophobic interaction between the proteins and the two flavonoids was evidenced responsible for the stabilization, as sodium dodecyl sulfate could destroy the stabilization significantly (P<0.05. Casein and soybean protein provided greater stabilization than whey protein isolate. It is thus concluded that higher temperature and alkaline pH can enhance flavonoid loss, whereas coexisted proteins as flavonoid stabilizers can inhibit flavonoid degradation.

A protein-dye hybrid system as a narrow range tunable intracellular pH sensor.

Science.gov (United States)

Anees, Palapuravan; Sudheesh, Karivachery V; Jayamurthy, Purushothaman; Chandrika, Arunkumar R; Omkumar, Ramakrishnapillai V; Ajayaghosh, Ayyappanpillai

2016-11-18

Accurate monitoring of pH variations inside cells is important for the early diagnosis of diseases such as cancer. Even though a variety of different pH sensors are available, construction of a custom-made sensor array for measuring minute variations in a narrow biological pH window, using easily available constituents, is a challenge. Here we report two-component hybrid sensors derived from a protein and organic dye nanoparticles whose sensitivity range can be tuned by choosing different ratios of the components, to monitor the minute pH variations in a given system. The dye interacts noncovalently with the protein at lower pH and covalently at higher pH, triggering two distinguishable fluorescent signals at 700 and 480 nm, respectively. The pH sensitivity region of the probe can be tuned for every unit of the pH window resulting in custom-made pH sensors. These narrow range tunable pH sensors have been used to monitor pH variations in HeLa cells using the fluorescence imaging technique.

Precipitation and ultimate pH effect on chemical and gelation properties of protein prepared by isoelectric solubilization/precipitation process from pale, soft, exudative (PSE)-like chicken breast meat1.

Science.gov (United States)

Zhao, X; Xing, T; Chen, X; Han, M-Y; Li, X; Xu, X-L; Zhou, G-H

2017-05-01

Pale, soft, exudative (PSE)-like chicken breast is considered deteriorated raw material in the poultry meat industry that has inferior processing ability. The chemical and gelation properties of PSE-like chicken breast meat paste were studied. These pastes were prepared by the pH adjustment method and protein isolation using the isoelectric solubilization/precipitation (ISP) process from PSE-like chicken meat. The ISP-isolated samples were solubilized at pH 11.0 and recovered at pH 5.5 and 6.2. The ultimate pH of the ISP-isolated protein and meat paste was adjusted to 6.2 and 7.0. The ultimate pH in this article referred to the final pH of the extracted protein and meat paste. Higher reactive sulfhydryl content and surface hydrophobicity were found in the precipitation at pH 6.2 than at pH 5.5. However, various ultimate pH values showed no significant influence on the surface hydrophobicity. The hardness of gel, as measured by textural profile analysis, was improved using 6.2 as the precipitation pH compared with pH 5.5. The viscoelastic modulus (G΄) of gel pastes prior to the thermal gelation was higher with ISP treatment. However, lower G΄ was seen after thermal gelation compared with the control. Dynamic rheological measurement demonstrated a different gel-forming mechanism for protein precipitated at pH values of 5.5 and 6.2 compared with the meat paste. The cooking loss showed that the recovered protein failed to form a gel with good water-retention capacity unless the ultimate pH was adjusted to 7.0. Gels made from protein extracted by the ISP method had higher yellowness and lower redness values, probably due to protein denaturation. Precipitation at pH 6.2 formed a harder gel with lower water-retention ability than that at pH 5.5, and this result was possibly due to higher surface hydrophobicity and S-S bridge formation. Overall, network characteristics of ISP-treated protein gels were strongly dependent on precipitation pH and ultimate pH. © 2016

Similarity of salt influences on the pH of buffers, polyelectrolytes, and proteins.

Science.gov (United States)

Voinescu, Alina E; Bauduin, Pierre; Pinna, M Cristina; Touraud, Didier; Ninham, Barry W; Kunz, Werner

2006-05-04

Changes in pH induced by the addition of electrolytes to buffers, polyelectrolytes (a polycarboxy polymethylene and a polyethyleneimine), and proteins (casein, whey, and lysozyme) solutions are explored systematically. The two buffer systems are triethanolamine/triethanolammonium chloride and citric acid/sodium citrate. These are chosen because of the similarity of their acid-base equilibria with those of amino acids predominant in most proteins, that is, amino acids that include carboxylate or ammonium groups in their structures. The pH of triethanolamine and of citrate buffers respectively increases and decreases when salt is added. At low electrolyte concentrations (buffer solutions. It is even possible to qualitatively predict these changes in protein solutions simply from the primary protein structure. At least in the systems considered here, the specific ion effects on pH seem to correlate with the bulk activity coefficients of the added electrolytes, at least at moderate salt concentrations.

Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1.

Science.gov (United States)

Berry, Robert E; Yang, Fei; Shokhireva, Tatiana K; Amoia, Angela M; Garrett, Sarah A; Goren, Allena M; Korte, Stephanie R; Zhang, Hongjun; Weichsel, Andrzej; Montfort, William R; Walker, F Ann

2015-01-20

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer-dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and (1)H{(15)N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The "closed loop" form of the A-B and G-H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.

Extracting Tenebrio molitor protein while preventing browning: effect of pH and NaCl on protein yield

NARCIS (Netherlands)

Yi, L.; Boekel, van T.; Lakemond, C.M.M.

2017-01-01

The potential of insects as an alternative protein source for food applications was investigated by studying the effect of pH and NaCl on extraction yield of water-soluble proteins from Tenebrio molitor, while preventing browning due to polyphenol oxidation. Minimum protein solubility (29.6%) was at

Functionality of whey proteins covalently modified by allyl isothiocyanate. Part 1 physicochemical and antibacterial properties of native and modified whey proteins at pH 2 to 7

NARCIS (Netherlands)

Keppler, Julia Katharina; Martin, Dierk; Garamus, Vasil M.; Berton-Carabin, Claire; Nipoti, Elia; Coenye, Tom; Schwarz, Karin

2017-01-01

Whey protein isolate (WPI) (∼75% β-lactoglobulin (β-LG)) is frequently used in foods as a natural emulsifying agent. However, at an acidic pH value, its emulsification capacity is greatly reduced. The covalent attachment of natural electrophilic hydrophobic molecules to WPI proteins is a

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor.

Science.gov (United States)

Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

2011-07-06

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.

Catalyst of D. radiodurans LexA protein and its induced catalysis at alkaline pH activated by RecA

International Nuclear Information System (INIS)

Kong Xiangrong; Liu Fenju; Su Liaoyuan

2003-01-01

It is to study the catalytic reaction of LexA at alkaline pH. It has been observed that reaction has taken place under two conditions of D. radiodurans and LexA. Methods: The method of SDS polypropylene amine was adopted to make dyeing with R-250. Results: The result showed that there were no reactions when LexA protein turned to be at pH 7.0 and pH 8.0, the entire LexA protein was decreased a little at pH 9.0, while at pH 10.0, LexA protein started to decompose greatly, the fractions of protein can be seen obviously in the electrophoresis belt. Conclusion: The D. radiodurans and LexA protein will automatically drop at 37 degree C when D radiodurans LexA is at higher pH value, and its dependence on alkaline pH conditions reflects that the catalytic needs to be activated by the reaction of de- pH indicator of LexA protein ionization

Synergistic enhancement in the co-gelation of salt-soluble pea proteins and whey proteins.

Science.gov (United States)

Wong, Douglas; Vasanthan, Thava; Ozimek, Lech

2013-12-15

This paper investigated the enhancement of thermal gelation properties when salt-soluble pea proteins were co-gelated with whey proteins in NaCl solutions, using different blend ratios, total protein concentrations, pH, and salt concentrations. Results showed that the thermal co-gelation of pea/whey proteins blended in ratio of 2:8 in NaCl solutions showed synergistic enhancement in storage modulus, gel hardness, paste viscosity and minimum gelation concentrations. The highest synergistic enhancement was observed at pH 6.0 as compared with pH 4.0 and 8.0, and at the lower total protein concentration of 10% as compared with 16% and 22% (w/v), as well as in lower NaCl concentrations of 0.5% and 1.0% as compared with 1.5%, 2.0%, 2.5%, and 3.0% (w/v). The least gelation concentrations were also lower in the different pea/whey protein blend ratios than in pure pea or whey proteins, when dissolved in 1.0% or 2.5% (w/v) NaCl aqueous solutions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Interaction proteomics analysis of polycomb proteins defines distinct PRC1 complexes in mammalian cells

DEFF Research Database (Denmark)

Vandamme, Julien; Völkel, Pamela; Rosnoblet, Claire

2011-01-01

Polycomb group (PcG) proteins maintain transcriptional repression of hundreds of genes involved in development, signaling or cancer using chromatin-based epigenetic mechanisms. Biochemical studies in Drosophila have revealed that PcG proteins associate in at least two classes of protein complexes...... known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Drosophila core PRC1 is composed of four subunits, Polycomb (Pc), Sex combs extra (Sce), Polyhomeotic (Ph), and Posterior sex combs (Psc). Each of these proteins has multiple orthologs in vertebrates classified respectively as the CBX, RING...... in order to identify interacting partners of CBX family proteins under the same experimental conditions. Our analysis identified with high confidence about 20 proteins co-eluted with CBX2 and CBX7 tagged proteins, about 40 with CBX4, and around 60 with CBX6 and CBX8. We provide evidences that the CBX...

Characterization of Two 20kDa-Cement Protein (cp20k) Homologues in Amphibalanus amphitrite

KAUST Repository

He, Li-Sheng

2013-05-22

The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k) in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT), suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing. © 2013 He et al.

Characterization of Two 20kDa-Cement Protein (cp20k) Homologues in Amphibalanus amphitrite

KAUST Repository

He, Li-Sheng; Zhang, Gen; Qian, Pei-Yuan

2013-01-01

The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k) in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT), suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing. © 2013 He et al.

Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold

Science.gov (United States)

Mazzer, Alice R.; Perraud, Xavier; Halley, Jennifer; O’Hara, John; Bracewell, Daniel G.

2015-01-01

Protein A chromatography is a near-ubiquitous method of mAb capture in bioprocesses. The use of low pH buffer for elution from protein A is known to contribute to product aggregation. Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly. This paper presents a well-defined method for investigating this problem. An IgG4 was incubated in elution buffer after protein A chromatography (typical of the viral inactivation hold) and the quantity of monomer in neutralised samples was determined by size exclusion chromatography; elution buffers of different pH values predetermined to induce aggregation of the IgG4 were used. Rate constants for monomer decay over time were determined by fitting exponential decay functions to the data. Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH. Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested. PMID:26346187

Effect of pH 5 enzyme from liver on the protein synthesis by mammary gland subcellular fractions in vitro

International Nuclear Information System (INIS)

Singh, Jaspal; Singh, Ajit; Ganguli, N.C.

1976-01-01

The effect of pH 5 enzyme fraction of liver on the protein synthesizing activity of the subcellular fractions of the mammary gland has been investigated. Results indicate that (1) lactating liver pH 5 enzyme stimulates protein synthesis which is enhanced by the addition of ATP-generating system and (2) the enzyme fractions from the non-lactating liver inhibits the protein synthesis by mammary fractions, but in some cases like mitochondrial and supernatant fractions of mammary it elevates the synthesis when supplemented with ATP-generating system. Chlorella protein hydrolysate- 14 C was used as a tracer and rabits were used as experimental animals. (M.G.B.)

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

Science.gov (United States)

Kadamur, Ganesh

2016-01-01

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

pH induced protein-scaffold biosynthesis of tunable shape gold nanoparticles

International Nuclear Information System (INIS)

Zhang Xiaorong; He Xiaoxiao; Wang Kemin; Ren Fang; Qin Zhihe

2011-01-01

In this paper, a pH-inductive protein-scaffold biosynthesis of shape-tunable crystalline gold nanoparticles at room temperature has been developed. By simple manipulation of the reaction solution's pH, anisotropic gold nanoparticles including spheres, triangles and cubes could be produced by incubating an aqueous solution of sodium tetrachloroaurate with Dolichomitriopsis diversiformis biomasses after immersion in ultrapure Millipore water overnight. A moss protein with molecular weight of about 71 kDa and pI of 4.9 was the primary biomolecule involved in the biosynthesis of gold nanoparticles. The secondary configuration of the proteins by CD spectrum implied that the moss protein could display different secondary configurations including random coil, α-helix and intermediate conformations between random coil and α-helix for the experimental pH solution. The growth process of gold nanoparticles further showed that the moss protein with different configurations provided the template scaffold for the shape-controlled biosynthesis of gold nanoparticles. The constrained shape of the gold nanoparticles, however, disappeared in boiled moss extract. The gold nanoparticles with designed morphology were successfully reconstructed using the moss protein purified from the gold nanoparticles. Structural characterizations by SEM, TEM and SAED showed that the triangular and cubic gold nanoparticles were single crystalline.

Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

Science.gov (United States)

Yoo, Chul; Patwa, Tasneem H.; Kreunin, Paweena; Miller, Fred R.; Huber, Christian G.; Nesvizhskii, Alexey I.; Lubman, David M.

2012-01-01

A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 μg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis. PMID:17206599

Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients

NARCIS (Netherlands)

Bischoff, Rainer; Roecklin, D.; Roitsch, C.

1992-01-01

Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase

How to stop e-mail spam, spyware, malware, computer viruses, and hackers from ruining your computer or network the complete guide for your home and work

CERN Document Server

Brown, Bruce

2010-01-01

It seems like everywhere you go on the Internet, there is spam, spyware, and the risk of viruses infecting your computer and ruining your online experience. In businesses alone, according to Nucleus Research Inc. spam costs more than 712 per employee each year in productivity and computing resources and the estimation on money lost by businesses due to computer viruses ranges between 100 million and 2 billion annually depending on how the total is calculated. This complete, revolutionary book has compiled all of the vital information you need to make sure that you are able to combat the risk

Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state.

Science.gov (United States)

Iwai, Wakari; Yagi, Daichi; Ishikawa, Takuya; Ohnishi, Yuki; Tanaka, Ichiro; Niimura, Nobuo

2008-05-01

To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5-8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25-40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme.

The GRP1 PH domain, like the AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P(3).

Science.gov (United States)

Pilling, Carissa; Landgraf, Kyle E; Falke, Joseph J

2011-11-15

During the appearance of the signaling lipid PI(3,4,5)P(3), an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P(3)-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P(2) and bind the rare PI(3,4,5)P(3) target lipid with sufficiently high affinity. Our previous study of the E17K mutant of the protein kinase B (AKT1) PH domain, together with evidence from Carpten et al. [Carpten, J. D., et al. (2007) Nature 448, 439-444], revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P(2), thereby playing an essential role in specific PI(3,4,5)P(3) targeting [Landgraf, K. E., et al. (2008) Biochemistry 47, 12260-12269]. The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P(3)-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P(2) affinity and constitutive plasma membrane targeting. To test this hypothesis, we investigated the E345 residue, a putative sentry glutamate, of the general receptor for phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into the GRP1 PH domain enhances PI(4,5)P(2) affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in the AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P(2) releases the E345K GRP1 PH domain into the cytoplasm, and the efficiency of this release increases when Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K [Carpten, J. D., et al. (2007) Nature 448, 439-444; Lindhurst, M. J., et al

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain.

Science.gov (United States)

Prashek, Jennifer; Bouyain, Samuel; Fu, Mingui; Li, Yong; Berkes, Dusan; Yao, Xiaolan

2017-08-25

De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this study we show that isolated PH and START domains interact with each other. The crystal structure of a PH-START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH-START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine-rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

Science.gov (United States)

da Rosa-Garzon, Nathália Gonsales; Laure, Hélen Julie; Souza-Motta, Cristina Maria de; Rosa, José César; Cabral, Hamilton

2017-08-09

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

Voltage and pH sensing by the voltage-gated proton channel, HV1.

Science.gov (United States)

DeCoursey, Thomas E

2018-04-01

Voltage-gated proton channels are unique ion channels, membrane proteins that allow protons but no other ions to cross cell membranes. They are found in diverse species, from unicellular marine life to humans. In all cells, their function requires that they open and conduct current only under certain conditions, typically when the electrochemical gradient for protons is outwards. Consequently, these proteins behave like rectifiers, conducting protons out of cells. Their activity has electrical consequences and also changes the pH on both sides of the membrane. Here we summarize what is known about the way these proteins sense the membrane potential and the pH inside and outside the cell. Currently, it is hypothesized that membrane potential is sensed by permanently charged arginines (with very high p K a ) within the protein, which results in parts of the protein moving to produce a conduction pathway. The mechanism of pH sensing appears to involve titratable side chains of particular amino acids. For this purpose their p K a needs to be within the operational pH range. We propose a 'counter-charge' model for pH sensing in which electrostatic interactions within the protein are selectively disrupted by protonation of internally or externally accessible groups. © 2018 The Author.

Voltage and pH sensing by the voltage-gated proton channel, HV1

Science.gov (United States)

2018-01-01

Voltage-gated proton channels are unique ion channels, membrane proteins that allow protons but no other ions to cross cell membranes. They are found in diverse species, from unicellular marine life to humans. In all cells, their function requires that they open and conduct current only under certain conditions, typically when the electrochemical gradient for protons is outwards. Consequently, these proteins behave like rectifiers, conducting protons out of cells. Their activity has electrical consequences and also changes the pH on both sides of the membrane. Here we summarize what is known about the way these proteins sense the membrane potential and the pH inside and outside the cell. Currently, it is hypothesized that membrane potential is sensed by permanently charged arginines (with very high pKa) within the protein, which results in parts of the protein moving to produce a conduction pathway. The mechanism of pH sensing appears to involve titratable side chains of particular amino acids. For this purpose their pKa needs to be within the operational pH range. We propose a ‘counter-charge’ model for pH sensing in which electrostatic interactions within the protein are selectively disrupted by protonation of internally or externally accessible groups. PMID:29643227

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

Science.gov (United States)

Kadamur, Ganesh; Ross, Elliott M

2016-05-20

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

International Nuclear Information System (INIS)

Peters, Fabian; Maestre-Martinez, Mitcheell; Leonov, Andrei; Kovačič, Lidija; Becker, Stefan; Boelens, Rolf; Griesinger, Christian

2011-01-01

Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer.

Monomeric banana lectin at acidic pH overrules conformational stability of its native dimeric form.

Directory of Open Access Journals (Sweden)

Javed M Khan

Full Text Available Banana lectin (BL is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS binding, size exclusion chromatography (SEC and dynamic light scattering (DLS. During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml at pH 2.0 while single peak (61.45 ml at pH 7.4. The hydrodynamic radii (R h of native BL was 2.9 nm while at pH 2.0 two species were found with R h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high Cm and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.

Cation modulation of hemoglobin interaction with sodium n-dodecyl sulphate (SDS) iv: magnesium modulation at pH 7.20

OpenAIRE

Ali Akbar Moosavi-Movahedi; Ferdinand C. Chilaka; Charles O. Nwamba

2016-01-01

We investigate the interaction of Mg2+ (0–2.30 mM) and sodium n-dodecyl sulfate (SDS) with hemoglobins (Hbs) A and S at pH 7.20. SDS was used to model both membranes (0.60 mM SDS) and proteases (5.0 mM SDS). Via UV-visible spectroscopy, second derivative and difference second derivative spectroscopy, we interrogated for difference(s) in the interaction of these ligands with the proteins that can account for the HbS resistance to malaria parasite while been prone to sickling. Our results show ...

Constant pH molecular dynamics of proteins in explicit solvent with proton tautomerism.

Science.gov (United States)

Goh, Garrett B; Hulbert, Benjamin S; Zhou, Huiqing; Brooks, Charles L

2014-07-01

pH is a ubiquitous regulator of biological activity, including protein-folding, protein-protein interactions, and enzymatic activity. Existing constant pH molecular dynamics (CPHMD) models that were developed to address questions related to the pH-dependent properties of proteins are largely based on implicit solvent models. However, implicit solvent models are known to underestimate the desolvation energy of buried charged residues, increasing the error associated with predictions that involve internal ionizable residue that are important in processes like hydrogen transport and electron transfer. Furthermore, discrete water and ions cannot be modeled in implicit solvent, which are important in systems like membrane proteins and ion channels. We report on an explicit solvent constant pH molecular dynamics framework based on multi-site λ-dynamics (CPHMD(MSλD)). In the CPHMD(MSλD) framework, we performed seamless alchemical transitions between protonation and tautomeric states using multi-site λ-dynamics, and designed novel biasing potentials to ensure that the physical end-states are predominantly sampled. We show that explicit solvent CPHMD(MSλD) simulations model realistic pH-dependent properties of proteins such as the Hen-Egg White Lysozyme (HEWL), binding domain of 2-oxoglutarate dehydrogenase (BBL) and N-terminal domain of ribosomal protein L9 (NTL9), and the pKa predictions are in excellent agreement with experimental values, with a RMSE ranging from 0.72 to 0.84 pKa units. With the recent development of the explicit solvent CPHMD(MSλD) framework for nucleic acids, accurate modeling of pH-dependent properties of both major class of biomolecules-proteins and nucleic acids is now possible. © 2013 Wiley Periodicals, Inc.

RHEOLOGY OF CHICKPEA PROTEIN CONCENTRATE DISPERSIONS

Directory of Open Access Journals (Sweden)

Aurelia Ionescu

2011-12-01

Full Text Available Chickpea proteins are used as ingredients in comminuted sausage products and many oriental textured foods. Rheological behaviour of chickpea protein concentrate was studied using a controlled stress rheometer. The protein dispersion prepared with phosphate buffer at pH 7.0 presented non-Newtonian shear thinning behaviour and rheological data well fitted to the Sisko, Carreau and Cross models. The viscoelastic properties of the chickpea protein suspensions were estimated by measuring the storage and loss moduli in oscillatory frequency conditions (0.1-10 Hz at 20°C. Moreover, thermally induced gelation of the chickpea proteins (16, 24 and 36% was studied at pH 7.0 and 4.5 in the temperature range 50 to 100oC and salt concentration ranging from 0 to 1 M. Gelling behaviour was quantified by means of dynamic rheological measurements. Gels formation was preceded by the decrease of storage modulus and loss moduli, coupled with the increase of the phase angle (delta. The beginning of thermal gelation was influenced by protein concentration, pH and salt level. In all studied cases, storage modulus increased rapidly in the temperature range 70-90°C. All rheological parameters measured at 90°C were significantly higher at pH 4.5 compared to pH 7.0.

Investigation of variation of additional enthalpy of proteins with respect to pH by statistical mechanical methods

International Nuclear Information System (INIS)

Oylumoglu, G.

2005-01-01

In this study variation of additional enthalpy with respect to pH has been investigated by the statistical mechanical methods.. To bring up the additional effect, the partition function of the proteins are calculated by single protein molecule approximation. From the partition function, free energies of the proteins are obtained and by this way additional free energy has been used in the calculation of the terms in the thermodynamical quantity. Additional enthalpy H D has been obtained by taking effective electric field E and constant dipole moment M as thermodynamical variables and using Maxwell Equations. In the presented semi phenomenological theory, necessary data are taken from the experimental study of P.L. Privalov. The variation in the additional enthalpy H D has been investigated in the pH interval of 1-5 and the results of the calculations are discussed for Lysozyme

Unsolicited email, does Europe allow spam? : The state of the art of the European legislation with regard to unsolicited commercial communications

NARCIS (Netherlands)

Schaub, M.Y.

2002-01-01

Unsolicited commercial emails, also referred to as junk-mail or spam, is considered a rude and irritating form of marketing. The sending of unsolicited commercial emails has appeared on the European political agenda more then once. The discussion on this issue evolves around the question whether an

Thermochemistry of the reactions of PH +2 ( 1A 1) and PH +2 ( 3B 1) with CO. A G2 molecular orbital study

Science.gov (United States)

Esseffar, M.; Luna, A.; Mó, O.; Yáñez, M.

1994-06-01

The Gaussian-2 (G2) theoretical procedure, based on ab initio molecular orbital theory, is used to study the potential energy surfaces corresponding to gas phase reactions between PH +2 singlet and triplet state cations with carbon monoxide. Important differences between singlets and triplets, both regarding their bonding and their stabilities have been found. The most outstanding result is that, although the first 3B 1 excited state of PH +2 is only about 20 kcal/mol above the 1A 1 ground state, the singlet global minimum of the [H 2, P, C, O] + potential energy surface lies 61 kcal/mol below the triplet global minimum. This is so because, in general, triplet state cations are ion—dipole complexes, while the singlets are covalently bound species. In agreement with experimental evidence, only the formation of the adduct is exothermic, while all processes yielding PO or PC containing species as well as the proton transfer reaction, are strongly endothermic. Estimates of the heats of formation of H 2PO + and HCP species are given.

Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state

International Nuclear Information System (INIS)

Iwai, Wakari; Yagi, Daichi; Ishikawa, Takuya; Ohnishi, Yuki; Tanaka, Ichiro; Niimura, Nobuo

2008-01-01

Hen egg-white lysozyme was crystallized over a wide pH range (2.5–8.0) and the quality of the crystals was characterized. Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5–8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH < 4.5 the border between the metastable region and the nucleation region shifted to the left (lower precipitant concentration) in the phase diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25–40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme

Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase.

Directory of Open Access Journals (Sweden)

Jason M Shapiro

Full Text Available We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90-110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1, heat shock protein 90 (HSP90, and Na+/K+ ATPase or Mg++ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation.

Understanding Early Post-Mortem Biochemical Processes Underlying Meat Color and pH Decline in the Longissimus thoracis Muscle of Young Blond d'Aquitaine Bulls Using Protein Biomarkers.

Science.gov (United States)

Gagaoua, Mohammed; Terlouw, E M Claudia; Micol, Didier; Boudjellal, Abdelghani; Hocquette, Jean-François; Picard, Brigitte

2015-08-05

Many studies on color biochemistry and protein biomarkers were undertaken in post-mortem beef muscles after ≥24 hours. The present study was conducted on Longissimus thoracis muscles of 21 Blond d'Aquitaine young bulls to evaluate the relationships between protein biomarkers present during the early post-mortem and known to be related to tenderness and pH decline and color development. pH values at 45 min, 3 h, and 30 h post-mortem were correlated with three, seven, and six biomarkers, respectively. L*a*b* color coordinates 24 h post-mortem were correlated with nine, five, and eight protein biomarkers, respectively. Regression models included Hsp proteins and explained between 47 and 59% of the variability between individuals in pH and between 47 and 65% of the variability in L*a*b* color coordinates. Proteins correlated with pH and/or color coordinates were involved in apoptosis or had antioxidative or chaperone activities. The main results include the negative correlations between pH45 min, pH3 h, and pHu and Prdx6, which may be explained by the antioxidative and phospholipase activities of this biomarker. Similarly, inducible Hsp70-1A/B and μ-calpain were correlated with L*a*b* coordinates, due to the protective action of Hsp70-1A/B on the proteolytic activities of μ-calpain on structural proteins. Correlations existed further between MDH1, ENO3, and LDH-B and pH decline and color stability probably due to the involvement of these enzymes in the glycolytic pathway and, thus, the energy status of the cell. The present results show that research using protein indicators may increase the understanding of early post-mortem biological mechanisms involved in pH and beef color development.

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change.

OpenAIRE

Masuda, Tetsuya; Ohta, Keisuke; Mikami, Bunzo; Kitabatake, Naofumi; Tani, Fumito

2012-01-01

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50 nM. Although the sweetness remains when thaumatin is heated at 80 °C for 4h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0Å. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of ...

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

Science.gov (United States)

Jutras, Philippe V; D'Aoust, Marc-André; Couture, Manon M-J; Vézina, Louis-Philippe; Goulet, Marie-Claire; Michaud, Dominique; Sainsbury, Frank

2015-09-01

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of extraction pH on heat-induced aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd).

Science.gov (United States)

Ruiz, Geraldine Avila; Xiao, Wukai; van Boekel, Martinus; Minor, Marcel; Stieger, Markus

2016-10-15

The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8 (E8), 9 (E9), 10 (E10) and 11 (E11)), followed by acid precipitation. The obtained protein isolates were freeze dried. The protein isolates E8 and E9 resulted in a lower protein yield as well as less protein denaturation. These isolates also had a higher protein purity, more protein bands at higher molecular weights, and a higher protein solubility in the pH range of 3-4.5, compared to the isolates E10 and E11. Heating the 10%w/w protein isolate suspensions E8 and E9 led to increased aggregation, and semi-solid gels with a dense microstructure were formed. The isolate suspensions E10 and E11, on the other hand, aggregated less, did not form self-supporting gels and had loose particle arrangements. We conclude that extraction pH plays an important role in determining the functionality of quinoa protein isolates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of hippocampus-dependent memory by the zinc finger protein Zbtb20 in mature CA1 neurons.

Science.gov (United States)

Ren, Anjing; Zhang, Huan; Xie, Zhifang; Ma, Xianhua; Ji, Wenli; He, David Z Z; Yuan, Wenjun; Ding, Yu-Qiang; Zhang, Xiao-Hui; Zhang, Weiping J

2012-10-01

The mammalian hippocampus harbours neural circuitry that is crucial for associative learning and memory. The mechanisms that underlie the development and regulation of this complex circuitry are not fully understood. Our previous study established an essential role for the zinc finger protein Zbtb20 in the specification of CA1 field identity in the developing hippocampus. Here, we show that conditionally deleting Zbtb20 specifically in mature CA1 pyramidal neurons impaired hippocampus-dependent memory formation, without affecting hippocampal architecture or the survival, identity and basal excitatory synaptic activity of CA1 pyramidal neurons. We demonstrate that mature CA1-specific Zbtb20 knockout mice exhibited reductions in long-term potentiation (LTP) and NMDA receptor (NMDAR)-mediated excitatory post-synaptic currents. Furthermore, we show that activity-induced phosphorylation of ERK and CREB is impaired in the hippocampal CA1 of Zbtb20 mutant mice. Collectively, these results indicate that Zbtb20 in mature CA1 plays an important role in LTP and memory by regulating NMDAR activity, and activation of ERK and CREB.

20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

Science.gov (United States)

Ji, M-M; Liu, A-Q; Sima, Y-H; Xu, S-Q

2016-10-01

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. © 2016 The Royal Entomological Society.

Concentration of total protein and degree of acidity (pH of saliva when fasting and after breakfasting

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Gemella Nur Illahi

2016-04-01

Full Text Available Background: While fasting, the mouth does not work to eat and drink so that the salivary glands become less active so saliva production decreased and there was a change in eating timewhich is relation to the mastication process that impact on changes in the degree of acidity (pH Objectives: To determine the concentration of total protein and the degree of acidity (pH of saliva when fasting and after breakfasting. Materials and Methods: The study was observational analytic design with longitudinal (follow up study conducted in the Hj. Halima Dg. Sikati Dental Hospital inKandea in July 2015, the sampling method was purposive sampling. Population was 35 clinical students at the Department of Dental Public Health, Faculty of Dentistry Hasanuddin University with a total sample of 16 students who fit the criteria of the study subjects. To calculate the total protein of saliva concentration using Kyltecautoanalyzerand pH meter to measure the acidity of saliva. Data was analyzed was using SPSS version 17.0 (paired t-test, p <0.05. Results: The mean of total protein (% while fasting by 0135% ± 0.026 and the mean total protein (% after breakfasting at 0.179% ± 0.035, while the average degree of acidity (pH during fasting at 7.26 ± 0:24 and the average degree of acidity (pH after breakfasting at 7.66 ± 0.23 with p-value (0.000. Conclusions: An increase in the total protein concentration and acidity (pH after breakfasting.

Characterization of pH titration shifts for all the nonlabile proton resonances in a protein by two-dimensional NMR: The case of mouse epidermal growth factor

International Nuclear Information System (INIS)

Kohda, Daisuke; Sawada, Toshie; Inagaki, Fuyuhiko

1991-01-01

The pH titration shifts for all the nonlabile proton resonances in a 53-residue protein (mouse epidermal growth factor) were measured in the p 2 H range 1.5-9 with two-dimensional (2D) 1 H NMR. The 2D NMR pH titration experiment made it possible to determine the pK values for all the ionizable group which were titrated in the pH range 1.5-9 in the protein. The pK values of the nine ionizable groups (α-amino group, four Asp, two Glu, one His, and α-carboxyl group) were found to be near their normal values. The 2D titration experiment also provided a detailed description of the pH-dependent behavior of the proton chemical shifts and enabled us to characterize the pH-dependent changes of protein conformation. Analysis of the pH-dependent shifts of ca. 200 proton resonances offered evidence of conformational changes in slightly basic pH solution: The deprotonation of the N-terminal α-amino group induced a widespread conformational change over the β-sheet structure in the protein, while the effects of deprotonation of the His22 imidazole group were relatively localized. The authors found that the 2D NMR pH titration experiment is a powerful tool for investigating the structural and dynamic properties of proteins

Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis.

Directory of Open Access Journals (Sweden)

Xing-Chun Zhao

Full Text Available Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs. Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB-sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP. Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases.

PhEXPA1, a Petunia hybrida expansin, is involved in cell wall metabolism and in plant architecture specification.

Science.gov (United States)

Dal Santo, Silvia; Fasoli, Marianna; Cavallini, Erika; Tornielli, Giovanni Battista; Pezzotti, Mario; Zenoni, Sara

2011-12-01

Expansins are wall-loosening proteins that induce wall stress relaxation and irreversible wall extension in a pH-dependent manner. Despite a substantial body of work has been performed on the characterization of many expansins genes in different plant species, the knowledge about their precise biological roles during plant development remains scarce. To yield insights into the expansion process in Petunia hybrida, PhEXPA1, an expansin gene preferentially expressed in petal limb, has been characterized. The constitutive overexpression of PhEXPA1 significantly increased expansin activity, cells size and organ dimensions. Moreover, 35S::PhEXPA1 transgenic plants exhibited an altered cell wall polymer composition and a precocious timing of axillary meristem development compared with wild-type plants. These findings supported a previous hypothesis that expansins are not merely structural proteins involved in plant cell wall metabolism but they also take part in many plant development processes. Here, to support this expansins dual role, we discuss about differential cell wall-related genes expressed in PhEXPA1 expression mutants and gradients of altered petunia branching pattern. © 2011 Landes Bioscience

Crystallization and preliminary X-ray analysis of coagulation factor IX-binding protein from habu snake venom at pH 6.5 and 4.6

International Nuclear Information System (INIS)

Suzuki, Nobuhiro; Shikamoto, Yasuo; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

2004-01-01

Crystals of habu coagulation factor IX-binding protein have been obtained at pH 6.5 and 4.6 and characterized by X-ray diffraction. Coagulation factor IX-binding protein isolated from Trimeresurus flavoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca 2+ -binding site with differing affinity (K d values of 14 and 130 µM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the affinity of the low-affinity site was reduced considerably. In order to identify which site has high affinity and also to investigate the Ca 2+ -releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29 Å resolution, respectively; the former crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 60.7, b = 63.5, c = 66.9 Å, β = 117.0°, while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8 Å, β = 110.4°

Effect of extraction pH on heat-induced aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd)

NARCIS (Netherlands)

Ruiz, Geraldine Avila; Xiao, Wukai; Boekel, van Tiny; Minor, Marcel; Stieger, Markus

2016-01-01

The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8

Adsorption of Hydrophobin-Protein Mixtures at the Air-Water Interface: The Impact of pH and Electrolyte.

Science.gov (United States)

Tucker, Ian M; Petkov, Jordan T; Penfold, Jeffrey; Thomas, Robert K; Cox, Andrew R; Hedges, Nick

2015-09-15

The adsorption of the proteins β-casein, β-lactoglobulin, and hydrophobin, and the protein mixtures of β-casein/hydrophobin and β-lactoglobulin/hydrophobin have been studied at the air-water interface by neutron reflectivity, NR. Changing the solution pH from 7 to 2.6 has relatively little impact on the adsorption of hydrophobin or β-lactoglobulin, but results in a substantial change in the structure of the adsorbed layer of β-casein. In β-lactoglobulin/hydrophobin mixtures, the adsorption is dominated by the hydrophobin adsorption, and is independent of the hydrophobin or β-lactoglobulin concentration and solution pH. At pH 2.6, the adsorption of the β-casein/hydrophobin mixtures is dominated by the hydrophobin adsorption over the range of β-casein concentrations studied. At pH 4 and 7, the adsorption of β-casein/hydrophobin mixtures is dominated by the hydrophobin adsorption at low β-casein concentrations. At higher β-casein concentrations, β-casein is adsorbed onto the surface monolayer of hydrophobin, and some interpenetration between the two proteins occurs. These results illustrate the importance of pH on the intermolecular interactions between the two proteins at the interface. This is further confirmed by the impact of PBS, phosphate buffered saline, buffer and CaCl2 on the coadsorption and surface structure. The results provide an important insight into the adsorption properties of protein mixtures and their application in foam and emulsion stabilization.

Ammonium ion transport by the AMT/Rh homolog TaAMT1;1 is stimulated by acidic pH

DEFF Research Database (Denmark)

Søgaard, Rikke; Alsterfjord, Magnus; Macaulay, Nanna

2009-01-01

It is unclear how ammonia is transported by proteins from the Amt/Mep/Rh superfamily. We investigated this for the ammonium transporter TaAMT1;1 from wheat expressed in Xenopus oocytes by two-electrode voltage clamp and radio-labeled uptakes. Inward currents were activated by NH (4......) (+) or methylammonium ions (MeA(+)). Importantly, currents increased fivefold when the external pH was decreased from 7.4 to 5.5; this type of pH dependence is unique and is a strong indication of NH (4) (+) or MeA(+) transport. This was confirmed by the close correlation between the uptake of radio-labeled Me......A(+) and MeA(+)-induced currents. Homology models of members of the Amt/Mep/Rh superfamily exhibited major divergences in their cytoplasmic regions. A point mutation in this region of TaAMT1;1 abolished the pH sensitivity and decreased the apparent affinities for NH (4) (+) and MeA(+). We suggest a model...

Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins

International Nuclear Information System (INIS)

Shirokane, Michio; Sawano, Yoriko; Miyazono, Ken-ichi; Nagata, Koji; Tanokura, Masaru

2007-01-01

PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution. PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P2 1 2 1 2 1 , with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (V M = 2.4 Å 3 Da −1 ) and had a solvent content of 48%

Condensation of rye chromatin in somatic interphase nuclei of Ph1 and ph1b wheat

Czech Academy of Sciences Publication Activity Database

Kopecký, David; Allen, D.C.; Duchoslav, M.; Doležel, Jaroslav; Lukaszewski, A.J.

2007-01-01

Roč. 119, 3-4 (2007), s. 263-267 ISSN 1424-8581 R&D Projects: GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50380511 Source of funding: V - iné verejné zdroje Keywords : hexaploid wheat * Ph1 and ph1b * rye chromatin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.402, year: 2007

Stress corrosion cracking in 17-4PH and 17-7PH stainless steels in NaCl and NaOH (20%) a 90 deg C

International Nuclear Information System (INIS)

Gaona-Tiburcio, C.; Almeraya-Calderon, F.; Martinez-Villafane, A.

2000-01-01

One of the problems that affects to the electric industry is the not programmed stoppages in the power plants, due to the failure of any main component: boiler, turbine and generator. In the turbine, the combined action of a corrosive agent (humid polluted vapor) and a mechanical effort, generally will result in Stress Corrosion Cracking (SCC). In this work the SCC susceptibility of the precipitation hardening stainless steels 17-4PH and 17-17PH, thoroughly used in steam turbine blades of power stations is analyzed. The specimens were tested in the presence of NaCl and NaOH(20%) to 90 deg C and different pH. The CERT test (Constant Extension Rate Test) was used, at 10''-6 s''-1, supplementing it with electrochemical noise, the aim was to identify the conditions of maximum susceptibility and the performance of the studied materials. The fractographic analysis revealed ductile and brittle fracture. Intergranular cracking, characteristic of the anodic dissolution mechanisms of the materials was observed. Nevertheless, the main mechanism responsible the failure was hydrogen embrittlement. (Author) 6 refs

Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida.

Science.gov (United States)

Zenoni, Sara; Fasoli, Marianna; Tornielli, Giovanni Battista; Dal Santo, Silvia; Sanson, Andrea; de Groot, Peter; Sordo, Sara; Citterio, Sandra; Monti, Francesca; Pezzotti, Mario

2011-08-01

• Expansins are cell wall proteins required for cell enlargement and cell wall loosening during many developmental processes. The involvement of the Petunia hybrida expansin A1 (PhEXPA1) gene in cell expansion, the control of organ size and cell wall polysaccharide composition was investigated by overexpressing PhEXPA1 in petunia plants. • PhEXPA1 promoter activity was evaluated using a promoter-GUS assay and the protein's subcellular localization was established by expressing a PhEXPA1-GFP fusion protein. PhEXPA1 was overexpressed in transgenic plants using the cauliflower mosaic virus (CaMV) 35S promoter. Fourier transform infrared (FTIR) and chemical analysis were used for the quantitative analysis of cell wall polymers. • The GUS and GFP assays demonstrated that PhEXPA1 is present in the cell walls of expanding tissues. The constitutive overexpression of PhEXPA1 significantly affected expansin activity and organ size, leading to changes in the architecture of petunia plants by initiating premature axillary meristem outgrowth. Moreover, a significant change in cell wall polymer composition in the petal limbs of transgenic plants was observed. • These results support a role for expansins in the determination of organ shape, in lateral branching, and in the variation of cell wall polymer composition, probably reflecting a complex role in cell wall metabolism. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

Cold-adapted influenza and recombinant adenovirus vaccines induce cross-protective immunity against pH1N1 challenge in mice.

Directory of Open Access Journals (Sweden)

Mark R Soboleski

Full Text Available The rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1 highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus.BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca influenza viruses from 1977 or recombinant adenoviruses (rAd expressing 1934 nucleoprotein (NP and consensus matrix 2 (M2 (NP+M2-rAd. Antibodies against the M2 ectodomain (M2e were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus.Cross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic.

A histidine-rich linker region in peptidylglycine α-amidating monooxygenase has the properties of a pH sensor.

Science.gov (United States)

Vishwanatha, Kurutihalli; Bäck, Nils; Mains, Richard E; Eipper, Betty A

2014-05-02

Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.

Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

Energy Technology Data Exchange (ETDEWEB)

DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar; Heath, Richard J.; White, Stephen W.; Russell, Charles J. (Tennessee-HSC); (SJCH)

2012-12-10

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 {angstrom} resolution and two structures of HP HA at 2.95 and 3.10 {angstrom} resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

Finding a needle in a haystack: the role of electrostatics in target lipid recognition by PH domains.

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Craig N Lumb

Full Text Available Interactions between protein domains and lipid molecules play key roles in controlling cell membrane signalling and trafficking. The pleckstrin homology (PH domain is one of the most widespread, binding specifically to phosphatidylinositol phosphates (PIPs in cell membranes. PH domains must locate specific PIPs in the presence of a background of approximately 20% anionic lipids within the cytoplasmic leaflet of the plasma membrane. We investigate the mechanism of such recognition via a multiscale procedure combining Brownian dynamics (BD and molecular dynamics (MD simulations of the GRP1 PH domain interacting with phosphatidylinositol (3,4,5-trisphosphate (PI(3,4,5P₃. The interaction of GRP1-PH with PI(3,4,5P₃ in a zwitterionic bilayer is compared with the interaction in bilayers containing different levels of anionic 'decoy' lipids. BD simulations reveal both translational and orientational electrostatic steering of the PH domain towards the PI(3,4,5P₃-containing anionic bilayer surface. There is a payoff between non-PIP anionic lipids attracting the PH domain to the bilayer surface in a favourable orientation and their role as 'decoys', disrupting the interaction of GRP1-PH with the PI(3,4,5P₃ molecule. Significantly, approximately 20% anionic lipid in the cytoplasmic leaflet of the bilayer is nearly optimal to both enhance orientational steering and to localise GRP1-PH proximal to the surface of the membrane without sacrificing its ability to locate PI(3,4,5P₃ within the bilayer plane. Subsequent MD simulations reveal binding to PI(3,4,5P₃, forming protein-phosphate contacts comparable to those in X-ray structures. These studies demonstrate a computational framework which addresses lipid recognition within a cell membrane environment, offering a link between structural and cell biological characterisation.

Fluorescent S-layer fusion proteins

International Nuclear Information System (INIS)

Kainz, B.

2010-01-01

This work describes the construction and characterisation of fluorescent S-layer fusion proteins used as building blocks for the fabrication of nanostructured monomolecular biocoatings on silica particles with defined fluorescence properties. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the pH-dependant cyan, green and yellow variant of the green fluorescent protein (GFP) and the red fluorescent protein mRFP1. These fluorescent S-layer fusion proteins, acting as scaffold and optical sensing element simultaneously, were able to reassemble in solution and on silica particles forming 2D nanostructures with p2 lattice symmetry (a=11 ±0.5 nm, b=14 ±0.4 nm, g=80 ±1 o ). The pH-dependant fluorescence behaviour was studied with fluorimetry, confocal microscopy and flow cytometry. These fluorescent S-layer fusion proteins can be used as pH-sensor. 50% of the fluorescence intensity decreases at their calculated pKa values (pH6 - pH5). The fluorescence intensity of the GFP variants vanished completely between pH4 and pH3 whereas the chromophore of the red protein mRFP1 was only slightly affected in acidic conditions. At the isoelectric point of the S-layer coated silica particles (pH4.6 ±0.2) an increase in particle aggregation was detected by flow cytometry. The cyan and yellow fluorescent proteins were chosen to create a bi-fluorescent S-layer tandem fusion protein with the possibility for resonance energy transfer (FRET). A transfer efficiency of 20% and a molecular distance between the donor (ECFP) and acceptor (YFP) chromophores of around 6.2 nm could be shown. This bi-fluorescent ECFP-SgsE-YFP tandem fusion protein was able to reassemble on solid surfaces. The remarkable combination of fluorescence and self-assembly and the design of bi-functional S-layer tandem fusion protein matrices makes them to a promising tool in nanobiotechnology. (author) [de

pH controlled gating of toxic protein pores by dendrimers

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Mandal, Taraknath; Kanchi, Subbarao; Ayappa, K. G.; Maiti, Prabal K.

2016-06-01

Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent bacterial strains, on a target cell membrane is a challenging and active area of research. Here we demonstrate that PAMAM dendrimers can act as effective pH controlled gating devices once the pore has been formed. We have used fully atomistic molecular dynamics (MD) simulations to characterize the cytolysin A (ClyA) protein pores modified with fifth generation (G5) PAMAM dendrimers. Our results show that the PAMAM dendrimer, in either its protonated (P) or non-protonated (NP) states can spontaneously enter the protein lumen. Protonated dendrimers interact strongly with the negatively charged protein pore lumen. As a consequence, P dendrimers assume a more expanded configuration efficiently blocking the pore when compared with the more compact configuration adopted by the neutral NP dendrimers creating a greater void space for the passage of water and ions. To quantify the effective blockage of the protein pore, we have calculated the pore conductance as well as the residence times by applying a weak force on the ions/water. Ionic currents are reduced by 91% for the P dendrimers and 31% for the NP dendrimers. The preferential binding of Cl- counter ions to the P dendrimer creates a zone of high Cl- concentration in the vicinity of the internalized dendrimer and a high concentration of K+ ions in the transmembrane region of the pore lumen. In addition to steric effects, this induced charge segregation for the P dendrimer effectively blocks ionic transport through the pore. Our investigation shows that the bio-compatible PAMAM dendrimers can potentially be used to develop therapeutic protocols based on the pH sensitive gating of pores formed by pore forming toxins to mitigate bacterial infections.Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent

Molecular interactions of mussel protective coating protein, mcfp-1, from Mytilus californianus.

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Lu, Qingye; Hwang, Dong Soo; Liu, Yang; Zeng, Hongbo

2012-02-01

Protective coating of the byssus of mussels (Mytilus sp.) has been suggested as a new paradigm of medical coating due to its high extensibility and hardness co-existence without their mutual detriment. The only known biomacromolecule in the extensible and tough coating on the byssus is mussel foot protein-1 (mfp-1), which is made up with positively charged residues (~20 mol%) and lack of negatively charged residues. Here, adhesion and molecular interaction mechanisms of Mytilus californianus foot protein-1 (mcfp-1) from California blue mussel were investigated using a surface forces apparatus (SFA) in buffer solutions of different ionic concentrations (0.2-0.7 M) and pHs (3.0-5.5). Strong and reversible cohesion between opposed positively charged mcfp-1 films was measured in 0.1 M sodium acetate buffer with 0.1 M KNO(3). Cohesion of mcfp-1 was gradually reduced with increasing the ionic strength, but was not changed with pH variations. Oxidation of 3,4-dihydroxyphenylalanine (DOPA) residues of mcfp-1, a key residue for adhesive and coating proteins of mussel, didn't change the cohesion strength of mcfp-1 films, but the addition of chemicals with aromatic groups (i.e., aspirin and 4-methylcatechol) increased the cohesion. These results suggest that the cohesion of mcfp-1 films is mainly mediated by cation-π interactions between the positively charged residues and benzene rings of DOPA and other aromatic amino acids (~20 mol% of total amino acids of mcfp-1), and π-π interactions between the phenyl groups in mcfp-1. The adhesion mechanism obtained for the mcfp-1 proteins provides important insight into the design and development of functional biomaterials and coatings mimicking the extensible and robust mussel cuticle coating. Copyright © 2011 Elsevier Ltd. All rights reserved.

Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins

Energy Technology Data Exchange (ETDEWEB)

Shirokane, Michio; Sawano, Yoriko; Miyazono, Ken-ichi; Nagata, Koji; Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

2007-06-01

PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution. PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (V{sub M} = 2.4 Å{sup 3} Da{sup −1}) and had a solvent content of 48%.

Sequentially Integrated Optimization of the Conditions to Obtain a High-Protein and Low-Antinutritional Factors Protein Isolate from Edible Jatropha curcas Seed Cake.

Science.gov (United States)

León-López, Liliana; Dávila-Ortiz, Gloria; Jiménez-Martínez, Cristian; Hernández-Sánchez, Humberto

2013-01-01

Jatropha curcas seed cake is a protein-rich byproduct of oil extraction which could be used to produce protein isolates. The purpose of this study was the optimization of the protein isolation process from the seed cake of an edible provenance of J. curcas by an alkaline extraction followed by isoelectric precipitation method via a sequentially integrated optimization approach. The influence of four different factors (solubilization pH, extraction temperature, NaCl addition, and precipitation pH) on the protein and antinutritional compounds content of the isolate was evaluated. The estimated optimal conditions were an extraction temperature of 20°C, a precipitation pH of 4, and an amount of NaCl in the extraction solution of 0.6 M for a predicted protein content of 93.3%. Under these conditions, it was possible to obtain experimentally a protein isolate with 93.21% of proteins, 316.5 mg 100 g(-1) of total phenolics, 2891.84 mg 100 g(-1) of phytates and 168 mg 100 g(-1) of saponins. The protein content of the this isolate was higher than the content reported by other authors.

Discovery of human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) immunocontraceptive epitopes and their effects on fertility in male and female mice.

Science.gov (United States)

Chen, Xuemei; Liu, Xiaodong; Ren, Xiuhua; Li, Xuewu; Wang, Li; Zang, Weidong

2016-03-01

The key goals of immunocontraception research are to obtain full contraceptive effects using vaccines administered to both males and females. Current research concerning human anti-sperm contraceptive vaccines is focused on delineating infertility-related epitopes to avoid autoimmune disease. We constructed phage-display peptide libraries to select epitope peptides derived from human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) using sera collected from infertile women harbouring anti-sperm antibodies. Following five rounds of selection, positive colonies were reconfirmed for reactivity with the immunoinfertile sera. We biopanned and analysed the chemical properties of four epitope peptides, named P82, Sa6, Sa37 and Sa76. Synthetic peptides were made and coupled to either bovine serum albumin (BSA) or ovalbumin. We used the BSA-conjugated peptides to immunise BALB/c mice and examined the effects on fertility in female and male mice. The synthetic peptides generated a sperm-specific antibody response in female and male mice that caused a contraceptive state. The immunocontraceptive effect was reversible and, with the disappearance of peptide-specific antibodies, there was complete restoration of fertility. Vaccinations using P82, Sa6 and Sa76 peptides resulted in no apparent side effects. Thus, it is efficient and practical to identify epitope peptide candidates by phage display. These peptides may find clinical application in the specific diagnosis and treatment of male and female infertility and contraceptive vaccine development.

Growth rate analysis and protein identification of Kappaphycus alvarezii (Rhodophyta, Gigartinales under pH induced stress culture

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Mian Zi Tee

2015-11-01

Full Text Available Environmental pH is one of the factors contributing to abiotic stress which in turn influences the growth and development of macroalgae. This study was conducted in order to assess the growth and physiological changes in Kappaphycus alvarezii under different pH conditions: pHs 6, ∼8.4 (control and 9. K. alvarezii explants exhibited a difference in the daily growth rate (DGR among the different pH treatments (p ≤ 0.05. The highest DGR was observed in control culture with pH ∼8.4 followed by alkaline (pH 9 and acidic (pH 6 induced stress cultures. Protein expression profile was generated from different pH induced K. alvarezii cultures using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE followed by protein identification and analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS and Mascot software. Ribulose bisphosphate carboxylase (Rubisco large chain was identified to be up-regulated under acidic (pH 6 condition during the second and fourth week of culture. The findings indicated that Rubisco can be employed as a biomarker for pH induced abiotic stress. Further study on the association between the expression levels of Rubisco large chain and their underlying mechanisms under pH stress conditions is recommended.

Binding of Gallic Acid and Epigallocatechin Gallate to Heat-Unfolded Whey Proteins at Neutral pH Alters Radical Scavenging Activity of in Vitro Protein Digests.

Science.gov (United States)

Cao, Yanyun; Xiong, Youling L

2017-09-27

Preheated (80 °C for 9 min) whey protein isolate (HWPI) was reacted with 20, 120, and 240 μmol/g (protein basis) gallic acid (GA) or epigallocatechin gallate (EGCG) at neutral pH and 25 °C. Isothermal titration calorimetry and fluorometry showed a similar trend that GA binding to HWPI was moderate but weaker than EGCG binding. However, the shift of maximal fluorescence emission wavelength in opposite directions in response to GA (blue) and EGCG (red) suggests discrepant binding patterns. Electrophoresis results showed that EGCG induced formation of HWPI complexes while GA only had a marginal effect. Both free and phenolic-bound HWPI exhibited mild antiradical activity. However, when subjected to in vitro digestion, synergistic radical-scavenging activity was produced between the phenolics and peptides with the highest synergism being observed on 120 μmol/g phenolics.

Protein Complexation and pH Dependent Release Using Boronic Acid Containing PEG-Polypeptide Copolymers.

Science.gov (United States)

Negri, Graciela E; Deming, Timothy J

2017-01-01

New poly(L-lysine)-b-poly(ethylene glycol) copolypeptides have been prepared, where the side-chain amine groups of lysine residues are modified to contain ortho-amine substituted phenylboronic acid, i.e., Wulff-type phenylboronic acid (WBA), groups to improve their pH responsive, carbohydrate binding properties. These block copolymers form nanoscale complexes with glycosylated proteins that are stable at physiological pH, yet dissociate and release the glycoproteins under acidic conditions, similar to those found in endosomal and lysosomal compartments within cells. These results suggest that WBA modified polypeptide copolymers are promising for further development as degradable carriers for intracellular protein delivery. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spectroscopic determination of pH

International Nuclear Information System (INIS)

Faanu, A.; Glover, E.T.; Bailey, E.; Rochelle, C.

2009-01-01

A technique of measuring pH at temperature range of 20 - 70 0 C and high pressure conditions of 1 - 200 atmospheres has been developed by relating the ratio of absorbance peaks of indicator solutions (basic and acidic) as a function of pH, using ultraviolet-visible spectrophotometer. The pH values of the buffer solutions measured at 20 0 C and 70 0 C indicated slight temperature dependence, while the pressure had no effect. The pH of the buffer solutions increased with temperature with relative standard deviations in the range 0.4 - 0.5 % at 95 % confidence interval. The possible causes of the temperature dependence were attributed to changes in pH values as the temperature changed. (au)

Interactions in heated milk model systems with different ratios of nanoparticulated whey protein at varying pH

DEFF Research Database (Denmark)

Liu, Guanchen; Jæger, Tanja C.; Nielsen, Søren B.

2017-01-01

To better understand the interactions between nanoparticulated whey protein (NWP) and other milk proteins during acidification, milk model systems were diluted to 0.5% protein concentration and adjusted to pH of 6.0-4.5 following homogenisation and heat treatment. The diluted systems with different...... concentrations of NWP (0-0.5%) were characterised in terms of particle size, viscosity, surface charge and hydrophobicity. When pH was adjusted to 5.5, aggregation was initiated at levels of NWP (0.25-0.5%) leading to significant increase in particle size and viscosity. Pure NWP (0.5%) showed largest initial...

Optimised purification and characterisation of lipid transfer protein 1 (LTP1) and its lipid-bound isoform LTP1b from barley malt.

Science.gov (United States)

Nieuwoudt, Melanie; Lombard, Nicolaas; Rautenbach, Marina

2014-08-15

In beer brewing, brewers worldwide strive to obtain product consistency in terms of flavour, colour and foam. Important proteins contributing to beer foam are lipid transfer proteins (LTPs), in particular LTP1 and its lipid-bound isoform LTP1b, which are known to transport lipids in vivo and prevent lipids from destabilising the beer foam. LTP1 and LTP1b were successfully purified using only five purification steps with a high purified protein yield (160 mg LTP1 and LTP1b from 200 g barley). Circular dichroism of LTP1 and LTP1b confirmed that both proteins are highly tolerant to high temperatures (>90 °C) and are pH stable, particularly at a neutral to a more basic pH. Only LTP1 exhibited antiyeast and thermo-stable lytic activity, while LTP1b was inactive, indicating that the fatty acid moiety compromised the antimicrobial activity of LTP1. This lack in antiyeast activity and the positive foam properties of LTP1b would benefit beer fermentation and quality. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of pH on the hinge region of influenza viral protein: a combined constant pH and well-tempered molecular dynamics study

Science.gov (United States)

Pathak, Arup Kumar

2018-05-01

Despite the knowledge that the influenza protein, hemagglutinin, undergoes a large conformational change at low pH during the process of fusion with the host cell, its molecular mechanism remains elusive. The present constant pH molecular dynamics (CpHMD) study identifies the residues responsible for large conformational change in acidic condition. Based on the pKa calculations, it is predicted that His-106 is much more responsible for the large conformational change than any other residues in the hinge region of hemagglutinin protein. Potential of mean force profile from well-tempered meta-dynamics (WT-MtD) simulation is also generated along the folding pathway by considering radius of gyration (R gyr) as a collective variable (CV). It is very clear from the present WT-MtD study, that the initial bending starts at that hinge region, which may trigger other conformational changes. Both the protein–protein and protein–water HB time correlation functions are monitored along the folding pathway. The protein–protein (full or hinge region) HB time correlation functions are always found to be stronger than those of the protein–water time correlation functions. The dynamical balance between protein–protein and protein–water HB interactions favors the stabilization of the folded state.

Cold-Adapted Influenza and Recombinant Adenovirus Vaccines Induce Cross-Protective Immunity against pH1N1 Challenge in Mice

Science.gov (United States)

Soboleski, Mark R.; Gabbard, Jon D.; Price, Graeme E.; Misplon, Julia A.; Lo, Chia-Yun; Perez, Daniel R.; Ye, Jianqiang; Tompkins, S. Mark; Epstein, Suzanne L.

2011-01-01

Background The rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1) highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus. Methodology/Principal Findings BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca) influenza viruses from 1977 or recombinant adenoviruses (rAd) expressing 1934 nucleoprotein (NP) and consensus matrix 2 (M2) (NP+M2-rAd). Antibodies against the M2 ectodomain (M2e) were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus. Conclusion/Significance Cross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic. PMID:21789196

Gravity-driven pH adjustment for site-specific protein pKa measurement by solution-state NMR

Science.gov (United States)

Li, Wei

2017-12-01

To automate pH adjustment in site-specific protein pKa measurement by solution-state NMR, I present a funnel with two caps for the standard 5 mm NMR tube. The novelty of this simple-to-build and inexpensive apparatus is that it allows automatic gravity-driven pH adjustment within the magnet, and consequently results in a fully automated NMR-monitored pH titration without any hardware modification on the NMR spectrometer.

Graphene-based biomimetic materials targeting urine metabolite as potential cancer biomarker: application over different conductive materials for potentiometric transduction

Science.gov (United States)

Truta, Liliana A.A.N.A.; Ferreira, Nádia S.; Sales, M. Goreti F.

2015-01-01

This works presents a novel surface Smart Polymer Antibody Material (SPAM) for Carnitine (CRT, a potential biomarker of ovarian cancer), tested for the first time as ionophore in potentiometric electrodes of unconventional configuration. The SPAM material consisted of a 3D polymeric network created by surface imprinting on graphene layers. The polymer was obtained by radical polymerization of (vinylbenzyl)trimethylammonium chloride and 4-styrenesulfonic acid (signaling the binding sites), and vinyl pivalate and ethylene glycol dimethacrylate (surroundings). Non-imprinted material (NIM) was prepared as control, by excluding the template from the procedure. These materials were then used to produce several plasticized PVC membranes, testing the relevance of including the SPAM as ionophore, and the need for a charged lipophilic additive. The membranes were casted over solid conductive supports of graphite or ITO/FTO. The effect of pH upon the potentiometric response was evaluated for different pHs (2-9) with different buffer compositions. Overall, the best performance was achieved for membranes with SPAM ionophore, having a cationic lipophilic additive and tested in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 5.1. Better slopes were achieved when the membrane was casted on conductive glass (−57.4mV/decade), while the best detection limits were obtained for graphite-based conductive supports (3.6×10−5mol/L). Good selectivity was observed against BSA, ascorbic acid, glucose, creatinine and urea, tested for concentrations up to their normal physiologic levels in urine. The application of the devices to the analysis of spiked samples showed recoveries ranging from 91% (± 6.8%) to 118% (± 11.2%). Overall, the combination of the SPAM sensory material with a suitable selective membrane composition and electrode design has lead to a promising tool for point-of-care applications. PMID:26456975

Side-chain dynamics of a detergent-solubilized membrane protein: Measurement of tryptophan and glutamine hydrogen-exchange rates in M13 coat protein by 1H NMR spectroscopy

International Nuclear Information System (INIS)

O'Neil, J.D.J.; Sykes, B.D.

1989-01-01

M13 coat protein is a small (50 amino acids) lipid-soluble protein that becomes an integral membrane protein during the infection stage of the life cycle of the M13 phage and is therefore used as a model membrane protein. To study side-chain dynamics in the protein, the authors have measured individual hydrogen-exchange rates for a primary amide in the side chain of glutamine-15 and for the indole amine of tryptophan-26. The protein was solubilized with the use of perdeuteriated sodium dodecyl sulfate (SDS), and hydrogen-exchange rates were measured by using 1 H nuclear magnetic resonance spectroscopy. The glutamine-15 syn proton exchanged at a rate identical with that in glutamine model peptides except that the pH corresponding to minimum exchange was elevated by about 1.5 pH units. The tryptophan-26 indole amine proton exchange was biphasic, suggesting that two populations of tryptophan-26 exist. It is suggested that the two populations may reflect protein dimerization or aggregation in the SDS micelles. The pH values of minimum exchange for tryptophan-26 in both environments were also elevated by 1.3-1.9 pH units. This phenomenon is reproduced when small tryptophan- and glutamine-containing hydrophobic peptides are dissolved in the presence of SDS micelles. The electrostatic nature of this phenomenon is proven by showing that the minimum pH for exchange can be reduced by dissolving the hydrophobic peptides in the positively charged detergent micelle dodecyltrimethylammonium bromide

pH Modulates the Binding of EGR1 Transcription Factor to DNA

Science.gov (United States)

Mikles, David C.; Bhat, Vikas; Schuchardt, Brett J.; Deegan, Brian J.; Seldeen, Kenneth L.; McDonald, Caleb B.; Farooq, Amjad

2013-01-01

EGR1 transcription factor orchestrates a plethora of signaling cascades involved in cellular homeostasis and its down-regulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with increasing pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as H382 by virtue of the fact that its substitution to non-ionizable residues abolishes pH-dependence of the binding of EGR1 to DNA. Notably, H382 inserts into the major groove of DNA and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, H382 is predominantly conserved across other members of EGR1 family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating protein-DNA interactions central to this family of transcription factors. Collectively, our findings uncover an unexpected but a key step in the molecular recognition of EGR1 family of transcription factors and suggest that they may act as sensors of pH within the intracellular environment. PMID:23718776

pH Dependent Antimicrobial Peptides and Proteins, Their Mechanisms of Action and Potential as Therapeutic Agents

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Erum Malik

2016-11-01

Full Text Available Antimicrobial peptides (AMPs are potent antibiotics of the innate immune system that have been extensively investigated as a potential solution to the global problem of infectious diseases caused by pathogenic microbes. A group of AMPs that are increasingly being reported are those that utilise pH dependent antimicrobial mechanisms, and here we review research into this area. This review shows that these antimicrobial molecules are produced by a diverse spectrum of creatures, including vertebrates and invertebrates, and are primarily cationic, although a number of anionic examples are known. Some of these molecules exhibit high pH optima for their antimicrobial activity but in most cases, these AMPs show activity against microbes that present low pH optima, which reflects the acidic pH generally found at their sites of action, particularly the skin. The modes of action used by these molecules are based on a number of major structure/function relationships, which include metal ion binding, changes to net charge and conformational plasticity, and primarily involve the protonation of histidine, aspartic acid and glutamic acid residues at low pH. The pH dependent activity of pore forming antimicrobial proteins involves mechanisms that generally differ fundamentally to those used by pH dependent AMPs, which can be described by the carpet, toroidal pore and barrel-stave pore models of membrane interaction. A number of pH dependent AMPs and antimicrobial proteins have been developed for medical purposes and have successfully completed clinical trials, including kappacins, LL-37, histatins and lactoferrin, along with a number of their derivatives. Major examples of the therapeutic application of these antimicrobial molecules include wound healing as well as the treatment of multiple cancers and infections due to viruses, bacteria and fungi. In general, these applications involve topical administration, such as the use of mouth washes, cream formulations

Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology.

Science.gov (United States)

Kottom, Theodore J; Limper, Andrew H

2011-10-01

Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates. Copyright © 2011 John Wiley & Sons, Ltd.

Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass - Prediction of viscosity through protein-protein interaction measurements

DEFF Research Database (Denmark)

Neergaard, Martin S; Kalonia, Devendra S; Parshad, Henrik

2013-01-01

The purpose of this work was to explore the relation between protein-protein interactions (PPIs) and solution viscosity at high protein concentration using three monoclonal antibodies (mAbs), two of the IgG4 subclass and one of the IgG1 subclass. A range of methods was used to quantify the PPI...... low or high protein concentration determined using DLS. The PPI measurements were correlated with solution viscosity (measured by DLS using polystyrene nanospheres and ultrasonic shear rheology) as a function of pH (4-9) and ionic strength (10, 50 and 150mM). Our measurements showed that the highest...... solution viscosity was observed under conditions with the most negative kD, the highest apparent radius and the lowest net charge. An increase in ionic strength resulted in a change in the nature of the PPI at low pH from repulsive to attractive. In the neutral to alkaline pH region the mAbs behaved...

Evaluation of Saccharomyces cerevisiae GAS1 with respect to its involvement in tolerance to low pH and salt stress.

Science.gov (United States)

Matsushika, Akinori; Suzuki, Toshihiro; Goshima, Tetsuya; Hoshino, Tamotsu

2017-08-01

We previously showed that overexpression of IoGAS1, which was isolated from the multiple stress-tolerant yeast Issatchenkia orientalis, endows Saccharomyces cerevisiae cells with the ability to grow and ferment under acidic and high-salt conditions. The deduced amino acid sequence of the IoGAS1 gene product exhibits 60% identity with the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity. However, the functional roles of ScGAS1 in stress tolerance and pH regulation remain unclear. In the present study, we characterized ScGAS1 regarding its roles in tolerance to low pH and high salt concentrations. Transcriptional analysis indicated that, as for the IoGAS1 gene, ScGAS1 expression was pH dependent, with maximum expression at pH 3.0; the presence of salt increased endogenous expression of both GAS1 genes at almost all pH levels. These results suggested that ScGAS1, like IoGAS1, is involved in a novel acid- and salt-stress adaptation mechanism in S. cerevisiae. Overexpression of ScGAS1 in S. cerevisiae improved growth and ethanol production from glucose under acid stress without added salt, although the stress tolerance of the ScGAS1-overexpressing strain was inferior to that of the IoGAS1-overexpressing strain. However, overexpression of ScGAS1 did not result in increased tolerance of S. cerevisiae to combined acid and salt stress, even though ScGAS1 appears to be a salt-responsive gene. Thus, ScGAS1 is directly implicated in tolerance to low pH but does not confer salinity tolerance, supporting the view that ScGAS1 and IoGAS1 have overlapping yet distinct roles in stress tolerance in yeast. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The effect of emulsifying salts on the turbidity of a diluted milk system with varying pH and protein concentration.

Science.gov (United States)

Culler, M D; Saricay, Y; Harte, F M

2017-06-01

Solutions of 10 commonly used emulsifying salts (ES) listed in the Code of Federal Regulations (21CFR133.179) for pasteurized process cheese were tested for their effect on the turbidity of a diluted milk system at different pH and protein concentrations to characterize the conditions that affect micellar structure. Emulsifying salt solutions were made by mixing the ES in a 1-in-20 dilution of water in skim milk ultrafiltrate (3 kDa molecular weight cut-off) to obtain ES concentrations from 0 to 248 mM. Skim milk was added to solutions containing nanopure water, skim milk ultrafiltrate, and a specific ES ranging in concentration from 0 to 248 mM and pH 5, 5.8, 6.8, 7.8, and 8.8. The turbidity of the samples was measured as the optical density at 400 nm immediately after mixing (time, t = 0), after 30 s (t = 30s), and after 30 min (t = 30min). Emulsifying salts were found to cause a decrease in the turbidity of the system, which was modeled using an exponential decay model, where C* represents a threshold salt concentration at which rapid dissociation occurs. At pH values 5.8 and 6.8, the ES caused the greatest decrease in turbidity of the diluted milk system. At pH 5, the ES had the least effect on the turbidity of the system. Sodium hexametaphosphate was found to have the strongest dissociative effect, with a C* value of 0.33 mM for t = 0 at pH 6.8. In contrast, the largest C* value calculated at pH 6.8 was monosodium phosphate at 278.22 mM. Increased time resulted in lower C* values. The model established for this study can be used to predict the dissociation of casein micelles in the presence of various types of ES. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH

Energy Technology Data Exchange (ETDEWEB)

Cingolani, Gino, E-mail: cingolag@upstate.edu; Andrews, Dewan [Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210 (United States); Casjens, Sherwood [Department of Pathology, Division of Cell Biology and Immunology, University of Utah Medical School, Salt Lake City, UT 84112 (United States); Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210 (United States)

2006-05-01

The crystallogenesis of bacteriophage P22 tail-fiber gp26 is described. To study possible pH-induced conformational changes in gp26 structure, native trimeric gp26 has been crystallized at acidic pH (4.6) and a chimera of gp26 fused to maltose-binding protein (MBP-gp26) has been crystallized at neutral and alkaline pH (7-10). Gp26 is one of three phage P22-encoded tail accessory factors essential for stabilization of viral DNA within the mature capsid. In solution, gp26 exists as an extended triple-stranded coiled-coil protein which shares profound structural similarities with class I viral membrane-fusion protein. In the cryo-EM reconstruction of P22 tail extracted from mature virions, gp26 forms an ∼220 Å extended needle structure emanating from the neck of the tail, which is likely to be brought into contact with the cell’s outer membrane when the viral DNA-injection process is initiated. To shed light on the potential role of gp26 in cell-wall penetration and DNA injection, gp26 has been crystallized at acidic, neutral and alkaline pH. Crystals of native gp26 grown at pH 4.6 diffract X-rays to 2.0 Å resolution and belong to space group P2{sub 1}, with a dimer of trimeric gp26 molecules in the asymmetric unit. To study potential pH-induced conformational changes in the gp26 structure, a chimera of gp26 fused to maltose-binding protein (MBP-gp26) was generated. Hexagonal crystals of MBP-gp26 were obtained at neutral and alkaline pH using the high-throughput crystallization robot at the Hauptman–Woodward Medical Research Institute, Buffalo, NY, USA. These crystals diffract X-rays to beyond 2.0 Å resolution. Structural analysis of gp26 crystallized at acidic, neutral and alkaline pH is in progress.

Functional properties of pumpkin (Cucurbita pepo seed protein isolate and hydrolysate

Directory of Open Access Journals (Sweden)

Bučko Sandra Đ.

2016-01-01

Full Text Available Pumpkin seed protein isolate (PSPI was enzymatically hydrolysed by pepsin to obtain pumpkin seed protein hydrolysate, PSPH. Investigation on solubility, interfacial and emulsifying properties of both PSPI and PSPH was conducted under different conditions of pH (3-8 and ionic strength (0-1 mol/dm3 NaCl. PSPI had the lowest solubility, i.e. isoelectric point (pI, at pH 5. PSPH had higher solubility than PSPI over whole range of pH and ionic strengths tested. Decrease in surface and interfacial tension evidenced that both PSPI and PSPH adsorb at air/protein solution and oil/protein solution interface. Emulsions (20 % oil in water stabilized by 1 g/100cm3 PSPI or PSPH solution were prepared at pH 3, 5 and 8 and ionic strength of 0 and 0.5 mol/dm3 NaCl. PSPH stabilized emulsions from coalescence at all pH and ionic strengths tested. PSPI was able to stabilize emulsions at pH 3 and 0 mol/dm3 NaCl, and at pH 8 regardless of ionic strength, while emulsions at pH 5 and both 0 and 0.5 mol/dm3 NaCl and at pH 3 when ionic strength was increased separated to oil and serum layer immediately after preparation. All emulsions were susceptible to creaming instability. [Projekat Ministarstva nauke Republike Srbije, br. III 46010

Protein Alterations in Infiltrating Ductal Carcinomas of the Breast as Detected by Nonequilibrium pH Gradient Electrophoresis and Mass Spectrometry

Directory of Open Access Journals (Sweden)

Maria Kabbage

2008-01-01

Full Text Available Improvement of breast-cancer detection through the identification of potential cancer biomarkers is considered as a promising strategy for effective assessment of the disease. The current study has used nonequilibrium pH gradient electrophoresis with subsequent analysis by mass spectrometry to identify protein alterations in invasive ductal carcinomas of the breast from Tunisian women. We have identified multiple protein alterations in tumor tissues that were picked, processed, and unambiguously assigned identities by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF. The proteins identified span a wide range of functions and are believed to have potential clinical applications as cancer biomarkers. They include glycolytic enzymes, molecular chaperones, cytoskeletal-related proteins, antioxydant enzymes, and immunologic related proteins. Among these proteins, enolase 1, phosphoglycerate kinase 1, deoxyhemoglobin, Mn-superoxyde dismutase, α-B-crystallin, HSP27, Raf kinase inhibitor protein, heterogeneous nuclear ribonucleoprotein A2/B1, cofilin 1, and peptidylprolyl isomerase A were overexpressed in tumors compared with normal tissues. In contrast, the IGHG1 protein, the complement C3 component C3c, which are two newly identified protein markers, were downregulated in IDCA tissues.

Fibril formation from pea protein and subsequent gel formation.

Science.gov (United States)

Munialo, Claire Darizu; Martin, Anneke H; van der Linden, Erik; de Jongh, Harmen H J

2014-03-19

The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20 h at pH 2.0. Following heating of pea proteins, it was observed that all of the proteins were hydrolyzed into peptides and that 50% of these peptides were assembled into fibrils. Changes on a structural level in pea proteins were studied using circular dichroism, transmission electron microscopy, and particle size analysis. During the fibril assembly process, an increase in aggregate size was observed, which coincided with an increase in thioflavin T binding, indicating the presence of β-sheet aggregates. Fibrils made using pea proteins were more branched and curly. Gel formation of preformed fibrils was induced by slow acidification from pH 7.0 to a final pH of around pH 5.0. The ability of pea protein-based fibrillar gels to fracture during an amplitude sweep was comparable to those of soy protein and whey protein-based fibrillar gels, although gels prepared from fibrils made using pea protein and soy protein were weaker than those of whey protein. The findings show that fibrils can be prepared from pea protein, which can be incorporated into protein-based fibrillar gels.

Characterization of functional antibody and memory B-cell responses to pH1N1 monovalent vaccine in HIV-infected children and youth.

Directory of Open Access Journals (Sweden)

Donna J Curtis

Full Text Available We investigated immune determinants of antibody responses and B-cell memory to pH1N1 vaccine in HIV-infected children.Ninety subjects 4 to <25 years of age received two double doses of pH1N1 vaccine. Serum and cells were frozen at baseline, after each vaccination, and at 28 weeks post-immunization. Hemagglutination inhibition (HAI titers, avidity indices (AI, B-cell subsets, and pH1N1 IgG and IgA antigen secreting cells (ASC were measured at baseline and after each vaccination. Neutralizing antibodies and pH1N1-specific Th1, Th2 and Tfh cytokines were measured at baseline and post-dose 1.At entry, 26 (29% subjects had pH1N1 protective HAI titers (≥1:40. pH1N1-specific HAI, neutralizing titers, AI, IgG ASC, IL-2 and IL-4 increased in response to vaccination (p<0.05, but IgA ASC, IL-5, IL-13, IL-21, IFNγ and B-cell subsets did not change. Subjects with baseline HAI ≥1:40 had significantly greater increases in IgG ASC and AI after immunization compared with those with HAI <1:40. Neutralizing titers and AI after vaccination increased with older age. High pH1N1 HAI responses were associated with increased IgG ASC, IFNγ, IL-2, microneutralizion titers, and AI. Microneutralization titers after vaccination increased with high IgG ASC and IL-2 responses. IgG ASC also increased with high IFNγ responses. CD4% and viral load did not predict the immune responses post-vaccination, but the B-cell distribution did. Notably, vaccine immunogenicity increased with high CD19+CD21+CD27+% resting memory, high CD19+CD10+CD27+% immature activated, low CD19+CD21-CD27-CD20-% tissue-like, low CD19+CD21-CD27-CD20-% transitional and low CD19+CD38+HLADR+% activated B-cell subsets.HIV-infected children on HAART mount a broad B-cell memory response to pH1N1 vaccine, which was higher for subjects with baseline HAI≥1:40 and increased with age, presumably due to prior exposure to pH1N1 or to other influenza vaccination/infection. The response to the vaccine was dependent

Using PyMOL to Explore the Effects of pH on Noncovalent Interactions between Immunoglobulin G and Protein A: A Guided-Inquiry Biochemistry Activity.

Science.gov (United States)

Roche Allred, Zahilyn D; Tai, Heeyoung; Bretz, Stacey Lowery; Page, Richard C

2017-11-01

Students' understandings of foundational concepts such as noncovalent interactions, pH and pK a are crucial for success in undergraduate biochemistry courses. We developed a guided-inquiry activity to aid students in making connections between noncovalent interactions and pH/pK a . Students explore these concepts by examining the primary and tertiary structures of immunoglobulin G (IgG) and Protein A. Students use PyMOL, an open source molecular visualization application, to (1) identify hydrogen bonds and salt bridges between and within the proteins at physiological pH and (2) apply their knowledge of pH/pK a to association rate constant data for these proteins at pH 4 and pH 11. The laboratory activity was implemented within a one semester biochemistry laboratory for students majoring in allied health disciplines, engineering, and biological sciences. Several extensions for more advanced students are discussed. Students' overall performance highlighted their ability to successfully complete tasks such as labeling and identifying noncovalent interactions and revealed difficulties with analyzing noncovalent interactions under varying pH/pK a conditions. Students' evaluations after completing the activity indicated they felt challenged but also recognized the potential of the activity to help them gain meaningful understanding of the connections between noncovalent interactions, pH, pK a , and protein structure. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):528-536, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

Science.gov (United States)

Li, Cynthia H; Narhi, Linda O; Wen, Jie; Dimitrova, Mariana; Wen, Zai-qing; Li, Jenny; Pollastrini, Joseph; Nguyen, Xichdao; Tsuruda, Trace; Jiang, Yijia

2012-12-18

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

Effect of incubation time of sago (metroxylon sago) waste by local microorganism ″ginta″ on ph, crude protein, and crude fiber content

Science.gov (United States)

Ginting, Nurzainah; Pase, E.

2018-03-01

This study aims to examine the effect of incubation times of sago waste by local microorganism (MOL) “Ginta” to the crude protein and crude fiber content in relation to finding a cheap and good quality ruminants feed alternative. Incubation times were 0 hours to 144 hours. The data obtained were analyzed using Completely Randomize Design consisting of seven treatments and three replications. The result showed that the duration of incubation of sago waste by local microorganism (MOL) “Ginta” caused pH reduction, improved crude protein and crude fiber content. pH reduction was from 7.03 at 0 hour to 4.05 at 144 hours incubation. The highest increased in crude protein was H6U3 (5.58%) : 144 hours incubation and the lowest was H0U2 (3.22%) : 0 hour incubation while the highest crude fiber was H0U1 (19.99%) : 0 hour incubation and the lowest was H6U3 (18.23%) : 144 hours incubation. It can be concluded that incubation of sago waste triggered lower pH, higher crude protein and lower crude fiber than uninoculated. A recommendation could be given on using MOL ‘Ginta” in order to produce a cheap and good quality ruminans feed alternative.

Emulsion properties of pork myofibrillar protein in combination with microbial transglutaminase and calcium alginate under various pH conditions.

Science.gov (United States)

Hong, Geun Pyo; Min, Sang-Gi; Chin, Koo Bok

2012-01-01

In this study, the effects of microbial transglutaminase (MTG) and calcium alginate (CA) systems in combination with soybean oil on the emulsion properties of porcine myofibrillar protein (MP) were evaluated under various pH conditions. MTG was shown to improve emulsifying capacity and creaming stability, which increased with increasing pH values up to 6.5. The CA did not influence emulsifying capacity, but it improved the creaming stability of the MP-stabilized emulsions. Both MTG and CA enhanced the rheological properties, but their effects on the physical characteristics of the protein evidenced an opposite trend in relation to pH, i.e., the MTG system improved both the emulsion and gelling properties with increasing pH, whereas the CA system was effective when the pH was lowered. By combining the two MP gelling systems, a stable and pH-insensible emulsion could be produced. Copyright © 2011 Elsevier Ltd. All rights reserved.

Changes in pH and NADPH regulate the DNA binding activity of neuronal PAS domain protein 2, a mammalian circadian transcription factor.

Science.gov (United States)

Yoshii, Katsuhiro; Tajima, Fumihisa; Ishijima, Sumio; Sagami, Ikuko

2015-01-20

Neuronal PAS domain protein 2 (NPAS2) is a core clock transcription factor that forms a heterodimer with BMAL1 to bind the E-box in the promoter of clock genes and is regulated by various environmental stimuli such as heme, carbon monoxide, and NAD(P)H. In this study, we investigated the effects of pH and NADPH on the DNA binding activity of NPAS2. In an electrophoretic mobility shift (EMS) assay, the pH of the reaction mixture affected the DNA binding activity of the NPAS2/BMAL1 heterodimer but not that of the BMAL1/BMAL1 homodimer. A change in pH from 7.0 to 7.5 resulted in a 1.7-fold increase in activity in the absence of NADPH, and NADPH additively enhanced the activity up to 2.7-fold at pH 7.5. The experiments using truncated mutants revealed that N-terminal amino acids 1-61 of NPAS2 were sufficient to sense the change in both pH and NADPH. We further analyzed the kinetics of formation and DNA binding of the NPAS2/BMAL1 heterodimer at various pH values. In the absence of NADPH, a change in pH from 6.5 to 8.0 decreased the KD(app) value of the E-box from 125 to 22 nM, with an 8-fold increase in the maximal level of DNA binding for the NPAS2/BMAL1 heterodimer. The addition of NADPH resulted in a further decrease in KD(app) to 9 nM at pH 8.0. Furthermore, NPAS2-dependent transcriptional activity in a luciferase assay using NIH3T3 cells also increased with the pH of the culture medium. These results suggest that NPAS2 has a role as a pH and metabolite sensor in regulating circadian rhythms.

A Busy period analysis of the level dependent PH/PH/1/K queue

NARCIS (Netherlands)

Al Hanbali, Ahmad

2011-01-01

In this paper, we study the transient behavior of a level dependent single server queuing system with a waiting room of finite size during the busy period. The focus is on the level dependent PH/PH/1/K queue. We derive in closed form the joint transform of the length of the busy period, the number

pH Sensing and Regulation in Cancer

OpenAIRE

Mehdi eDamaghi; Jonathan W. Wojtkowiak; Robert J. Gillies

2013-01-01

Cells maintain intracellular pH (pHi) within a narrow range (7.1-7.2) by controlling membrane proton pumps and transporters whose activity is set by intra-cytoplasmic pH sensors. These sensors have the ability to recognize and induce cellular responses to maintain the intracellular pH, often at the expense of acidifying the extracellular pH. In turn, extracellular acidification impacts cells via specific acid-sensing ion channels (ASICs) and proton-sensing G-protein coupled receptors (GPCRs...

Role of MCT1 and CAII in skeletal muscle pH homeostasis, energetics, and function: in vivo insights from MCT1 haploinsufficient mice

KAUST Repository

Chatel, Benjamin; Bendahan, David; Hourdé , Christophe; Pellerin, Luc; Lengacher, Sylvain; Magistretti, Pierre J.; Le Fur, Yann; Vilmen, Christophe; Bernard, Monique; Messonnier, Laurent A.

2017-01-01

The purpose of this study was to investigate the effects of a partial suppression of monocarboxylate transporter (MCT)-1 on skeletal muscle pH, energetics, and function (MCT1(+/-) mice). Twenty-four MCT1(+/-) and 13 wild-type (WT) mice were subjected to a rest-exercise-recovery protocol, allowing assessment of muscle energetics (by magnetic resonance spectroscopy) and function. The study included analysis of enzyme activities and content of protein involved in pH regulation. Skeletal muscle of MCT1(+/-) mice had lower MCT1 (-61%; P < 0.05) and carbonic anhydrase (CA)-II (-54%; P < 0.05) contents. Although intramuscular pH was higher in MCT1(+/-) mice at rest (P < 0.001), the mice showed higher acidosis during the first minute of exercise (P < 0.01). Then, the pH time course was similar among groups until exercise completion. MCT1(+/-) mice had higher specific peak (P < 0.05) and maximum tetanic (P < 0.01) forces and lower fatigability (P < 0.001) when compared to WT mice. We conclude that both MCT1 and CAII are involved in the homeostatic control of pH in skeletal muscle, both at rest and at the onset of exercise. The improved muscle function and resistance to fatigue in MCT1(+/-) mice remain unexplained.-Chatel, B., Bendahan, D., Hourdé, C., Pellerin, L., Lengacher, S., Magistretti, P., Fur, Y. L., Vilmen, C., Bernard, M., Messonnier, L. A. Role of MCT1 and CAII in skeletal muscle pH homeostasis, energetics, and function: in vivo insights from MCT1 haploinsufficient mice.

Role of MCT1 and CAII in skeletal muscle pH homeostasis, energetics, and function: in vivo insights from MCT1 haploinsufficient mice

KAUST Repository

Chatel, Benjamin

2017-03-03

The purpose of this study was to investigate the effects of a partial suppression of monocarboxylate transporter (MCT)-1 on skeletal muscle pH, energetics, and function (MCT1(+/-) mice). Twenty-four MCT1(+/-) and 13 wild-type (WT) mice were subjected to a rest-exercise-recovery protocol, allowing assessment of muscle energetics (by magnetic resonance spectroscopy) and function. The study included analysis of enzyme activities and content of protein involved in pH regulation. Skeletal muscle of MCT1(+/-) mice had lower MCT1 (-61%; P < 0.05) and carbonic anhydrase (CA)-II (-54%; P < 0.05) contents. Although intramuscular pH was higher in MCT1(+/-) mice at rest (P < 0.001), the mice showed higher acidosis during the first minute of exercise (P < 0.01). Then, the pH time course was similar among groups until exercise completion. MCT1(+/-) mice had higher specific peak (P < 0.05) and maximum tetanic (P < 0.01) forces and lower fatigability (P < 0.001) when compared to WT mice. We conclude that both MCT1 and CAII are involved in the homeostatic control of pH in skeletal muscle, both at rest and at the onset of exercise. The improved muscle function and resistance to fatigue in MCT1(+/-) mice remain unexplained.-Chatel, B., Bendahan, D., Hourdé, C., Pellerin, L., Lengacher, S., Magistretti, P., Fur, Y. L., Vilmen, C., Bernard, M., Messonnier, L. A. Role of MCT1 and CAII in skeletal muscle pH homeostasis, energetics, and function: in vivo insights from MCT1 haploinsufficient mice.

SOCS-1 localizes to the microtubule organizing complex-associated 20S proteasome.

Science.gov (United States)

Vuong, Bao Q; Arenzana, Teresita L; Showalter, Brian M; Losman, Julie; Chen, X Peter; Mostecki, Justin; Banks, Alexander S; Limnander, Andre; Fernandez, Neil; Rothman, Paul B

2004-10-01

The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unknown. In this report, SOCS-1 is found to colocalize and biochemically copurify with the microtubule organizing complex (MTOC) and its associated 20S proteasome. The SOCS-1 SH2 domain is required for the localization of SOCS-1 to the MTOC. Overexpression of SOCS-1 targets Jak1 in an SH2-dependent manner to a perinuclear distribution resembling the MTOC-associated 20S proteasome. Analysis of MTOCs fractionated from SOCS-1-deficient cells demonstrates that SOCS-1 may function redundantly to regulate the localization of Jak1 to the MTOC. Nocodazole inhibits the protein turnover of SOCS-1, demonstrating that the minus-end transport of SOCS-1 to the MTOC-associated 20S proteasome is required to regulate SOCS-1 protein levels. These data link SOCS-1 directly with the proteasome pathway and suggest another function for the SH2 domain of SOCS-1 in the regulation of Jak/STAT signaling.

Evaluation of the pH- and Thermal Stability of the Recombinant Green Fluorescent Protein (GFP) in the Presence of Sodium Chloride

Science.gov (United States)

Ishii, Marina; Kunimura, Juliana Sayuri; Jeng, Hélio Tallon; Vessoni Penna, Thereza Christina; Cholewa, Olivia

The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0-7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9-30% NaCl or without and incubated at 80-95°C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84 ±0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94±0.60) was half that observed in phosphate buffer (pH 6.08±0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80°C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65±0.17) were directly dependent on the NaCl concentration and 5-10 times higher than D-values for GFP in WFI at 80°C. GFP pH-and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change

Energy Technology Data Exchange (ETDEWEB)

Masuda, Tetsuya, E-mail: t2masuda@kais.kyoto-u.ac.jp [Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Department of Natural Resources, Graduate School of Global Environmental Studies, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Ohta, Keisuke [Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Department of Natural Resources, Graduate School of Global Environmental Studies, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Kitabatake, Naofumi [Department of Foods and Human Nutrition, Notre Dame Seishin University, Okayama 700-8516 (Japan); Tani, Fumito [Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Department of Natural Resources, Graduate School of Global Environmental Studies, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan)

2012-03-02

Highlights: Black-Right-Pointing-Pointer Structure of a recombinant thaumatin at pH 8.0 determined at a resolution of 1.0 A. Black-Right-Pointing-Pointer Substantial fluctuations of a loop in domain II was found in the structure at pH 8.0. Black-Right-Pointing-Pointer B-factors for Lys137, Lys163, and Lys187 were significantly affected by pH change. Black-Right-Pointing-Pointer An increase in mobility might play an important role in the heat-induced aggregation. -- Abstract: Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50 nM. Although the sweetness remains when thaumatin is heated at 80 Degree-Sign C for 4 h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0 A. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of a C{alpha} atom to be substantially greater in the large disulfide-rich region of domain II, especially residues 154-164, suggesting that a loop region in domain II to be affected by solvent conditions. Furthermore, B-factors of Lys137, Lys163, and Lys187 were significantly affected by pH change, suggesting that a striking increase in the mobility of these lysine residues, which could facilitate a reaction with a free sulfhydryl residue produced via the {beta}-elimination of disulfide bonds by heating at a pH above 7.0. The increase in mobility of lysine residues as well as a loop region in domain II might play an important role in the heat-induced aggregation of thaumatin above pH 7.0.

Ph1 chromosomes and bcr gene rearrangements in chronic myelocytic leukemia patients developed from atomic bomb survivors

International Nuclear Information System (INIS)

Tanaka, Kimio; Takechi, Miho; Shigeta, Chiharu; Sakatani, Keiko; Oguma, Nobuo; Kamada, Nanao; Takimoto, Yasuo; Kuramoto, Atsushi

1989-01-01

This study compared findings of chronic myelocytic leukemia (CML) in A-bomb survivors (n=8) developing CML within 10 years after the bombing and in non-exposed CML patients (n=14). Both Ph 1 chromosomes and bcr rearrangement were observed in all patients in both exposed and non-exposed groups. There was no significant difference in distribution sites of bcr rearrangement between the groups. These results suggest that bcr-abl chimera mRNA and chimera protein associated with Ph 1 chromosomes have an important role in the development of CML among A-bomb survivors, as well as among non-exposed patients. (N.K.)

The Effect of NaOH Concentration on pH, Egg White Protein Content and Yolk Colour Pidan Egg

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Herly Evanuarini

2017-11-01

Full Text Available The purpose of this research was to determine the best treatment NaOH addition on pidan eggs. The materials used for this research was pidan made from duck egg, NaOH, salt, black tea and water. The method was used experiment laboratory and Completely Randomized Design (CRD using 4 treatments and 4 replications. The treatments were T0 (control, T1 (1.4%, T2 (2.8% and T3 (4.2%. The data were analyzed by Analysis of Variance (ANOVA if there was significantly continued by Duncan’s Multiple Range Test (DMRT. The result showed that NaOH concentration on pidan eggs gave significant effect (P<0.05 on albumen protein content, gave highly significant (P<0.01 on pH value and yolk colour. The conclusion of this research was 4.2% NaOH addition on pidan egg was the best treatment with gave result yolk and albumen pH: 10.69; 10.25, albumen protein content 26.89%, egg yolk colour L* (lightness, a* (redness, b* (yellowness:  26.89; 11.33, and 26.77. The suggestion of this research was ussed different immersion time on pidan egg production.

Speed associated with plasma pH, oxygen content, total protein and urea in an 80 km race.

Science.gov (United States)

Hoffman, R M; Hess, T M; Williams, C A; Kronfeld, D S; Griewe-Crandell, K M; Waldron, J E; Graham-Thiers, P M; Gay, L S; Splan, R K; Saker, K E; Harris, P A

2002-09-01

To test the hypothesis that endurance performance may be related quantitatively to changes in blood, we measured selected blood variables then determined their reference ranges and associations with speed during an 80 km race. The plan had 46 horses in a 2 x 2 factorial design testing a potassium-free electrolyte mix and a vitamin supplement. Blood samples were collected before the race, at 21, 37, 56 and 80 km, and 20 min after finishing, for assay of haematocrit, plasma pH, pO2, pCO2, [Na+], [K+], [Ca++], [Mg++], [Cl-], lactate, glucose, urea, cortisol, alpha-tocopherol, ascorbate, creatine kinase, aspartate amino transferase, lipid hydroperoxides, total protein, albumin and creatinine, and erythrocyte glutathione and glutathione peroxidase. Data from 34 finishers were analysed statistically. Reference ranges for resting and running horses were wide and overlapping and, therefore, limiting with respect to evaluation of individual horses. Speed correlations were most repeatable, with variables reflecting blood oxygen transport (enabling exercise), acidity and electrolytes (limiting exercise) and total protein (enabling then, perhaps, limiting). Stepwise regressions also included plasma urea concentration (limiting). The association of speed with less plasma acidity and urea suggests the potential for fat adaptation and protein restriction in endurance horses, as found previously in Arabians performing repeated sprints. Conditioning horses fed fat-fortified and protein-restricted diets may not only improve performance but also avoid grain-associated disorders.

Ethylene regulates phosphorus remobilization and expression of a phosphate transporter (PhPT1) during petunia corolla senescence

Science.gov (United States)

Chapin, Laura J.; Jones, Michelle L.

2009-01-01

The programmed degradation of macromolecules during petal senescence allows the plant to remobilize nutrients from dying to developing tissues. Ethylene is involved in regulating the timing of nucleic acid degradation in petunia, but it is not clear if ethylene has a role in the remobilization of phosphorus during petal senescence. To investigate ethylene's role in nutrient remobilization, the P content of petals (collectively called the corolla) during early development and senescence was compared in ethylene-sensitive wild type Petunia×hybrida ‘Mitchell Diploid’ (MD) and transgenic petunias with reduced sensitivity to ethylene (35S::etr1-1). When compared to the total P content of corollas on the day of flower opening (the early non-senescing stage), P in MD corollas had decreased 74% by the late stage of senescence (advanced wilting). By contrast, P levels were only reduced by an average of 32% during etr1-1 corolla (lines 44568 and Z00-35-10) senescence. A high-affinity phosphate transporter, PhPT1 (PhPht1;1), was cloned from senescing petunia corollas by RT-PCR. PhPT1 expression was up-regulated during MD corolla senescence and a much smaller increase was detected during the senescence of etr1-1 petunia corollas. PhPT1 mRNA levels showed a rapid increase in detached corollas (treated at 1 d after flower opening) following treatment with low levels of ethylene (0.1 μl l-1). Transcripts accumulated in the presence of the protein synthesis inhibitor, cycloheximide, indicating that PhPT1 is a primary ethylene response gene. PhPT1 is a putative phosphate transporter that may function in Pi translocation during senescence. PMID:19380421

Investigation of the protective effect of whey proteins on lactococcal phages during heat treatment at various pH.

Science.gov (United States)

Geagea, Hany; Gomaa, Ahmed I; Remondetto, Gabriel; Moineau, Sylvain; Subirade, Muriel

2015-10-01

The incorporation of whey protein concentrates (WPC) into cheese is a risky process due to the potential contamination with thermo-resistant phages of lactic acid bacteria (LAB). Furthermore, whey proteins can protect phages during heat treatment, thereby increasing the above risk. The main objective of this work was to understand this protective effect in order to better control LAB phages and maximize whey recycling in the cheese industry. First, the inactivation of a previously characterized thermo-resistant lactococcal virulent phage (P1532) was investigated at 95 °C in WPC, in individual whey components β-lactoglobulin, α-lactalbumin, and bovine serum albumin as well as under different heat and pH conditions. The structural changes of the tested proteins were also monitored by transmission FTIR spectroscopy. Phage inactivation results indicated that the protective effect of whey proteins was pH and time dependent at 95 °C and was not restricted to one component. FTIR spectra suggest that the protection is related to protein molecular structures and to the level of protein aggregates, which was more pronounced in acidic conditions. Moreover, the molecular structure of the three proteins tested was differently influenced by pH and the duration of the heat treatment. This work confirms the protective effect of WPC on phages during heat treatment and offers the first hint to explain such phenomenon. Finding the appropriate treatment of WPC to reduce the phage risk is one of the keys to improving the cheese manufacturing process. Copyright © 2015 Elsevier B.V. All rights reserved.

Genetically encoded pH sensor for tracking surface proteins through endocytosis.

Science.gov (United States)

Grover, Anmol; Schmidt, Brigitte F; Salter, Russell D; Watkins, Simon C; Waggoner, Alan S; Bruchez, Marcel P

2012-05-14

Traffic cam: a tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of β(2)-adrenergic receptors. It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Calcium affecting protein expression in longan under simulated acid rain stress.

Science.gov (United States)

Pan, Tengfei; Li, Yongyu; Ma, Cuilan; Qiu, Dongliang

2015-08-01

Longan (Dimocarpus longana Lour. cv. Wulongling) of uniform one-aged seedlings grown in pots were selected to study specific proteins expressed in leaves under simulated acid rain (SiAR) stress and exogenous Ca(2+) regulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there was a protein band specifically expressed under SiAR of pH 2.5 stress for 15 days with its molecular weight of about 23 kD. A 17 kD protein band specifically expressed after SiAR stress 5 days. Compared with pH 2.5, the pH 3.5 of SiAR made a less influence to protein expression. Two-dimensional electrophoresis (2-DE) results showed that six new specific proteins including C4 (20.2 kD pI 6.0), F (24 kD pI 6.35), B3 (22.3 kD pI 6.35), B4 (23.5 kD pI 6.5), C5 (21.8 kD pI 5.6), and C6 (20.2 kD pI 5.6) specifically expressed. C4 always expressed during SiAR stress. F expressed under the stress of pH 2.5 for 15 days and expressed in all pH SiAR stress for 20 days. The expression of proteins including B3, C5, and C6 was related to pH value and stress intensity of SiAR. The expression of B4 resulted from synergistic effects of SiAR and Ca. The expression of G1 (Mr 19.3 kD, pI 4.5), G2 (Mr 17.8 kD, pI 4.65), G3 (Mr 16.6 kD, pI 4.6), and G4 (Mr 14.7 kD, pI 4.4) enhanced under the treatment of 5 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM chlorpromazine (CPZ). These proteins showed antagonistic effects and might be relative to the Ca-calmodulin (Ca-CaM) system of longan in response to SiAR stress.

Effect of temperature and pH on the actiity of ribulose 1,5-diphosphate carboxylase from the thermophilic hydrogen bacterium Pseudomonas thermophila

Energy Technology Data Exchange (ETDEWEB)

Romanova, A K; Emnova, E E; Zykalova, K A

1980-01-01

The activity of ribulose 1,5-diphosphate (RDP) carboxylase was found in the soluble fraction of the cytoplasm from sonicated Pseudomonas thermophila K-2 cells. The enzyme is relatively thermolabile and completey loses its activity at 80/sup 0/C. The activity of RDP carboxylase at 60/sup 0/C increases by 40% during the first 10 min of heating in the presence of Mg/sup 2 +/ ions, bicarbonate and dithiothreitol, and again decreases if the enzyme is heated over 20 min. The optimum temperature of the enzyme is 50 to 55/sup 0/C. The specific activity of the enzyme in fresh preparations under these conditions reaches 0.22 unit per 1 mg of protein in the extract. The calculated value of the activation energy for RDP carboxylase is 6.4 keal.mole/sup -1/, but 11.6 kcal.mole/sup -1/ in frozen preparations. The optimal pH is 7.0 to 7.3 depending on the buffer. The temperature optimum for the enzyme action does not depend on pH within the range of 7.3 to 8.8. Therefore, RDP carboxylase of Ps, thermophila K-2 differs from RDP carboxylases of mesophilic cultures studied earlier by a higher susceptibility to a decrease in temeprature (the enzyme activity is negligible at 30/sup 0/C), by a lower value of the activation energy at suboptimal temperatures, and by a lower pH optimum of the enzyme action.

Genetic interactions of MAF1 identify a role for Med20 in transcriptional repression of ribosomal protein genes.

Directory of Open Access Journals (Sweden)

Ian M Willis

2008-07-01

Full Text Available Transcriptional repression of ribosomal components and tRNAs is coordinately regulated in response to a wide variety of environmental stresses. Part of this response involves the convergence of different nutritional and stress signaling pathways on Maf1, a protein that is essential for repressing transcription by RNA polymerase (pol III in Saccharomyces cerevisiae. Here we identify the functions buffering yeast cells that are unable to down-regulate transcription by RNA pol III. MAF1 genetic interactions identified in screens of non-essential gene-deletions and conditionally expressed essential genes reveal a highly interconnected network of 64 genes involved in ribosome biogenesis, RNA pol II transcription, tRNA modification, ubiquitin-dependent proteolysis and other processes. A survey of non-essential MAF1 synthetic sick/lethal (SSL genes identified six gene-deletions that are defective in transcriptional repression of ribosomal protein (RP genes following rapamycin treatment. This subset of MAF1 SSL genes included MED20 which encodes a head module subunit of the RNA pol II Mediator complex. Genetic interactions between MAF1 and subunits in each structural module of Mediator were investigated to examine the functional relationship between these transcriptional regulators. Gene expression profiling identified a prominent and highly selective role for Med20 in the repression of RP gene transcription under multiple conditions. In addition, attenuated repression of RP genes by rapamycin was observed in a strain deleted for the Mediator tail module subunit Med16. The data suggest that Mediator and Maf1 function in parallel pathways to negatively regulate RP mRNA and tRNA synthesis.

Protein hydrolysates from the alga Chlorella vulgaris 87/1 with potentialities in immuno nutrition

International Nuclear Information System (INIS)

Morris, Humberto J; Carrillo, Olimpia; Almarales, Angel; Bermudez, Rosa C; Alonso, Maria E; Borges, Leonardo; Quintana, Maria M; Fontaine, Roberto; Llaurado, Gabriel; Hernandez, Martha

2009-01-01

Chlorella vulgaris (Chlorophyta, Chlorophyceae) has received a particular attention in the programmes of microalgae utilisation in biotechnology. Enzymatic hydrolysis of cell proteins represents a very promising method to increase protein digestibility and thus, for obtaining hydrolysates with improved nutritional and functional properties. However, this technology has been little approached and the biological evaluation of hydrolysates has had a strictly nutritional nature. The design of hydrolysis conditions that combined for the first time, the use of C.vulgaris 87/1 treated with ethanol and pancreatin at pH values of 7.5-8.0, led to a product with a degree of hydrolysis of 20-22% and yields of 50-55%, characterised by a high digestibility (97.2%) and nitrogen solubility over a wide pH range (2.0-10.0). Hydrolysis curves were fitted to an exponential model, common to many food proteins. The bulk of the product dry matter consists of soluble peptides and free amino acids (47.7%) with three main peptides of molecular masses between 2 and 5 kDa. The oral administration of Chlorella hydrolysate (500 mg/kg) to undernourished Balb/c mice provided benefits in terms of liver protein metabolism and the induction of anabolic processes in gut mucosa. The hydrolysate also enhanced the immunological recovery, as judged by the stimulation of haemopoiesis, monocyte macrophage system activation, as well as humoral and cell mediated immune functions, like T-dependent antibody response and the reconstitution of delayed-type hypersensitivity (DTH) response. These results represent the first findings in the world concerning the immunomodulating effects of a microalgae protein hydrolysate. (author)

Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

NARCIS (Netherlands)

Peters, Fabian; Maestre-Martinez, M.; Leonov, A.; Kovacic, L.; Becker, S.; Boelens, R.; Griesinger, C.

2011-01-01

Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large

Amide temperature coefficients in the protein G B1 domain

International Nuclear Information System (INIS)

Tomlinson, Jennifer H.; Williamson, Mike P.

2012-01-01

Temperature coefficients have been measured for backbone amide 1 H and 15 N nuclei in the B1 domain of protein G (GB1), using temperatures in the range 283–313 K, and pH values from 2.0 to 9.0. Many nuclei display pH-dependent coefficients, which were fitted to one or two pK a values. 1 H coefficients showed the expected behaviour, in that hydrogen-bonded amides have less negative values, but for those amides involved in strong hydrogen bonds in regular secondary structure there is a negative correlation between strength of hydrogen bond and size of temperature coefficient. The best correlation to temperature coefficient is with secondary shift, indicative of a very approximately uniform thermal expansion. The largest pH-dependent changes in coefficient are for amides in loops adjacent to sidechain hydrogen bonds rather than the amides involved directly in hydrogen bonds, indicating that the biggest determinant of the temperature coefficient is temperature-dependent loss of structure, not hydrogen bonding. Amide 15 N coefficients have no clear relationship with structure.

Computational Studies of pH Sensing Design Principles in Proteins

Science.gov (United States)

Garrido Ruiz, Diego

Changes in pH are important regulatory signals for biological function, under physiological and pathological conditions. Recent advances in computer simulations strategies have made the exploration of the effects of charge titrations on protein function possible. In this work, I make use of these strategies to investigate the thermodynamic coupling between conformation and protonation states that give rise to pH-dependent function. As motivation for the rest of the work, I start by presenting a collaborative investigation on a pH-sensing mutant of the EGFR tyrosine kinase common to a set of distinct cancers. From then, I reduce the complexity of the systems under study to build models where exact enumeration of states is possible to inquire about the nature of the couplings between protonation states and conformation. Finally, I discuss detailed simulations of pH-sensing proteins for which I use the expectations and insights generated with simple models to identify and interpret couplings of interest for pH-dependent behavior.

Salivary protein concentration, flow rate, buffer capacity and pH estimation: A comparative study among young and elderly subjects, both normal and with gingivitis and periodontitis.

Science.gov (United States)

Shaila, Mulki; Pai, G Prakash; Shetty, Pushparaj

2013-01-01

To evaluate the salivary protein concentration in gingivitis and periodontitis patients and compare the parameters like salivary total protein, salivary albumin, salivary flow rate, pH, buffer capacity and flow rate in both young and elderly patients with simple methods. One hundred and twenty subjects were grouped based on their age as young and elderly. Each group was subgrouped (20 subjects) as controls, gingivitis and periodontitis. Unstimulated whole saliva was collected from patients and flow rate was noted down during collection of the sample. Salivary protein estimation was done using the Biuret method and salivary albumin was assessed using the Bromocresol green method. pH was estimated with a pHmeter and buffering capacity was analyzed with the titration method. Student's t-test, Fisher's test (ANOVA) and Tukey HSD (ANOVA) tests were used for statistical analysis. A very highly significant rise in the salivary total protein and albumin concentration was noted in gingivitis and periodontitis subjects of both young and elderly. An overall decrease in salivary flow rate was observed among the elderly, and also the salivary flow rate of women was significantly lower than that of men. Significant associations between salivary total protein and albumin in gingivitis and periodontitis were found with simple biochemical tests. A decrease in salivary flow rate among elderly and among women was noted.

Identifying immunogenic CD4+ T-cell epitopes of Myeloid cell leukemia 1 using overlapping 20-mer peptides spanning the whole protein

DEFF Research Database (Denmark)

Woodworth, Joshua S.; Agger, Else Marie; Hansen, Paul Robert

2015-01-01

) small-molecule inhibitors [6] and (iii) peptide inhibitors [7]. In recent years, therapeutic vaccination with synthetic peptides derived from anti-apoptotic proteins such as Mcl-1 has emerged as a promising strategy against hematological cancers. In this study, 34 overlapping 20-mer peptides, spanning...

Development of antifertility vaccine using sperm specific proteins

Directory of Open Access Journals (Sweden)

A H Bandivdekar

2014-01-01

Full Text Available Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4, protein hyaluronidase (PH-20, and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80kDa HSA is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80kDa HSA (Peptide NT and its peptides (Peptides 1, 2, 3 and 4 obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80kDA HAS suggest that the synthetic Peptide-1 of 80kDa HSA is the promising candidate for development of male contraceptive vaccine.

Major immunogenic proteins of phocid herpes-viruses and their relationships to proteins of canine and feline herpesviruses.

Science.gov (United States)

Harder, T C; Harder, M; de Swart, R L; Osterhaus, A D; Liess, B

1998-04-01

The immunogenic proteins of cells infected with the alpha- or the gamma-herpesvirus of seals, phocid herpesvirus-1 and -2 (PhHV-1, -2), were examined in radioimmunoprecipitation assays as a further step towards the development of a PhHV-1 vaccine. With sera obtained from convalescent seals of different species or murine monoclonal antibodies (Mabs), at least seven virus-induced glycoproteins were detected in lysates of PhHV-1-infected CrFK cells. A presumably disulphide-linked complex composed of glycoproteins of 59, 67 and 113/120 kDa, expressed on the surface of infected cells, was characterized as a major immunogenic infected cell protein of PhHV-1. This glycoprotein complex has previously been identified as the proteolytically cleavable glycoprotein B homologue of PhHV-1 (14). At least three distinct neutralization-relevant epitopes were operationally mapped, by using Mabs, on the glycoprotein B of PhHV-1. Among the infected cell proteins of the antigenically closely related feline and canine herpesvirus, the glycoprotein B equivalent proved to be the most highly conserved glycoprotein. Sera obtained from different seal species from Arctic, Antarctic, and European habitats did not precipitate uniform patterns of infected cell proteins from PhHV-1-infected cell lysates although similar titres of neutralizing antibodies were displayed. Thus, antigenic differences among the alphaherpesvirus species prevalent in the different pinniped populations cannot be excluded. PhHV-2 displayed a different pattern of infected cell proteins and only limited cross-reactivity to PhHV-1 at the protein level was detected, which is in line with its previous classification as a distinct species, based on nucleotide sequence analysis, of the gammaherpesvirus linenge. A Mab raised against PhHV-2 and specific for a major glycoprotein of 117 kDa, cross reacted with the glycoprotein B of PhHV-1. The 117-kDa glycoprotein could represent the uncleaved PhHV-2 glycoprotein B homologue.

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases.

Science.gov (United States)

Tsonev, Latchezar I; Hirsh, Allen G

2008-07-25

pISep is a major new advance in low ionic strength ion exchange chromatography. It enables the formation of externally controlled pH gradients over the very broad pH range from 2 to 12. The gradients can be generated on either cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Associated pISep software makes possible the calculation of either linear, nonlinear or combined, multi-step, multi-slope pH gradients. These highly reproducible pH gradients, while separating proteins and glycoproteins in the order of their electrophoretic pIs, provide superior chromatographic resolution compared to salt. This paper also presents a statistical mechanical model for protein binding to ion exchange stationary phases enhancing the electrostatic interaction theory for the general dependence of retention factor k, on both salt and pH simultaneously. It is shown that the retention factors computed from short time isocratic salt elution data of a model protein can be used to accurately predict its salt elution concentration in varying slope salt elution gradients formed at varying isocratic pH as well as the pH at which it will be eluted from an anionic exchange column by a pISep pH gradient in the absence of salt.

Molten Globule-Like Partially Folded State of Bacillus licheniformis α-Amylase at Low pH Induced by 1,1,1,3,3,3-Hexafluoroisopropanol

Directory of Open Access Journals (Sweden)

Adyani Azizah Abd Halim

2014-01-01

Full Text Available Effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP on acid-denatured Bacillus licheniformis α-amylase (BLA at pH 2.0 was investigated by far-UV CD, intrinsic fluorescence, and ANS fluorescence measurements. Addition of increasing HFIP concentrations led to an increase in the mean residue ellipticity at 222 nm (MRE222 nm up to 1.5 M HFIP concentration beyond which it sloped off. A small increase in the intrinsic fluorescence and a marked increase in the ANS fluorescence were also observed up to 0.4 M HFIP concentration, both of which decreased thereafter. Far- and near-UV CD spectra of the HFIP-induced state observed at 0.4 M HFIP showed significant retention of the secondary structures closer to native BLA but a disordered tertiary structure. Increase in the ANS fluorescence intensity was also observed with the HFIP-induced state, suggesting exposure of the hydrophobic clusters to the solvent. Furthermore, thermal denaturation of HFIP-induced state showed a non-cooperative transition. Taken together, all these results suggested that HFIP-induced state of BLA represented a molten globule-like state at pH 2.0.

Karakteristik Protein dan Nitrogen Non Protein Daging Ikan Cucut Lanyam (Charcharhinus limbatus (Characteristics of Protein and Non Protein Nitrogen in Lanyam Shark Muscle

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Yuspihana Fitrial

2017-02-01

Based on protein solubility of Lanyam muscle at pH 1.5 to 12 obtained two points which is minimum solubility at pH 4.5 and pH 9. Based on the classification Osborn, Lanyam muscle contained albumin (28.64%, globulin (13:44%, prolamin (03.29%, glutelin (33.70%. Observation of non-protein nitrogen levels indicated that the washing process was very effective to reduce non-protein nitrogen levels up to 62.34% and urea levels up to 58% . Differential Scanning Calorimetry Study of Lanyam mince showed two types of protein that has a different stability to heat and after added 2.5% NaCl formed a peak which is a fusion of both these proteins

Protein fouling in carbon nanotubes enhanced ultrafiltration membrane: Fouling mechanism as a function of pH and ionic strength

KAUST Repository

Lee, Jieun; Jeong, Sanghyun; Ye, Yun; Chen, Vicki; Vigneswaran, Saravanamuthu; Leiknes, TorOve; Liu, Zongwen

2016-01-01

The protein fouling behavior was investigated in the filtration of the multiwall carbon nanotube (MWCNT) composite membrane and commercial polyethersulfone ultrafiltration (PES-UF) membrane. The effect of solution chemistry such as pH and ionic strength on the protein fouling mechanism was systematically examined using filtration model such as complete pore blocking, intermediate pore blocking and cake layer formation. The results showed that the initial permeate flux pattern and fouling behavior of the MWCNT composite membrane were significantly influenced by pH and ionic strength while the effect of PES-UF membrane on flux was minimal. In a lysozyme (Lys) filtration, the severe pore blocking in the MWCNT membrane was made by the combined effect of intra-foulant interaction (Lys-Lys) and electrostatic repulsion between the membrane surface and the foulant at pH 4.7 and 10.4, and increasing ionic strength where the foulant-foulant interaction and membrane-fouling interaction were weak. In a bovine serum albumin (BSA) filtration, severe pore blocking was reduced by less deposition via the electrostatic interaction between the membrane and foulant at pH 4.7 and 10.4 and increasing ionic strength, at which the interaction between the membrane and BSA became weak. For binary mixture filtration, the protein fouling mechanism was more dominantly affected by foulant-foulant interaction (Lys-BSA, Lys-Lys, and BSA-BSA) at pH 7.0 and increase in ionic strength. This research demonstrates that MWCNT membrane fouling can be alleviated by changing pH condition and ionic strength based on the foulant-foulant interaction and the electrostatic interaction between the membrane and foulant.

Protein fouling in carbon nanotubes enhanced ultrafiltration membrane: Fouling mechanism as a function of pH and ionic strength

KAUST Repository

Lee, Jieun

2016-11-04

The protein fouling behavior was investigated in the filtration of the multiwall carbon nanotube (MWCNT) composite membrane and commercial polyethersulfone ultrafiltration (PES-UF) membrane. The effect of solution chemistry such as pH and ionic strength on the protein fouling mechanism was systematically examined using filtration model such as complete pore blocking, intermediate pore blocking and cake layer formation. The results showed that the initial permeate flux pattern and fouling behavior of the MWCNT composite membrane were significantly influenced by pH and ionic strength while the effect of PES-UF membrane on flux was minimal. In a lysozyme (Lys) filtration, the severe pore blocking in the MWCNT membrane was made by the combined effect of intra-foulant interaction (Lys-Lys) and electrostatic repulsion between the membrane surface and the foulant at pH 4.7 and 10.4, and increasing ionic strength where the foulant-foulant interaction and membrane-fouling interaction were weak. In a bovine serum albumin (BSA) filtration, severe pore blocking was reduced by less deposition via the electrostatic interaction between the membrane and foulant at pH 4.7 and 10.4 and increasing ionic strength, at which the interaction between the membrane and BSA became weak. For binary mixture filtration, the protein fouling mechanism was more dominantly affected by foulant-foulant interaction (Lys-BSA, Lys-Lys, and BSA-BSA) at pH 7.0 and increase in ionic strength. This research demonstrates that MWCNT membrane fouling can be alleviated by changing pH condition and ionic strength based on the foulant-foulant interaction and the electrostatic interaction between the membrane and foulant.

Polyamine stress at high pH in Escherichia coli K-12

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Tate Daniel P

2005-10-01

Full Text Available Abstract Background Polyamines such as spermine and spermidine are required for growth of Escherichia coli; they interact with nucleic acids, and they bind to ribosomes. Polyamines block porins and decrease membrane permeability, activities that may protect cells in acid. At high concentrations, however, polyamines impair growth. They impair growth more severely at high pH, probably due to their increased uptake as membrane-permeant weak bases. The role of pH is critical in understanding polyamine stress. Results The effect of polyamines was tested on survival of Escherichia coli K-12 W3110 in extreme acid or base (pH conditions outside the growth range. At pH 2, 10 mM spermine increased survival by 2-fold, and putrescine increased survival by 30%. At pH 9.8, however, E. coli survival was decreased 100-fold by 10 mM spermine, putrescine, cadaverine, or spermidine. At pH 8.5, spermine decreased the growth rate substantially, whereas little effect was seen at pH 5.5. Spermidine required ten-fold higher concentrations to impair growth. On proteomic 2-D gels, spermine and spermidine caused differential expression of 31 different proteins. During log-phase growth at pH 7.0, 1 mM spermine induced eight proteins, including PykF, GlpK, SerS, DeaD, OmpC and OmpF. Proteins repressed included acetate-inducible enzymes (YfiD, Pta, Lpd as well as RapA (HepA, and FabB. At pH 8.5, spermine induced additional proteins: TnaA, OmpA, YrdA and NanA (YhcJ and also repressed 17 proteins. Four of the proteins that spermine induced (GlpK, OmpA, OmpF, TnaA and five that were repressed (Lpd, Pta, SucB, TpiA, YfiD show similar induction or repression, respectively, in base compared to acid. Most of these base stress proteins were also regulated by spermidine, but only at ten-fold higher concentration (10 mM at high pH (pH 8.5. Conclusion Polyamines increase survival in extreme acid, but decrease E. coli survival in extreme base. Growth inhibition by spermine and

A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

Science.gov (United States)

Chang, Xiaoxiao; Donnelly, Linda; Sun, Daoyang; Rao, Jingping; Reid, Michael S.; Jiang, Cai-Zhong

2014-01-01

Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO), and ABA (NCED) biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA) and abiotic stresses (dehydration, NaCl and cold). Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence. PMID:24551088

A Petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence.

Science.gov (United States)

Chang, Xiaoxiao; Donnelly, Linda; Sun, Daoyang; Rao, Jingping; Reid, Michael S; Jiang, Cai-Zhong

2014-01-01

Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO), and ABA (NCED) biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA) and abiotic stresses (dehydration, NaCl and cold). Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence.

A Petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence.

Directory of Open Access Journals (Sweden)

Xiaoxiao Chang

Full Text Available Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO, and ABA (NCED biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29 was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA and abiotic stresses (dehydration, NaCl and cold. Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence.

Molecular Recognition of PTS-1 Cargo Proteins by Pex5p: Implications for Protein Mistargeting in Primary Hyperoxaluria

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Noel Mesa-Torres

2015-02-01

Full Text Available Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1, where alanine:glyoxylate aminotransferase (AGT undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3–4 orders of magnitude reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

Optimization of the protein concentration process from residual peanut oil-cake

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Gayol, M. F.

2013-12-01

Full Text Available The objective of this study was to find the best process conditions for preparing protein concentrate from residual peanut oil-cake (POC. The study was carried out on POC from industrial peanut oil extraction. Different protein extraction and precipitation conditions were used: water/ flour ratio (10:1, 20:1 and 30:1, pH (8, 9 and 10, NaCl concentration (0 and 0.5 M, extraction time (30, 60 and 120 min, temperature (25, 40 and 60 °C, extraction stages (1, 2 and 3, and precipitation pH (4, 4.5 and 5. The extraction and precipitation conditions which showed the highest protein yield were 10:1 water/flour ratio, extraction at pH 9, no NaCl, 2 extraction stages of 30 min at 40 °C and precipitation at pH 4.5. Under these conditions, the peanut protein concentrate (PC contained 86.22% protein, while the initial POC had 38.04% . POC is an alternative source of protein that can be used for human consumption or animal nutrition. Therefore, it adds value to an industry residue.El objetivo de este trabajo fue encontrar las mejores condiciones para obtener un concentrado de proteínas a partir de la torta residual de maní (POC. El estudio se llevó a cabo en POC provenientes de la extracción industrial de aceite de maní. Se utilizaron distintas condiciones para la extracción y precipitación de proteínas: relación agua / harina (10:1, 20:1 y 30:1, pH de extracción (8, 9 y 10, concentración de NaCl (0 y 0,5 M, tiempo de extracción (30, 60 y 120 min, temperatura (25, 40 y 60 °C, número de etapas de extracción (1, 2 y 3, y el pH de precipitación (4, 4,5 y 5. Las condiciones de extracción y de precipitación que mostraron mayor rendimiento de proteína fueron: relación de 10:1 en agua / harina, pH de extracción de 9, en ausencia de NaCl, 2 etapas de extracción de 30 min cada una a 40 °C y el pH de precipitación de 4,5. En estas condiciones, el concentrado de proteína de maní (PC fue de 86,22%, mientras que el porcentaje de proteínas de

The acid test of fluoride: how pH modulates toxicity.

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Ramaswamy Sharma

2010-05-01

Full Text Available It is not known why the ameloblasts responsible for dental enamel formation are uniquely sensitive to fluoride (F(-. Herein, we present a novel theory with supporting data to show that the low pH environment of maturating stage ameloblasts enhances their sensitivity to a given dose of F(-. Enamel formation is initiated in a neutral pH environment (secretory stage; however, the pH can fall to below 6.0 as most of the mineral precipitates (maturation stage. Low pH can facilitate entry of F(- into cells. Here, we asked if F(- was more toxic at low pH, as measured by increased cell stress and decreased cell function.Treatment of ameloblast-derived LS8 cells with F(- at low pH reduced the threshold dose of F(- required to phosphorylate stress-related proteins, PERK, eIF2alpha, JNK and c-jun. To assess protein secretion, LS8 cells were stably transduced with a secreted reporter, Gaussia luciferase, and secretion was quantified as a function of F(- dose and pH. Luciferase secretion significantly decreased within 2 hr of F(- treatment at low pH versus neutral pH, indicating increased functional toxicity. Rats given 100 ppm F(- in their drinking water exhibited increased stress-mediated phosphorylation of eIF2alpha in maturation stage ameloblasts (pH<6.0 as compared to secretory stage ameloblasts (pH approximately 7.2. Intriguingly, F(--treated rats demonstrated a striking decrease in transcripts expressed during the maturation stage of enamel development (Klk4 and Amtn. In contrast, the expression of secretory stage genes, AmelX, Ambn, Enam and Mmp20, was unaffected.The low pH environment of maturation stage ameloblasts facilitates the uptake of F(-, causing increased cell stress that compromises ameloblast function, resulting in dental fluorosis.

Effect of pH on the Heat-Induced Denaturation and Renaturation of Green Fluorescent Protein: A Laboratory Experiment

Science.gov (United States)

Flores, Rosa V.; Sola, Hilda M.; Torres, Juan C.; Torres, Rafael E.; Guzman, Ernick E.

2013-01-01

A fluorescence spectroscopy experiment is described where students integrated biochemistry and instrumental analysis, while characterizing the green fluorescent protein excitation and emission spectra in terms of its phenolic and phenolate chromophores. Students studied the combined effect of pH and temperature on the protein's fluorescence,…

Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH

NARCIS (Netherlands)

Ruiz, Geraldine Avila; Opazo-Navarrete, Mauricio; Meurs, Marlon; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Stieger, Markus; Janssen, Anja E.M.

2016-01-01

The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60,

SALIVARY PH CHANGES AFTER GIC RESTORATION ON DECIDUOUS TEETH

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Chandra Nila Sukma

2015-06-01

Full Text Available Glass Ionomer Cement (GIC is the most widely used material in pediatric dentistry. The purpose of this study was to analyze pH changes of saliva after GIC restoration on primary teeth. For this purpose, 20 primary canines which were restored with GIC 24 hours previously were plunged into 20 tubes containing each 1,5 ml pH 6,8 Fusayama artificial saliva and then stored in incubator at the temperature of 37°C. The pH changes were measured at 30, 60, and 90 minutes later with digital pH meter PH-201. It was revealed that the highest pH acceleration was at 30 minutes exposure an decrease thereafter and the lowest pH acceleration was at 90 minutes exposure. Statistical analysis was performed by Anova and Tukey HSD.

DNase 1 Retains Endodeoxyribonuclease Activity Following Gold Nanocluster Synthesis

Science.gov (United States)

2014-07-04

mM) was added to 2 mL of protein solution under vigorous stirring at 37 °C (Table 1). After 5 min, 200 μL of NaOH (1 M) was added to raise the pH to...12 for the 1, 5, and 10 mM HAuCl4 samples whereas 400 μL of NaOH (1M) was required to obtain a pH of 12 for the 20 mM HAuCl4 sample due to the... magnesium sulfate pH 5.0), containing 2 μg of dsDNA. The reaction was incubated at room temperature for 20 min followed by the addition of 2 units of

Effect of pH and Recombinant Barley (Hordeum vulgare L.) Endoprotease B2 on Degradation of Proteins in Soaked Barley

DEFF Research Database (Denmark)

Christensen, Jesper Bjerg; Dionisio, Giuseppe; Poulsen, Hanne Damgaard

2014-01-01

.3. Solubilized and degraded proteins evaluated by biuret, SDS-PAGE, and differential proteomics revealed that pH 4.3 had the greatest impact on both solubilization and degradation. In order to boost proteolysis, the recombinant barley endoprotease B2 (rec-HvEP-B2) was included after 8 h using the pH 4.3 regime......Nonfermented soaking of barley feedstuff has been established as an in vitro procedure prior to the feeding of pigs as it can increase protein digestibility. In the current study, two feed cultivars of barley (Finlissa and Zephyr) were soaked in vitro either nonbuffered or buffered at pH 3.6 and 4....... Proteolysis evaluated by SDS-PAGE and differential proteomics confirmed a powerful effect of adding rec-HvEP-B2 to the soaked barley, regardless of the genotype. Our study addresses the use of rec-HvEP-B2 as an effective feed enzyme protease. HvEP-B2 has the potential to increase the digestibility of protein...

Hemoglobin bioconjugates with surface-protected gold nanoparticles in aqueous media: The stability depends on solution pH and protein properties.

Science.gov (United States)

Del Caño, Rafael; Mateus, Lucia; Sánchez-Obrero, Guadalupe; Sevilla, José Manuel; Madueño, Rafael; Blázquez, Manuel; Pineda, Teresa

2017-11-01

The identification of the factors that dictate the formation and physicochemical properties of protein-nanomaterial bioconjugates are important to understand their behavior in biological systems. The present work deals with the formation and characterization of bioconjugates made of the protein hemoglobin (Hb) and gold nanoparticles (AuNP) capped with three different molecular layers (citrate anions (c), 6-mercaptopurine (MP) and ω-mercaptoundecanoic acid (MUA)). The main focus is on the behavior of the bioconjugates in aqueous buffered solutions in a wide pH range. The stability of the bioconjugates have been studied by UV-visible spectroscopy by following the changes in the localized surface resonance plasmon band (LSRP), Dynamic light scattering (DLS) and zeta-potential pH titrations. It has been found that they are stable in neutral and alkaline solutions and, at pH lower than the protein isoelectric point, aggregation takes place. Although the surface chemical properties of the AuNPs confer different properties in respect to colloidal stability, once the bioconjugates are formed their properties are dictated by the Hb protein corona. The protein secondary structure, as analyzed by Attenuated total reflectance infrared (ATR-IR) spectroscopy, seems to be maintained under the conditions of colloidal stability but some small changes in protein conformation take place when the bioconjugates aggregate. These findings highlight the importance to keep the protein structure upon interaction with nanomaterials to drive the stability of the bioconjugates. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization and Preparation of Broken Rice Proteins Modified by Proteases

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Lixia Hou

2010-01-01

Full Text Available Broken rice is an underutilized by-product of milling. Proteins prepared from broken rice by treatments with alkaline protease and papain have been characterized with regard to nutritional and functional properties. The protein content and the protein recovery were 56.45 and 75.45 % for alkaline protease treatment, and 65.45 and 46.32 % for papain treatment, respectively. Protease treatment increased the lysine and valine content, leading to a more balanced amino acid profile. Broken rice proteins had high emulsifying capacity, 58.3–71.6 % at neutral pH, and adequate water holding capacity, ranging from 1.96 to 2.93 g/g of proteins. At pH=7.0, the broken rice protein had the highest water holding capacity and the best interfacial activities (emulsifying capacity, emulsifying stability, foaming capacity and foaming stability, which may be the result of the higher solubility at pH=7.0. The interfacial activities increased with the increase in the mass fraction of broken rice proteins. The proteins prepared by the papain treatment had higher water holding capacity (p>0.05, emulsifying capacity (p0.05 than alkaline protease treatment at the same pH or mass fraction. To test the fortification of food products with broken rice proteins, pork sausages containing the proteins were prepared. Higher yield of the sausages was obtained with the increased content of broken rice proteins, in the range of 2.0–9.0 %. The results indicate that broken rice proteins have potential to be used as the protein fortification ingredient for food products.

Macronutrients and micronutrients of soybean oil bodies extracted at different pH.

Science.gov (United States)

Chen, Yeming; Cao, Yanyun; Zhao, Luping; Kong, Xiangzhen; Hua, Yufei

2014-07-01

In this study, the macronutrients and micronutrients of pH 6.8, 8.0, 9.5, and 11.0 extracted soybean oil bodies (OBs) were examined, revealing that soybean OBs might be used as a natural carrier for bioactive components (unsaturated fatty acids, phospholipid, tocopherol, and phytosterol). pH 6.8 extracted OBs (dry basis) contained 85.88% neutral lipid, 8.18% protein, and 5.85% polar lipid (mainly phospholipid) by gravimetric analysis. The percentage of neutral lipid was increased, while those of protein and polar lipid were decreased with increasing pH. Tocopherol (about 75 mg/100 g neutral lipid of OBs) was not affected, while phytosterol was decreased (136 to 110 mg/100 g neutral lipid of OBs) with increasing pH. The detectable total monosaccaride (galactosamine, glucosamine, and glucose) content of extracted OBs was low and also decreased (35.80 to 6.13 mg/100 g neutral lipid of OBs) with increasing pH. The protein of extracted OBs had higher percentage of essential amino acids than soybean protein isolate with tryptophan and methionine as limited amino acids. The fatty acid composition of extracted OBs was rich in linoleic acid (about 59%), oleic acid (about 20%), and α-linolenic acid (about 7%). Oil bodies (OBs) from soybean and other plant seeds are greatly examined owing to their potential utilizations in food ingredients. The determination of its macronutrients and micronutrients would be very meaningful for its efficient utilization in the future. © 2014 Institute of Food Technologists®

Polymeric pH nanosensor with extended measurement range bearing octaarginine as cell penetrating peptide

DEFF Research Database (Denmark)

Ke, Peng; Sun, Honghao; Liu, Mingxing

2016-01-01

A synthetic peptide octaarginine which mimics human immunodeficiency virus-1, Tat protein is used as cell penetrating moiety for new pH nanosensors which demonstrate enhanced cellular uptake and expanded measurement range from pH 3.9 to pH 7.3 by simultaneously incorporating two complemental pH-s......H-sensitive fluorophores in a same nanoparticle. The authors believe that this triple fluorescent pH sensor provides a new tool to pH measurements that can have application in cellular uptake mechanism study and new nanomedicine design.......A synthetic peptide octaarginine which mimics human immunodeficiency virus-1, Tat protein is used as cell penetrating moiety for new pH nanosensors which demonstrate enhanced cellular uptake and expanded measurement range from pH 3.9 to pH 7.3 by simultaneously incorporating two complemental p...

New insights into flavivirus biology: the influence of pH over interactions between prM and E proteins

Science.gov (United States)

Oliveira, Edson R. A.; de Alencastro, Ricardo B.; Horta, Bruno A. C.

2017-11-01

Diseases caused by flaviviruses, such as dengue and zika, are globally recognized as major threats. During infection, a critical point in their replicative cycle is the maturation step, which occurs throughout the cellular exocytic pathway. This step is a pH-dependent process that involves the modification of the viral envelope by converting prM (pre-membrane) into M (membrane) proteins with the release of a "pr peptide". After this reaction, the pr peptides remain bound to the viral envelope while the virions cross the acidic trans-Golgi network, and are released only at neutral pH after secretion of the virus particles. Despite this current knowledge, the molecular basis of the flavivirus maturation step is largely unknown. Here, based on the crystal structure of the dengue pr-E complex ("pr peptide" bound to virus envelope protein) and using molecular dynamics simulations, we found that the pH shift from acidic to neutral yields considerable structural changes in the system. Dynamic cross correlation maps and root mean square deviation analyses revealed that the pr-E junction is clearly unstable under neutral pH. Secondary structure analysis also revealed that the fusion loop region, present in the E protein, is sensitive to pH and tends to unstructure at a neutral environment. Moreover, we found that five residues present in the E protein, Gly102, His244, Thr70, Thr68 and Asn67 are critical to confer stability to the pr-E complex while inside the Golgi apparatus. This work brings details about the dynamical behavior of the pr-E system, helps to better understand the flavivirus biology and may also be of use in the development of novel antiviral strategies.

In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system.

Science.gov (United States)

Martinière, Alexandre; Bassil, Elias; Jublanc, Elodie; Alcon, Carine; Reguera, Maria; Sentenac, Hervé; Blumwald, Eduardo; Paris, Nadine

2013-10-01

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.5) and Arabidopsis thaliana root cells (ΔpH -2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H(+) ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

pH stability and comparative evaluation of ranaspumin-2 foam for application in biochemical reactors

International Nuclear Information System (INIS)

Choi, Hyo-Jick; Ebersbacher, Charles F; Quan, Fu-Shi; Montemagno, Carlo D

2013-01-01

Aqueous channels of foam represent a simplified, natural bioreactor on the micro-/nano-scale. Previous studies have demonstrated the feasibility and potential application of foams in replicating cellular process in vitro, but no research has been performed to establish a basis for designing stable and biocompatible foam formulations. Our research has been directed specifically to the evaluation of ranaspumin-2 (RSN-2), a frog foam nest protein. The strong surfactant activity of RSN-2 enabled us to produce foams using low protein concentration (1 mg ml −1 ) over a wide pH range (pH ≥ 3). Importantly, the RSN-2 formulation exhibited the best foam stability at a near neutral pH condition, which shows a potential for application to various biosynthesis applications. Model cellular systems such as liposomes and inactivated A/PR/8/34 influenza virus maintained their physicochemical stability and full hemagglutination activity, indicating biocompatibility of RSN-2 with both cellular membranes and proteins both in bulk solution and in foam. Moreover, the addition of RSN-2 did not exert any deteriorative effects on bacterial cell growth kinetics. In contrast, Tween 20, Triton X-100, and BSA did not show satisfactory performance in terms of foamability, foam stability, physicochemcial stability, and biochemical stability. Although our study has been limited to representative formulations composed of only surfactant molecules, a number of unique advantages make RSN-2 a promising candidate for in vitro foam biosynthesis. (paper)

[Fe(F20 TPP)Cl]-catalyzed amination with arylamines and {[Fe(F20 TPP)(NAr)](PhI=NAr)} + . Intermediate assessed by high-resolution ESI-MS and DFT calculations.

Science.gov (United States)

Liu, Yungen; Chen, Guo-Qiang; Tse, Chun-Wai; Guan, Xianguo; Xu, Zheng-Jiang; Huang, Jie-Sheng; Che, Chi-Ming

2015-01-01

Amination of CH bonds catalyzed by transition metal complexes via nitrene/imide insertion is an appealing strategy for CN bond formation, and the use of iminoiodinanes, or their in situ generated forms from 'PhI(OAc)2 +primary amides (such as sulfonamides, sulfamates, and carbamates)', as nitrogen sources for the amination reaction has been well documented. In this work, a 'metal catalyst+PhI(OAc)2 +primary arylamines' amination protocol has been developed using [Fe(F20 TPP)Cl] (H2 F20 TPP=meso-tetrakis(pentafluorophenyl)porphyrin) as a catalyst. This catalytic method is applicable for both intra- and intermolecular amination of sp(2) and sp(3) CH bonds (>27 examples), affording the amination products, including natural products such as rutaecarpine, in moderate-to-good yields. ESI-MS analysis and DFT calculations lend support for the involvement of {[Fe(F20 TPP)(NC6 H4 -p-NO2 )](PhI=NC6 H4 -p-NO2 )} + . intermediate in the catalysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Low pH, Aluminum, and Phosphorus Coordinately Regulate Malate Exudation through GmALMT1 to Improve Soybean Adaptation to Acid Soils1[W][OA

Science.gov (United States)

Liang, Cuiyue; Piñeros, Miguel A.; Tian, Jiang; Yao, Zhufang; Sun, Lili; Liu, Jiping; Shaff, Jon; Coluccio, Alison; Kochian, Leon V.; Liao, Hong

2013-01-01

Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function. PMID:23341359

Two dimensional gel electrophoresis using narrow pH 3-5.6 immobilised pH gradient strips identifies potential novel disease biomarkers in plasma or serum

OpenAIRE

sprotocols

2015-01-01

Authors: Bevin Gangadharan & Nicole Zitzmann ### Abstract Two-dimensional gel electrophoresis (2-DE) is a protein separation technique often used to separate plasma or serum proteins in an attempt to identify novel biomarkers. This protocol describes how to run 2-DE gels using narrow pH 3-5.6 immobilised pH gradient strips to separate 2 mg of serum proteins. pH 3-6 ampholytes are used to enhance the solubility of proteins in this pH range before the serum proteins are separated in the...

Activity of encapsulated Lactobacillus bulgaricus in alginate-whey protein microspheres

Directory of Open Access Journals (Sweden)

Meng-Yan Chen

2014-10-01

Full Text Available In this work, alginate-whey protein was used as wall materials for encapsulating Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus. The characteristics of encapsulated and free L. bulgaricus showed that the free L. bulgaricus lost viability after 1 min exposure to simulated gastric fluid (SGF at pH 2.0 and 2.5. However, the viability of encapsulated L. bulgaricus did not decrease in SGF at pH 2.5 for 2 h incubation. The viable numbers of encapsulated L. bulgaricus decreased less than 1.0 log unit for 2 h incubation in SGF at pH 2.0. For bile stability, only 1.2 log units and 2.0 log units viability of the encapsulated L. bulgaricus was lost in 1 and 2% bile for 1 h exposure, respectively, compared with no survival of free L. bulgaricus under the same conditions. Encapsulated L. bulgaricus was completely released from the microspheres in simulated intestinal fluid (SIF, pH 6.8 in 3 h. The viability of the encapsulated L. bulgaricus retained more 8.0 log CFU/g after stored at 4°C for four weeks. However, for free L. bulgaricus, only around 3.0 log CFU/mL was found at the same storage conditions. Results showed that the encapsulation could improve the stability of L. bulgaricus.

Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th‐century pandemics

Science.gov (United States)

Pasricha, Gunisha; Mishra, Akhilesh C.; Chakrabarti, Alok K.

2012-01-01

Please cite this paper as: Pasricha et al. (2012) Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the Influenza A virus subtypes responsible for the 20th‐century pandemics. Influenza and Other Respiratory Viruses 7(4), 497–505. Background  PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. Methods  Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. Results  Analysis showed that 96·4% of the H5N1 influenza viruses harbored full‐length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th‐century pandemic influenza viruses contained full‐length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human‐ and avian host‐specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. Conclusions  Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host‐specific evolution of the virus

Protein Profiling Reveals Novel Proteins in Pollen and Pistil of W22 (ga1; Ga1 in Maize

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Jin Yu

2014-05-01

Full Text Available Gametophytic factors mediate pollen-pistil interactions in maize (Zea mays L. and play active roles in limiting gene flow among maize populations and between maize and teosinte. This study was carried out to identify proteins and investigate the mechanism of gametophytic factors using protein analysis. W22 (ga1; which did not carry a gametophytic factor and W22 (Ga1, a near iso-genic line, were used for the proteome investigation. SDS-PAGE was executed to investigate proteins in the pollen and pistil of W22 (ga1 and W22 (Ga1. A total of 44 differentially expressed proteins were identified in the pollen and pistil on SDS-PAGE using LTQ-FTICR MS. Among the 44 proteins, a total of 24 proteins were identified in the pollen of W22 (ga1 and W22 (Ga1 whereas 20 differentially expressed proteins were identified from the pistil of W22 (ga1 and W22 (Ga1. However, in pollen, 2 proteins were identified only in the W22 (ga1 and 12 proteins only in the W22 (Ga1 whereas 10 proteins were confirmed from the both of W22 (ga1 and W22 (Ga1. In contrary, 10 proteins were appeared only in the pistil of W22 (ga1 and 7 proteins from W22 (Ga1 while 3 proteins confirmed in the both of W22 (ga1 and W22 (Ga1. Moreover, the identified proteins were generally involved in hydrolase activity, nucleic acid binding and nucleotide binding. These results help to reveal the mechanism of gametophytic factors and provide a valuable clue for the pollen and pistil research in maize.

Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

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Guy Caljon

Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

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Ellen C Kammula

Full Text Available HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

Correlation of transmissive fractures in holes OL-PH1, ONK-PH2 .. ONK-PH7 and ONKALO tunnel fractures

International Nuclear Information System (INIS)

Palmen, J; Nummela, J.; Ahokas, H.

2011-02-01

In a preceding study Posiva flow logging (PFL) with a 0.5 m test interval and 10 cm steps has been used together with optical drillhole images and core logging fracture data for the exact determination of the depth of hydraulically conductive fractures in pilot holes. The fracture traces has been mapped from the ONKALO tunnel walls as a part of the systematic mapping. The mapping results has been digitized to a 3D tunnel layout in Surpac Vision programme. The data integrity and fracture trace uniqueness has been verified by Datactica Oy and further collected to a database (Rakokanta D atactica P osiva20091119.mdb). Water leakage of the mapped fractures exists as an attribute field for each fracture, but the value of the attribute has not been assessed conclusively. Those fractures mapped with leakage attribute have been defined as flowing, dripping, wet, or damp where the attribute is recorded. The fractures with no leakage attribute value appear to be dry (not leaking) or the information is not available (assessment was not performed). The water leaking surfaces on ONKALO tunnel wall have been mapped sequentially and conclusively (twice a year) as a part of the Olkiluoto monitoring program (OMO) using an equal five step measure as used with fracture traces in systematic mapping. The PFL results correlated with core logging fracture data from pilot holes OL-PH1 and ONK-PH2 .. ONK-PH7 were in this work further correlated with the fractures mapped from the ONKALO tunnel walls. Each hydraulically conductive fracture of OL-PH1 and ONK-PH2 - ONK-PH7 was investigated and linked to ONKALO fracture of a coherent orientation and matching location, where such fracture trace was available. The main objective of the work was to identify the ONKALO fractures which correspond to the flow from fracture(s) identified with the PFL method in pilot holes and to collect basic information about the occurrence, frequency and orientation of water bearing fractures along ONKALO tunnel

Thermal, chemical and pH induced unfolding of turmeric root lectin: modes of denaturation.

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Himadri Biswas

Full Text Available Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol-1 and 14.90 Kcal mol-1 for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔCp is 3.42 Kcal mol-1 K-1 for dimer. The small ΔCp for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.

Signaling alkaline pH stress in the yeast Saccharomyces cerevisiae through the Wsc1 cell surface sensor and the Slt2 MAPK pathway.

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Serrano, Raquel; Martín, Humberto; Casamayor, Antonio; Ariño, Joaquín

2006-12-29

Alkalinization of the external environment represents a stress situation for Saccharomyces cerevisiae. Adaptation to this circumstance involves the activation of diverse response mechanisms, the components of which are still largely unknown. We show here that mutation of members of the cell integrity Pkc1/Slt2 MAPK module, as well as upstream and downstream elements of the system, confers sensitivity to alkali. Alkalinization resulted in fast and transient activation of the Slt2 MAPK, which depended on the integrity of the kinase module and was largely abolished by sorbitol. Lack of Wsc1, removal of specific extracellular and intracellular domains, or substitution of Tyr(303) in this putative membrane stress sensor rendered cells sensitive to alkali and considerably decreased alkali-induced Slt2 activation. In contrast, constitutive activation of Slt2 by the bck1-20 allele increased pH tolerance in the wsc1 mutant. DNA microarray analysis revealed that several genes encoding cell wall proteins, such as GSC2/FKS2, DFG5, SKT5, and CRH1, were induced, at least in part, by high pH in an Slt2-dependent manner. We observed that dfg5, skt5, and particularly dfg5 skt5 cells were alkali-sensitive. Therefore, our results show that an alkaline environment imposes a stress condition on the yeast cell wall. We propose that the Slt2-mediated MAPK pathway plays an important role in the adaptive response to this insult and that Wsc1 participates as an essential cell-surface pH sensor. Moreover, these results provide a new example of the complexity of the response of budding yeast to the alkalinization of the environment.

Studies on the functional properties of protein concentrate of Kappaphycus alvarezii (Doty) Doty - an edible seaweed.

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Suresh Kumar, K; Ganesan, K; Selvaraj, Kandasamy; Subba Rao, P V

2014-06-15

Protein concentrate (PC) of Kappaphycus alvarezii (cultivated on the West coast of India), was extracted and its functional properties were evaluated. The K. alvarezii PC contained 62.3 ± 1.62% proteins. At pH 12, the nitrogen solubility of this PC was 58.72 ± 1.68% in the presence of 0.5M NaCl. The emulsifying and foaming properties of this PC varied with time and pH. However, it formed remarkably stable emulsions with Jatropha oil after 720 min (i.e. E720=53.67 ± 1.59). On the other hand, maximum foaming ability (53.33 ± 2.31%) of the PC was recorded at pH 4.0. This PC had high oil (1.29 ± 0.20 ml oil/g PC) and water absorption capacity (2.22 0.04 ml H2O/g PC). DSC analysis revealed thermal transitions at about 109.25°C at neutral pH. The results obtained in this investigation suggest the suitability of K. alvarezii PC as an inexpensive source of protein; thus this PC could be incorporated into several value-added food products. Copyright © 2013 Elsevier Ltd. All rights reserved.

Diagnostic significance of pleural fluid pH and pCO2

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K.E. Sobhey

2015-10-01

Results: We conducted this study on 50 patients with pleural effusions of different causes. The patients were classified into 5 groups according to the cause. For all the patients, measurement of pleural pH, pCO2, pO2, HCO3, protein, LDH, glucose and WBC was done. We observed lowest pH in complicated parapneumonic effusion (empyema 6.80 ± 0.15 and highest pH was observed in transudative effusion 7.47 ± 0.07. Tuberculous effusion has pH lower than pH of malignant effusion 7.17 ± 0.017 and 7.39 ± 0.08, respectively. Post pleurodesis malignant effusion has pH lower than pH of malignant effusion 7.28 ± 0.17 and 7.39 ± 0.08, respectively. There is a strong inverse correlation between pH and pCO2, WBC, LDH and protein (r = −0.813 and p < 0.001, (r = −0.796 and p, 0.001, (r = −0.829 and p, 0.001 and (r = −.837 and p, 0.001, respectively. While there is a weak correlation between pH and glucose of pleural fluid (r = 0.249 and p = 0.066. The highest increase of PNL numbers was in empyema (20169 ± 8094.8 cells/cc.The highest increase of lymphocytes was in malignant effusions (4285.00 ± 2948.20 cells/cc and tuberculous effusion (3977.7 ± 3169 cells/cc.

Amyotrophic Lateral Sclerosis Type 20 - In Silico Analysis and Molecular Dynamics Simulation of hnRNPA1.

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Bruna Baumgarten Krebs

Full Text Available Amyotrophic Lateral Sclerosis (ALS is a fatal neurodegenerative disease that affects the upper and lower motor neurons. 5-10% of cases are genetically inherited, including ALS type 20, which is caused by mutations in the hnRNPA1 gene. The goals of this work are to analyze the effects of non-synonymous single nucleotide polymorphisms (nsSNPs on hnRNPA1 protein function, to model the complete tridimensional structure of the protein using computational methods and to assess structural and functional differences between the wild type and its variants through Molecular Dynamics simulations. nsSNP, PhD-SNP, Polyphen2, SIFT, SNAP, SNPs&GO, SNPeffect and PROVEAN were used to predict the functional effects of nsSNPs. Ab initio modeling of hnRNPA1 was made using Rosetta and refined using KoBaMIN. The structure was validated by PROCHECK, Rampage, ERRAT, Verify3D, ProSA and Qmean. TM-align was used for the structural alignment. FoldIndex, DICHOT, ELM, D2P2, Disopred and DisEMBL were used to predict disordered regions within the protein. Amino acid conservation analysis was assessed by Consurf, and the molecular dynamics simulations were performed using GROMACS. Mutations D314V and D314N were predicted to increase amyloid propensity, and predicted as deleterious by at least three algorithms, while mutation N73S was predicted as neutral by all the algorithms. D314N and D314V occur in a highly conserved amino acid. The Molecular Dynamics results indicate that all mutations increase protein stability when compared to the wild type. Mutants D314N and N319S showed higher overall dimensions and accessible surface when compared to the wild type. The flexibility level of the C-terminal residues of hnRNPA1 is affected by all mutations, which may affect protein function, especially regarding the protein ability to interact with other proteins.

Synthesis and characterization of pH sensitive carboxySNARF-1 nanoreactors

International Nuclear Information System (INIS)

Chen Yenchi; Ostafin, Agnes; Mizukami, Hiroshi

2010-01-01

A rapid response dual wavelength emission pH sensor consisting of carboxySNARF-1 nanoreactors has been synthesized and shown to provide accurate pH measurements even in complex biological media, where the unprotected pH responsive dyes have failed. The carboxySNARF-1 nanoreactor is made of a calcium phosphate shell covering phosphatidylcholine liposomes filled with the dye. Its mean diameter is 150 nm with dynamic light scattering, the shell thickness is 5-7 nm with TEM, and it contains about 10 dyes/particle. The nanoreactor's response time to pH change nearly equals that of the dye in solution. Its pH titration curves at two different wavelengths are equivalent to those of the dye in solution and fluorescence intensity ratio dependent pH analysis is possible using the modified Henderson-Hasselbalch equation. However, the pH dependent fluorescence ratios of the dye in solution in the presence of plasma and albumin are distorted, and application of the Henderson-Hasselbalch equation is not possible. We have found that the distortions may be restored using cSNARF-1 nanoreactors and the pK a of the dye in the nanoreactor then equals that in solution. These results suggest that the interference to the dye for the pH analyses with the environmental molecules may be reduced or prohibited by usage of cSNARF-1 nanoreactors.

Synthesis and characterization of pH sensitive carboxySNARF-1 nanoreactors

Energy Technology Data Exchange (ETDEWEB)

Chen Yenchi; Ostafin, Agnes [Department of Materials Science, University of Utah, Salt Lake City, UT (United States); Mizukami, Hiroshi, E-mail: a.ostafin@utah.edu [Department of Biological Science, Wayne State University, Detroit, MI (United States)

2010-05-28

A rapid response dual wavelength emission pH sensor consisting of carboxySNARF-1 nanoreactors has been synthesized and shown to provide accurate pH measurements even in complex biological media, where the unprotected pH responsive dyes have failed. The carboxySNARF-1 nanoreactor is made of a calcium phosphate shell covering phosphatidylcholine liposomes filled with the dye. Its mean diameter is 150 nm with dynamic light scattering, the shell thickness is 5-7 nm with TEM, and it contains about 10 dyes/particle. The nanoreactor's response time to pH change nearly equals that of the dye in solution. Its pH titration curves at two different wavelengths are equivalent to those of the dye in solution and fluorescence intensity ratio dependent pH analysis is possible using the modified Henderson-Hasselbalch equation. However, the pH dependent fluorescence ratios of the dye in solution in the presence of plasma and albumin are distorted, and application of the Henderson-Hasselbalch equation is not possible. We have found that the distortions may be restored using cSNARF-1 nanoreactors and the pK{sub a} of the dye in the nanoreactor then equals that in solution. These results suggest that the interference to the dye for the pH analyses with the environmental molecules may be reduced or prohibited by usage of cSNARF-1 nanoreactors.

Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor.

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Cook, Jonathan D; Soto-Montoya, Hazel; Korpela, Markus K; Lee, Jeffrey E

2015-07-24

Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

B cell markers in Ph1-positive acute lymphoblastic leukemia.

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Alimena, G; De Rossi, G; Gastaldi, R; Guglielmi, C; Mandelli, F

1980-01-01

A case of acute lymphoblastic leukemia (ALL) where the blast cells had B cell markers and displayed the presence of a typical Ph1 chromosome, originated by a standard t (9;22) translocation, is reported. Cytological and clinical aspects during the entire course of the disease were consistent with the diagnosis of ALL. Evidence of differentiation along a well-defined lymphoid cell line in a Ph1-positive cell confirms the presence of the Ph1 chromosome in conditions other than chronic granulocytic leukemia and shows that it possibly does not occur in an exclusively undifferentiated totipotent stem cell.

Gold Nanocluster-DNase 1 Hybrid Materials for DNA Contamination Sensing

Science.gov (United States)

2014-01-01

or 1 mM) was added to 2 mL of protein solution under vigorous stirring at 37 ̊ C. After 5 minutes 200 µL of NaOH (1 M) was added to raise the pH to...12 for the 1, 5, and 10 mM HAuCl4 samples whereas 400 µL of NaOH (1M) was required. The various protein/gold mixtures were then left to react for...1: AuNCs were carried out in a final volume of 20 µL buffer (100 mM sodium acetate, 6.25 mM magnesium sulfate pH 5.0), containing 2 µg of dsDNA

Temperature and pH influence adsorption of cellobiohydrolase onto lignin by changing the protein properties.

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Lu, Xianqin; Wang, Can; Li, Xuezhi; Zhao, Jian

2017-12-01

Non-productive adsorption of cellulase onto lignin restricted the movement of cellulase and also hindered the cellulase recycling in bioconversion of lignocellulose. In this study, effect of temperature and pH on adsorption and desorption of cellobiohydrolase (CBH) on lignin and its possible mechanism were discussed. It found that pH value and temperature influenced the adsorption and desorption behaviors of CBH on lignin. Different thermodynamic models suggested that the action between lignin and CBH was physical action. More CBH was adsorbed onto lignin, but lower initial adsorption velocity was detected at 50°C comparing with 4°C. Elevating pH value could improve desorption of cellulase from lignin. The changes of hydrophobicity and electric potential on protein surface may partially explain the impact of environmental conditions on the adsorption and desorption behaviors of CBH on lignin, and comparing to electrical interaction, the hydrophobicity may be the dominating factor influencing the behaviors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Involvement of the ornithine decarboxylase gene in acid stress response in probiotic Lactobacillus delbrueckii UFV H2b20.

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Ferreira, A B; Oliveira, M N V de; Freitas, F S; Paiva, A D; Alfenas-Zerbini, P; Silva, D F da; Queiroz, M V de; Borges, A C; Moraes, C A de

2015-01-01

Amino acid decarboxylation is important for the maintenance of intracellular pH under acid stress. This study aims to carry out phylogenetic and expression analysis by real-time PCR of two genes that encode proteins involved in ornithine decarboxylation in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress. Sequencing and phylogeny analysis of genes encoding ornithine decarboxylase and amino acid permease in L. delbrueckii UFV H2b20 showed their high sequence identity (99%) and grouping with those of L. delbrueckii subsp. bulgaricus ATCC 11842. Exposure of L. delbrueckii UFV H2b20 cells in MRS pH 3.5 for 30 and 60 min caused a significant increase in expression of the gene encoding ornithine decarboxylase (up to 8.1 times higher when compared to the control treatment). Increased expression of the ornithine decarboxylase gene demonstrates its involvement in acid stress response in L. delbrueckii UFV H2b20, evidencing that the protein encoded by that gene could be involved in intracellular pH regulation. The results obtained show ornithine decarboxylation as a possible mechanism of adaptation to an acidic environmental condition, a desirable and necessary characteristic for probiotic cultures and certainly important to the survival and persistence of the L. delbrueckii UFV H2b20 in the human gastrointestinal tract.

Cloning, Overexpression, and Characterization of a Metagenome-Derived Phytase with Optimal Activity at Low pH.

Science.gov (United States)

Tan, Hao; Wu, Xiang; Xie, Liyuan; Huang, Zhongqian; Gan, Bingcheng; Peng, Weihong

2015-06-01

A phytase gene was identified in a publicly available metagenome derived from subsurface groundwater, which was deduced to encode for a protein of the histidine acid phosphatase (HAP) family. The nucleotide sequence of the phytase gene was chemically synthesized and cloned, in order to further overexpress the phytase in Escherichia coli. Purified protein of the recombinant phytase demonstrated an activity for phytic acid of 298 ± 17 μmol P/min/mg, at the pH optimum of 2.0 with the temperature of 37 °C. Interestingly, the pH optimum of this phytase is much lower in comparison with most HAP phytases known to date. It suggests that the phytase could possess improved adaptability to the low pH condition caused by the gastric acid in livestock and poultry stomachs.

A SERS-based pH sensor utilizing 3-amino-5-mercapto-1,2,4-triazole functionalized Ag nanoparticles.

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Piotrowski, Piotr; Wrzosek, Beata; Królikowska, Agata; Bukowska, Jolanta

2014-03-07

We report the first use of 3-amino-5-mercapto-1,2,4-triazole (AMT) to construct a surface-enhanced Raman scattering (SERS) based pH nano- and microsensor, utilizing silver nanoparticles. We optimize the procedure of homogenous attachment of colloidal silver to micrometer-sized silica beads via an aminosilane linker. Such micro-carriers are potential optically trappable SERS microprobes. It is demonstrated that the SERS spectrum of AMT is strongly dependent on the pH of the surroundings, as the transformation between two different adsorption modes, upright (A form) and lying flat (B form) orientation, is provoked by pH variation. The possibility of tuning the nanosensor working range by changing the concentration of AMT in the surrounding solution is demonstrated. A strong correlation between the pH response of the nanosensor and the AMT concentration in solution is found to be controlled by the interactions between the surface and solution molecules. In the absence of the AMT monomer, the performance of both the nano- and microsensor is shifted substantially to the strongly acidic pH range, from 1.5 to 2.5 and from 1.0 to 2.0, respectively, which is quite unique even for SERS-based sensors.

[24-h intraesophageal pH determination in children allergic to cow's milk protein at a tertiary care hospital].

Science.gov (United States)

Ramírez-Mayans, J A; Toro-Monjaraz, E M; Romero-Trujillo, J; Cervantes-Bustamante, R; Zárate-Mondragón, F; Montijo-Barrios, E; Cadena-León, J; Cazares-Méndez, M

2014-01-01

Cow's milk protein allergy (CMPA) is being seen more frequently on a daily basis in pediatric consultations. It shares symptoms with gastroesophageal reflux (GER), which can complicate the differential diagnosis. To attempt to corroborate the presence of acid GER in children with CMPA, as well as to find a characteristic profile through the 24-hour pH monitoring study in children with GER and CMPA METHODS: The intraesophageal pH monitoring studies performed on 47 children with CMPA were reviewed. The measurements in all the studies were carried out within a 24-hour period using Digitrapper® equipment with a multi-use GeroFlex® catheter, after calibration with pH 7 and pH 1 buffer solutions. Of the 47 children, 23 were boys (32.4%) and 24 were girls (33.8%) and the mean age was 5±3.7 years. Fourteen of the 47 children (29%) presented with GER, according to the result of the 24-hour intraesophageal measurement. Only 2 of the 47 patients studied fit the phasic profile. The findings show the existing relation between the two pathologies. Nevertheless, it is important to determine the presence of non-acid or weak acid reflux, because their existence can increase this association. Copyright © 2013 Asociación Mexicana de Gastroenterología. Published by Masson Doyma México S.A. All rights reserved.

Unfolding studies of the cysteine protease baupain, a papain-like enzyme from leaves of Bauhinia forficata: effect of pH, guanidine hydrochloride and temperature.

Science.gov (United States)

Silva-Lucca, Rosemeire A; Andrade, Sheila S; Ferreira, Rodrigo Silva; Sampaio, Misako U; Oliva, Maria Luiza V

2013-12-24

Baupain belongs to the α+β class of proteins with a secondary structure-content of 44% α-helix, 16% β-sheet and 12% β-turn. The structural transition induced by pH was found to be noncooperative, with no important differences observed in the pH range from 3.0 to 10.5. At pH 2.0 the protein presented substantial non-native structure with strong ANS binding. Guanidine hydrochloride (GdnHCl)-induced unfolding did not change the protein structure significantly until 4.0 M, indicating the high rigidity of the molecule. The unfolding was cooperative, as seen by the sigmoidal transition curves with midpoints at 4.7±0.2 M and 5.0±0.2 M GdnHCl, as measured by CD and fluorescence spectroscopy. A red shift of 7 nm in intrinsic fluorescence was observed with 6.0 M GdnHCl. Temperature-induced unfolding of baupain was incomplete, and at least 35% of the native structure of the protein was retained, even at high temperature (90 °C). Baupain showed characteristics of a molten globule state, due to preferential ANS binding at pH 2.0 in comparison to the native form (pH 7.0) and completely unfolded (6.0 M GdnHCl) state. Combined with information about N-terminal sequence similarity, these results allow us to include baupain in the papain superfamily.

Negative Effect of Ellagic Acid on Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Cancer Cells.

Science.gov (United States)

Abdelazeem, Khalid N M; Singh, Yogesh; Lang, Florian; Salker, Madhuri S

2017-01-01

Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions, which is in part accomplished by Na+/H+ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of Ellagic acid on transport processes has, however, never been reported. The present study thus elucidated an effect of Ellagic acid on cytosolic pH (pHi), NHE1 transcript levels, NHE1 protein abundance, Na+/H+ exchanger activity, and lactate release. Experiments were performed in Ishikawa cells without or with prior Ellagic acid (20 µM) treatment. NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance by Western blotting, pHi utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, cell volume from forward scatter in flow cytometry, reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein fluorescence, glucose uptake utilizing 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose, and lactate concentration in the supernatant utilizing a colorimetric (570 nm)/ fluorometric enzymatic assay. A 48 hour treatment with Ellagic acid (20 µM) significantly decreased NHE1 transcript levels by 75%, NHE1 protein abundance by 95%, pHi from 7.24 ± 0.01 to 7.02 ± 0.01, Na+/H+ exchanger activity by 77%, forward scatter by 10%, ROS by 82%, glucose uptake by 58%, and lactate release by 15%. Ellagic acid (20µM) markedly down-regulates ROS formation and NHE1 expression leading to decreased Na+/H+ exchanger activity, pHi, glucose uptake and lactate release in endometrial cancer cells. Those effects presumably contribute to reprogramming and growth

Negative Effect of Ellagic Acid on Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Cancer Cells

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Khalid N. M. Abdelazeem

2017-04-01

Full Text Available Background/Aims: Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions, which is in part accomplished by Na+/H+ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of Ellagic acid on transport processes has, however, never been reported. The present study thus elucidated an effect of Ellagic acid on cytosolic pH (pHi, NHE1 transcript levels, NHE1 protein abundance, Na+/H+ exchanger activity, and lactate release. Methods: Experiments were performed in Ishikawa cells without or with prior Ellagic acid (20 µM treatment. NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance by Western blotting, pHi utilizing (2',7'-bis-(2-carboxyethyl-5-(and-6-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, cell volume from forward scatter in flow cytometry, reactive oxygen species (ROS from 2’,7’-dichlorodihydrofluorescein fluorescence, glucose uptake utilizing 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-ylamino-2-deoxyglucose, and lactate concentration in the supernatant utilizing a colorimetric (570 nm/ fluorometric enzymatic assay. Results: A 48 hour treatment with Ellagic acid (20 µM significantly decreased NHE1 transcript levels by 75%, NHE1 protein abundance by 95%, pHi from 7.24 ± 0.01 to 7.02 ± 0.01, Na+/H+ exchanger activity by 77%, forward scatter by 10%, ROS by 82%, glucose uptake by 58%, and lactate release by 15%. Conclusion: Ellagic acid (20µM markedly down-regulates ROS formation and NHE1 expression leading to decreased Na+/H+ exchanger activity, pHi, glucose uptake and lactate release in endometrial cancer cells. Those

FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

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Cobos Everardo

2005-01-01

Full Text Available Abstract Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20 with three members (FAM20A, FAM20B and FAM20C in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating

Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

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Sørensen, Brita Singers; Busk, Morten; Overgaard, Jens; Horsman, Michael R; Alsner, Jan

2015-01-01

The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis. Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein. Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined. We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo

OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice.

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Liu, Yaju; Xu, Yunyuan; Xiao, Jun; Ma, Qibin; Li, Dan; Xue, Zhen; Chong, Kang

2011-07-01

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA(3)) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA(1) and real-time PCR quantitative assay analyses confirmed that the decrease of GA(1) in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice. Copyright © 2011 Elsevier GmbH. All rights reserved.

Analysis and implementation of cross lingual short message service spam filtering using graph-based k-nearest neighbor

Science.gov (United States)

Ayu Cyntya Dewi, Dyah; Shaufiah; Asror, Ibnu

2018-03-01

SMS (Short Message Service) is on e of the communication services that still be the main choice, although now the phone grow with various applications. Along with the development of various other communication media, some countries lowered SMS rates to keep the interest of mobile users. It resulted in increased spam SMS that used by several parties, one of them for advertisement. Given the kind of multi-lingual documents in a message SMS, the Web, and others, necessary for effective multilingual or cross-lingual processing techniques is becoming increasingly important. The steps that performed in this research is data / messages first preprocessing then represented into a graph model. Then calculated using GKNN method. From this research we get the maximum accuracy is 98.86 with training data in Indonesian language and testing data in indonesian language with K 10 and threshold 0.001.

Effect of pH on structure, function, and stability of mitochondrial carbonic anhydrase VA.

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Idrees, Danish; Shahbaaz, Mohd; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2017-02-01

Mitochondrial carbonic anhydrase VA (CAVA) catalyzes the hydration of carbon dioxide to produce proton and bicarbonate which is primarily expressed in the mitochondrial matrix of liver, and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis, and neuronal transmission. To understand the effect of pH on the structure, function, and stability of CAVA, we employed spectroscopic techniques such as circular dichroism, fluorescence, and absorbance measurements in wide range of pH (from pH 2.0 to pH 11.5). CAVA showed an aggregation at acidic pH range from pH 2.0 to pH 5.0. However, it remains stable and maintains its secondary structure in the pH range, pH 7.0-pH 11.5. Furthermore, this enzyme has an appreciable activity at more than pH 7.0 (7.0 < pH ≤ 11.5) with maximum activity at pH 9.0. The maximal values of k cat and k cat /K m at pH 9.0 are 3.7 × 10 6  s -1 and 5.5 × 10 7  M -1  s -1 , respectively. However, this enzyme loses its activity in the acidic pH range. We further performed 20-ns molecular dynamics simulation of CAVA to see the dynamics at different pH values. An excellent agreement was observed between in silico and in vitro studies. This study provides an insight into the activity of CAVA in the pH range of subcellular environment.

Understanding thermostability and pH dependent properties of proteins

DEFF Research Database (Denmark)

Galberg, Pernille

The work performed in this thesis is part of a larger project (“Computational design of stable enzymes”) involving several research teams, which aimed to improve PROPKA (http://propka.ki.ku.dk) and to provide the scientific community with a computational protocol and associated PROPKA program......, which could be used for predicting mutations with expectation of increased thermostability at a certain pH value or a shifted pH activity optimum. The ability of a Bacillus circulans xylanase (BCX) mutant (N35D/A115E) to induce a decrease in pH activity optimum was evaluated by a pH dependent xylanase...

Genome-wide identification and characterization of stress-associated protein (SAP gene family encoding A20/AN1 zinc-finger proteins in Medicago truncatula

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Zhou Yong

2018-01-01

Full Text Available Stress associated proteins (SAPs play important roles in developmental processes, responses to various stresses and hormone stimulation in plants. However, little is known about the SAP gene family in Medicago truncatula. In this study, a total of 17 MtSAP genes encoding A20/AN1 zinc-finger proteins were characterized. Out of these 17 genes, 15 were distributed over all 8 chromosomes at different densities, and two segmental duplication events were detected. The phylogenetic analysis of these proteins and their orthologs from Arabidopsis and rice suggested that they could be classified into five out of the seven groups of SAP family genes, with genes in the same group showing similar structures and conserved domains. The cis-elements of the MtSAP promoters were studied, and many cis-elements related to stress and plant hormone responses were identified. We also investigated the stress-responsive expression patterns of the MtSAP genes under various stresses, including drought, exposure to NaCl and cold. The qRT-PCR results showed that numerous MtSAP genes exhibited transcriptional responses to multiple abiotic stresses. These results lay the foundation for further functional characterization of SAP genes. To the best of our knowledge, this is the first report of a genome-wide analysis of the SAP gene family in M. truncatula.

The PH Domain of PDK1 Exhibits a Novel, Phospho-Regulated Monomer-Dimer Equilibrium With Important Implications for Kinase Domain Activation: Single Molecule and Ensemble Studies†

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Ziemba, Brian P.; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J.

2013-01-01

Phosphoinositide-Dependent Kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4-5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric state(s) of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. The present study investigates the binding of purified WT and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single molecule and ensemble measurements. Single molecule analysis of the brightness of fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric, while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single molecule analysis of 2-D diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little protein penetration into the bilayer as observed for other PH domains. The 2-D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that enables greater protein insertion into

Modulation of phosducin-like protein 3 (PhLP3 levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.

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Nandini V L Hayes

Full Text Available Phosducin-like protein 3 (PhLP3 forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in β-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of β-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems.As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO cell lines. We show that over-expression of PhLP3 promotes an imbalance of α and β tubulin subunits, microtubule disassembly and cell death. In contrast, β-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state.Our results support the hypothesis that PhLP3 is important for the maintenance of β-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially

pH and temperature effects on the molecular conformation of the porcine pancreatic secretory trypsin inhibitor as detected by hydrogen-1 nuclear magnetic resonance

International Nuclear Information System (INIS)

De Marco, A.; Menegatti, E.; Guarneri, M.

1982-01-01

1 H NMR spectra of the porcine pancreatic secretory trypsin inhibitor (PSTI) have been recorded vs. pH and temperature. Of the two tyrosines, one titrates with a pK of 1.25, while the resonances from the other are pH insensitive in the investigated range 4.8 less than or equal to pH less than or equal to 12. This is consistent with PSTI having one Tyr solvent exposed (Try-20) and the other buried (Tyr-31). The resonances from the lysyl epsilon-CH 2 protons titrate with a pK of 10.95. The titration is accompanied by a pronounced line broadening, which starts near pH 8.5. Between pH 11.5 and pH 12 the epsilon-CH 2 resonances recover their low pH line width. Titration curves for the lysines and Tyr-20 reflect single proton ionization equilibria, suggesting that these residues do not interact among themselves. On the basis of double resonance experiments, combined with analysis of chemical shifts, spin-spin couplngs, and line widths, all methyl resonances are identified and followed as functions of pH and temperature. The γ-CH 3 doublet from the N-terminal Thr-1 is assigned by comparison between spectra of forms I and II of the inhibitor, the latter lacking the first four residues of form I. The β-CH 3 resonance from Ala-7 is also assigned. Proton resonance parameters of methyl groups are shown to afford useful NMR probes for the characterization of local nonbonded interactions, microenvironments, and mobilities

Estudio de corrosión bajo tensión en los aceros inoxidables 17-4PH y 17-7PH en presencia de NaCl y NaOH (20 % a 90 °C

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Gaona-Tiburcio, Citlalli

2000-04-01

Full Text Available One of the problems that affects to the electric industry is the not programmed stoppages in the power plants, due to the failure of any main component: boiler, turbine and generator. In the turbine, the combined action of a corrosive agent (humid polluted vapor and a mechanical effort generally will result in Stress Corrosion Cracking (SCC. In this work the SCC susceptibility of the precipitation hardening stainless steels 17-4PH and 17- 7PH, thoroughly used in steam turbine blades of power stations is analyzed. The specimens were tested in the presence of NaCl and NaOH (20 % to 90 °C and different pH. The CERT test (Constant Extension Rate Test was used, at 10^-6 s^-1 supplementing it with electrochemical noise; the aim was to identify the conditions of maximum susceptibility and the performance of the studied materials. The fractographic analysis revealed ductile and brittle fracture. Intergranular crackings, characteristic of the anodic dissolution mechanism of the material was observed. Nevertheless, the main mechanism responsible the failure was hydrogen embrittlement.

Uno de los problemas que afecta a la industria eléctrica es el de los paros no programados en las plantas generadoras de electricidad, debidos al fallo de algún componente principal: caldera, turbina y generador. En la turbina, la acción combinada de un agente corrosivo (vapor húmedo contaminado y un esfuerzo mecánico, generalmente provocará corrosión bajo tensión (CBT. En este trabajo se analiza la susceptibilidad a la CBT de los aceros inoxidables, endurecibles por precipitación, 17-4PH y 17-7PH, ampliamente usados en alabes de turbina de vapor de centrales termoeléctricas. Las muestras se ensayaron en presencia de NaCl y NaOH (20 % a 90 °C, y distintos valores de pH. Se empleó el ensayo CERT (Constant Extensión Rate Test, a velocidades de 10^-6 s^-1, complementándolo con ruido electroquímico, buscando

Millimeter/submillimeter Spectroscopy of PH2CN ({\\tilde{X}} 1A') and CH3PH2 ({\\tilde{X}} 1A'): Probing the Complexity of Interstellar Phosphorus Chemistry

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Halfen, D. T.; Clouthier, D. J.; Ziurys, L. M.

2014-11-01

Millimeter/submillimeter spectra of PH2CN ({\\tilde{X}} 1A') and CH3PH2 ({\\tilde{X}} 1A') have been recorded for the first time using direct absorption techniques. This work extends previous measurements of both molecules beyond the 10-50 GHz range. Both species were created in the presence of an AC discharge by the reaction of phosphorus vapor and either cyanogen and hydrogen (PH2CN) or methane (CH3PH2). Twelve rotational transitions of PH2CN were recorded over the region 305-422 GHz for asymmetry components Ka = 0 through 8. For CH3PH2, eight rotational transitions were measured from 210-470 GHz with Ka = 0 through 16; these spectra exhibited greater complexity due to the presence of internal rotation, which splits the Ka = 1, 2, and 3 asymmetry components into A and E states. Combined analyses of the millimeter/submillimeter and previous microwave data were performed for both molecules. For PH2CN, the spectra were fit with a Watson S-reduced asymmetric top Hamiltonian, resulting in more accurate rotational and centrifugal distortion constants. In the case of CH3PH2, an asymmetric top internal-rotation Hamiltonian was employed in the analysis, significantly improving the rotational and torsional parameters over previous microwave estimates. Searches for both molecules were subsequently conducted toward Sgr B2(N), using the 12 m telescope of the Arizona Radio Observatory (ARO). Neither species was identified, with abundance upper limits, relative to H2, of f (PH2CN/H2) PH2/H2) 2 and >200, respectively.

Linearização de curvas de titulação para determinação da capacidade tamponante da fibra de alimentos em ampla faixa de pH - DOI: 10.4025/actascianimsci.v32i1.5384 Linearization of the titration curves for determination of buffering capacity of feed fiber in a wide pH range - DOI: 10.4025/actascianimsci.v32i1.5384

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João Ricardo Dittrich

2009-12-01

Full Text Available Foi avaliado o método de linearização de curvas de titulação para determinação da capacidade tamponante (CT da fibra em detergente neutro (FDN de 13 alimentos. Foram utilizados dados individuais de dois experimentos, incluindo amostras de aveia branca, aveia preta, azevém, trevo branco e trevo vermelho (ensaio 1, além de amostras de milho, farelo de soja, palha de trigo e cinco misturas destes alimentos (ensaio 2. As titulações foram realizadas do pH inicial até o pH 2,0. A acidez titulável (AT foi medida como a quantidade de HCl necessária (mEq H+ g-1 de FDN para redução do pH inicial da solução até o pH 4,0; 3,0 e 2,0, e a medida taxa linear de tamponamento (TLT foi obtida como inverso da inclinação da regressão linear entre a medida linearizada do pH, calculada como EXP (1 pH-1, e a adição acumulada de HCl em cada intervalo. A transformação resultou em ajuste satisfatório à regressão linear para todos os materiais estudados, com r2 maior que 0,85 e erro-padrão residual menor que 0,059. Houve correlação positiva entre a AT e a TLT medidas nas diferentes faixas de pH. Diferentemente da AT, o método TLT permite obtenção da CT como taxa linear única, válida em qualquer intervalo de pH de 8,0 ao 2,0.A method for linearization of titration curves and its application to determine the buffering capacity (BC of neutral detergent fiber (NDF from 13 feeds and their mixtures was evaluated. Individual data from 2 essays, including samples of white oat, black oat, ryegrass, red clover and white clover (essay 1, and samples of corn, soybean meal, wheat straw and mixtures of these five feeds (essay 2. Titration was performed just to the pH 2.0. The titratable acidity was measured as the amount of HCl needed (mEq g-1 NDF to reduce the initial pH of the solution down to 4.0, 3.0 and 2.0, and the linear buffer (LB measure were determined as the inverse of the slope of the linear regression between linearized pH, calculated

Resistance of Streptococcus bovis to acetic acid at low pH: Relationship between intracellular pH and anion accumulation

Energy Technology Data Exchange (ETDEWEB)

Russell, J.B. (Cornell Univ., Ithaca, NY (USA))

1991-01-01

Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grown at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). Y{sub ATP} (grams of cells per mole at ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up ({sup 14}C)acetate and ({sup 14}C)benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation.

Resistance of Streptococcus bovis to acetic acid at low pH: Relationship between intracellular pH and anion accumulation

International Nuclear Information System (INIS)

Russell, J.B.

1991-01-01

Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grown at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). Y ATP (grams of cells per mole at ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up [ 14 C]acetate and [ 14 C]benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation

A pH- and temperature-responsive bioresorbable injectable hydrogel based on polypeptide block copolymers for the sustained delivery of proteins in vivo.

Science.gov (United States)

Turabee, Md Hasan; Thambi, Thavasyappan; Duong, Huu Thuy Trang; Jeong, Ji Hoon; Lee, Doo Sung

2018-02-27

Sustained delivery of protein therapeutics is limited owing to the fragile nature of proteins. Despite its great potential, delivery of proteins without any loss of bioactivity remains a challenge in the use of protein therapeutics in the clinic. To surmount this shortcoming, we report a pH- and temperature-responsive in situ-forming injectable hydrogel based on comb-type polypeptide block copolymers for the controlled delivery of proteins. Polypeptide block copolymers, composed of hydrophilic polyethylene glycol (PEG), temperature-responsive poly(γ-benzyl-l-glutamate) (PBLG), and pH-responsive oligo(sulfamethazine) (OSM), exhibit pH- and temperature-induced sol-to-gel transition behavior in aqueous solutions. Polypeptide block copolymers were synthesized by combining N-carboxyanhydride-based ring-opening polymerization and post-functionalization of the chain-end using N-hydroxy succinimide ester activated OSM. The physical properties of polypeptide-based hydrogels were tuned by varying the composition of temperature- and pH-responsive PBLG and OSM in block copolymers. Polypeptide block copolymers were non-toxic to human embryonic kidney cells at high concentrations (2000 μg mL -1 ). Subcutaneous administration of polypeptide block copolymer sols formed viscoelastic gel instantly at the back of Sprague-Dawley (SD) rats. The in vivo gels exhibited sustained degradation and were found to be bioresorbable in 6 weeks without any noticeable inflammation at the injection site. Anionic characteristics of hydrogels allow efficient loading of a cationic model protein, lysozyme, through electrostatic interaction. Lysozyme-loaded polypeptide block copolymer sols readily formed a viscoelastic gel in vivo and sustained lysozyme release for at least a week. Overall, the results demonstrate an elegant approach to control the release of certain charged proteins and open a myriad of therapeutic possibilities in protein therapeutics.

Bioactive L acidissima protein hydrolysates using Box-Behnken design.

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Sonawane, Sachin K; Arya, Shalini S

2017-07-01

This study examines the extraction and hydrolysis of proteins using single factor and Box-Behnken Design (BBD). From single factor tests, optimised extraction parameters were 1% alkali concentration, 40 °C temperature, 60 min time, and 1:20 solid to alkali ratio. Under these conditions; 924.31 mg/g of total protein was obtained from Limonia acidissima (L acidissima). The maximum degree of hydrolysis was 39.82% at pH 2, enzyme to substrate ratio 2.5% (w/w), and hydrolysis time was 42.41 min using BBD design. L acidissima seed protein hydrolysate showed 32.94% DPPH and 88.18% of ABTS activity at concentration of 100 µg/ml and 1 mg/ml, respectively. Reducing power of 0.16 and metal chelating activity of 87.39% was obtained from 5 mg/ml protein hydrolysates. This implied that L acidissima seed protein hydrolysate could be utilised in protein rich product or as protein supplements.

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis.

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Verweij, Walter; Spelt, Cornelis E; Bliek, Mattijs; de Vries, Michel; Wit, Niek; Faraco, Marianna; Koes, Ronald; Quattrocchio, Francesca M

2016-03-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. © 2016 American Society of Plant Biologists. All rights reserved.

Frequency Response of the Sample Vibration Mode in Scanning Probe Acoustic Microscope

International Nuclear Information System (INIS)

Ya-Jun, Zhao; Qian, Cheng; Meng-Lu, Qian

2010-01-01

Based on the interaction mechanism between tip and sample in the contact mode of a scanning probe acoustic microscope (SPAM), an active mass of the sample is introduced in the mass-spring model. The tip motion and frequency response of the sample vibration mode in the SPAM are calculated by the Lagrange equation with dissipation function. For the silicon tip and glass assemblage in the SPAM the frequency response is simulated and it is in agreement with the experimental result. The living myoblast cells on the glass slide are imaged at resonance frequencies of the SPAM system, which are 20kHz, 30kHz and 120kHz. It is shown that good contrast of SPAM images could be obtained when the system is operated at the resonance frequencies of the system in high and low-frequency regions

From proton nuclear magnetic resonance spectra to pH. Assessment of {sup 1}H NMR pH indicator compound set for deuterium oxide solutions

Energy Technology Data Exchange (ETDEWEB)

Tynkkynen, Tuulia, E-mail: tuulia.tynkkynen@uku.fi [Laboratory of Chemistry, Department of Biosciences, University of Kuopio, PO Box 1627, 70211 Kuopio (Finland); Tiainen, Mika; Soininen, Pasi; Laatikainen, Reino [Laboratory of Chemistry, Department of Biosciences, University of Kuopio, PO Box 1627, 70211 Kuopio (Finland)

2009-08-19

In this study, a protocol for pH determination from D{sub 2}O samples using {sup 1}H NMR pH indicator compounds was developed and assessed by exploring the pH-dependency of 13 compounds giving pH-dependent {sup 1}H NMR signals. The indicators cover the pH range from pH* 0 to 7.2. Equations to transform the indicator chemical shifts to pH estimates are given here for acetic acid, formic acid, chloroacetic acid, dichloroacetic acid, creatine, creatinine, glycine, histidine, 1,2,4-triazole, and TSP (2,2,3,3-tetradeutero-3-(trimethylsilyl)-propionic acid). To characterize the method in presence of typical solutes, the effects of common metabolites, albumin and ionic strength were also evaluated. For the ionic strengths, the effects were also modelled. The experiments showed that the use of pH sensitive {sup 1}H NMR chemical shifts allows the pH determination of typical metabolite solutions with accuracy of 0.01-0.05 pH units. Also, when the ionic strength is known with accuracy better than 0.1 mol dm{sup -3} and the solute concentrations are low, pH{sub nmr}{sup *} (the NMR estimate of pH) can be assumed to be within 0.05 pH units from potentiometrically determined pH.

Reduction of mercury (II) by humic substances-influence of pH, salinity of aquatic system

Digital Repository Service at National Institute of Oceanography (India)

Chakraborty, P.; Vudamala, K.; Coulibaly, M.; Ramteke, D.; Chennuri, K.; Lean, D.

60 80 pH 4.0 pH 7.0 pH 8.0 pH 9.0 Time (h-1) 0 20 40 60 80 100 120 140 160 0 10 20 30 40 pH 4.0 pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0 Figure 5 a b c Pe rc en ta ge o f t he to ta l d is so lv ed H g re du ce d b y H S an d tr ap ed in o xi di... humic substances (wt %) 0.42 0.44 0.46 0.48 0.50 0.52 0.54 0.56 Fr ac tio n (p er ce nt ag e) o f t ot al H g re du ce d by H S 20 30 40 50 60 70 Salinity = 5 PSU Salinity = 10 PSU Salinity = 15 PSU Salinity = 20PSU Salinity = 30 PSU 0.42 0...

Unfolding Studies of the Cysteine Protease Baupain, a Papain-Like Enzyme from Leaves of Bauhinia forficata: Effect of pH, Guanidine Hydrochloride and Temperature

Directory of Open Access Journals (Sweden)

Rosemeire A. Silva-Lucca

2013-12-01

Full Text Available Baupain belongs to the α+β class of proteins with a secondary structure-content of 44% α-helix, 16% β-sheet and 12% β-turn. The structural transition induced by pH was found to be noncooperative, with no important differences observed in the pH range from 3.0 to 10.5. At pH 2.0 the protein presented substantial non-native structure with strong ANS binding. Guanidine hydrochloride (GdnHCl-induced unfolding did not change the protein structure significantly until 4.0 M, indicating the high rigidity of the molecule. The unfolding was cooperative, as seen by the sigmoidal transition curves with midpoints at 4.7 ± 0.2 M and 5.0 ± 0.2 M GdnHCl, as measured by CD and fluorescence spectroscopy. A red shift of 7 nm in intrinsic fluorescence was observed with 6.0 M GdnHCl. Temperature-induced unfolding of baupain was incomplete, and at least 35% of the native structure of the protein was retained, even at high temperature (90 °C. Baupain showed characteristics of a molten globule state, due to preferential ANS binding at pH 2.0 in comparison to the native form (pH 7.0 and completely unfolded (6.0 M GdnHCl state. Combined with information about N-terminal sequence similarity, these results allow us to include baupain in the papain superfamily.

PINK1-Interacting Proteins: Proteomic Analysis of Overexpressed PINK1

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Aleksandar Rakovic

2011-01-01

Full Text Available Recent publications suggest that the Parkinson's disease- (PD- related PINK1/Parkin pathway promotes elimination of dysfunctional mitochondria by autophagy. We used tandem affinity purification (TAP, SDS-PAGE, and mass spectrometry as a first step towards identification of possible substrates for PINK1. The cellular abundance of selected identified interactors was investigated by Western blotting. Furthermore, one candidate gene was sequenced in 46 patients with atypical PD. In addition to two known binding partners (HSP90, CDC37, 12 proteins were identified using the TAP assay; four of which are mitochondrially localized (GRP75, HSP60, LRPPRC, and TUFM. Western blot analysis showed no differences in cellular abundance of these proteins comparing PINK1 mutant and control fibroblasts. When sequencing LRPPRC, four exonic synonymous changes and 20 polymorphisms in noncoding regions were detected. Our study provides a list of putative PINK1 binding partners, confirming previously described interactions, but also introducing novel mitochondrial proteins as potential components of the PINK1/Parkin mitophagy pathway.

Proteomic analysis of membrane microdomain-associated proteins in the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder reveals alterations in LAMP, STXBP1 and BASP1 protein expression.

LENUS (Irish Health Repository)

Behan, A T

2009-06-01

The dorsolateral prefrontal cortex (dlpfc) is strongly implicated in the pathogenesis of schizophrenia (SCZ) and bipolar disorder (BPD) and, within this region, abnormalities in glutamatergic neurotransmission and synaptic function have been described. Proteins associated with these functions are enriched in membrane microdomains (MM). In the current study, we used two complementary proteomic methods, two-dimensional difference gel electrophoresis and one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by reverse phase-liquid chromatography-tandem mass spectrometry (RP-LC-MS\\/MS) (gel separation liquid chromatography-tandem mass spectrometry (GeLC-MS\\/MS)) to assess protein expression in MM in pooled samples of dlpfc from SCZ, BPD and control cases (n=10 per group) from the Stanley Foundation Brain series. We identified 16 proteins altered in one\\/both disorders using proteomic methods. We selected three proteins with roles in synaptic function (syntaxin-binding protein 1 (STXBP1), brain abundant membrane-attached signal protein 1 (BASP1) and limbic system-associated membrane protein (LAMP)) for validation by western blotting. This revealed significantly increased expression of these proteins in SCZ (STXBP1 (24% difference; P<0.001), BASP1 (40% difference; P<0.05) and LAMP (22% difference; P<0.01)) and BPD (STXBP1 (31% difference; P<0.001), BASP1 (23% difference; P<0.01) and LAMP (20% difference; P<0.01)) in the Stanley brain series (n=20 per group). Further validation in dlpfc from the Harvard brain subseries (n=10 per group) confirmed increased protein expression in SCZ of STXBP1 (18% difference; P<0.0001), BASP1 (14% difference; P<0.0001) but not LAMP (20% difference; P=0.14). No significant differences in STXBP1, BASP1 or LAMP protein expression in BPD dlpfc were observed. This study, through proteomic assessments of MM in dlpfc and validation in two brain series, strongly implicates LAMP, STXBP1 and BASP1 in SCZ and supports

Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

Science.gov (United States)

Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

2016-01-01

Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells.

Effects of spray drying on physicochemical properties of milk protein-stabilised emulsions

NARCIS (Netherlands)

Sliwinski, E.L.; Lavrijsen, B.W.M.; Vollenbroek, J.M.; Stege, van der H.J.; Boekel, van M.A.J.S.; Wouters, J.T.M.

2003-01-01

The effect of spray drying and reconstitution has been studied for oil-in-water emulsions (20.6% maltodextrin, 20% soybean oil, 2.4% protein, 0.13 M NaCl, pH 6.7) with varying ratios of sodium caseinate and whey protein, but with equal size distribution (d(32) = 0.77 mum). When the concentration of

Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

Directory of Open Access Journals (Sweden)

Brita Singers Sørensen

Full Text Available The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect, which weakens the spatial linkage between hypoxia and acidosis.Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15 were treated with hypoxia, acidosis (pH 6.3, or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein.Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe, genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2, and Ribosomal protein L37 (RPL37. Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa and protein synthesis (both cell lines was observed when hypoxia and low pHe were combined.We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de

Stabilization of pH in solid-matrix hydroponic systems

Science.gov (United States)

Frick, J.; Mitchell, C. A.

1993-01-01

2-[N-morpholino]ethanesulfonic acid (MES) buffer or Amberlite DP-1 (cation-exchange resin beads) were used to stabilize substrate pH of passive-wicking, solid-matrix hydroponic systems in which small canopies of Brassica napus L. (CrGC 5-2, genome : ACaacc) were grown to maturity. Two concentrations of MES (5 or 10 mM) were included in Hoagland 1 nutrient solution. Alternatively, resin beads were incorporated into the 2 vermiculite : 1 perlite (v/v) growth medium at 6% or 12% of total substrate volume. Both strategies stabilized pH without toxic side effects on plants. Average seed yield rates for all four pH stabilization treatments (13.3 to 16.9 g m-2 day-1) were about double that of the control (8.2 g m-2 day-1), for which there was no attempt to buffer substrate pH. Both the highest canopy seed yield rate (16.9 g m-2 day-1) and the highest shoot harvest index (19.5%) occurred with the 6% resin bead treatment, even though the 10 mM MES and 12% bead treatments maintained pH within the narrowest limits. The pH stabilization methods tested did not significantly affect seed oil and protein contents.

Flow rate, pH and calcium concentration of saliva of children and adolescents with type 1 diabetes mellitus.

Science.gov (United States)

Moreira, A R; Passos, I A; Sampaio, F C; Soares, M S M; Oliveira, R J

2009-08-01

Alterations in salivary parameters may increase the caries risk in diabetic children, but, contradictory data on this issue have been reported. The aims of this study were to compare salivary parameters (flow rate, pH and calcium concentration) between healthy and type 1 diabetes mellitus (T1DM) individuals. The sample consisted of 7- to 18-year-old individuals divided into two groups: 30 subjects with T1DM (group A) and 30 healthy control subjects (group B). Fasting glucose levels were determined. Unstimulated and stimulated saliva was collected. The pH of unstimulated saliva was measured with paper strips and an electrode. Calcium concentrations in stimulated saliva were determined with a selective electrode. Group A individuals had inadequate blood glucose control (HbA(1C) >9%), with means +/- SD unstimulated salivary flow rate of 0.15 +/- 0.1 mL/min compared to 0.36 +/- 0.2 mL/min for group B (P salivary flow rate was similar by both groups and above 2.0 mL/min. Saliva pH was 6.0 +/- 0.8 for group A and significantly different from 7.0 +/- 0.6 for group B (P Salivary calcium was 14.7 +/- 8.1 mg/L for group A and significantly higher than 9.9 +/- 6.4 mg/L for group B (P salivary parameters favoring caries such as low saliva pH and unstimulated salivary flow rate were observed in T1DM individuals.

Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology.

Science.gov (United States)

Callaghan, Paul S; Siriwardana, Amila; Hassett, Matthew R; Roepe, Paul D

2016-03-31

Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.

The PH domain of phosphoinositide-dependent kinase-1 exhibits a novel, phospho-regulated monomer-dimer equilibrium with important implications for kinase domain activation: single-molecule and ensemble studies.

Science.gov (United States)

Ziemba, Brian P; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J

2013-07-16

Phosphoinositide-dependent kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology, this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric states of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. This study investigates the binding of purified wild-type (WT) and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single-molecule and ensemble measurements. Single-molecule analysis of the brightness of the fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single-molecule analysis of two-dimensional (2D) diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate as a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little penetration of the protein into the bilayer as observed for other PH domains. The 2D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that allows

Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

DEFF Research Database (Denmark)

Järås, Marcus; Johnels, Petra; Hansen, Nils Gunder

2010-01-01

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML...... will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test...

A spectroscopic study on stability of curcumin as a function of pH in silica nanoformulations, liposome and serum protein

Science.gov (United States)

Jain, Beena

2017-02-01

The effect of pH on the stability of curcumin formulated with different carriers has been studied spectroscopically. This was investigated by monitoring the absorption and emission kinetics and fluorescence decay time of four different curcumin formulations suspended in buffer with pH varying from 5 to 8.5. The carriers were organically modified silica NP (SiNP) having 3-amino propyl and/or vinyl groups, liposome and serum protein. The results reveal that stability of curcumin formulated with SiNP functionalized with 3-amino propyl group (SiNP-VA) is significantly higher as compared to SiNP with only vinyl group (SiNP-V) and buffer but lower as compared to serum protein and liposome. However, fluorescence quantum yield (QY) is highest in SiNP-VA among all the nano formulations at pH 7.4 and below, which is attributed to the excited state interaction of curcumin with the amino groups of SiNP-VA. Results suggest that SiNP-VA could be an effective carrier for curcumin, which may have applications for imaging and drug delivery.

Low pH, aluminum, and phosphorus coordinately regulate malate exudation through GmALMT1 to improve soybean adaptation to acid soils.

Science.gov (United States)

Liang, Cuiyue; Piñeros, Miguel A; Tian, Jiang; Yao, Zhufang; Sun, Lili; Liu, Jiping; Shaff, Jon; Coluccio, Alison; Kochian, Leon V; Liao, Hong

2013-03-01

Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function.

Plaque pH Changes Following Consumption of Two Types of Plain and Bulky Bread.

Science.gov (United States)

Mortazavi, Shiva; Noin, Sogol

2011-01-01

Consistency, backing process and content differences could influence cariogenic potential of foods. The aim was to compare plaque pH changes following consumption of two types of bread with different physical characteristics. In this clinical trial, interproximal plaque pH of 10 volunteers with high risk of dental caries (saliva Streptococcus mutans > 10(5), high dental caries experience, and average DMFT =6.10 ± 1.56) was measured. Plain traditionally backed "Sangak bread" and soft bulky "Baguette bread" and %10 sucrose solution were tested in a cross over designed experiment. Baseline plaque pH was recorded and followed by 1, 5, 10, 15, 20, and 30 minutes intervals. Data was analyzed using ANOVA and Tukey test (α = 0.05). Sucrose solution caused the most pronounced pH and ΔpH drop from 7.15 ± 0.33 at baseline to 6.78 ± 0.29. Means plaque pH of 10% sucrose solution and Baguette were not statistically different at 1, 20 and 30 minutes (P > 0.05). Mean plaque pH of Sangak and Baguette showed significant differences at 0, 1, 20 and30 minutes (P bread samples within first 10 minutes, pH increased and then started to decrease during tenth to fifteenth minutes. During all experiment phases, the mean pH of Baguette with less consistency and carbohydrate content and higher rate of starch gelatination was lower compared to Sangak.

The pH of chemistry assays plays an important role in monoclonal immunoglobulin interferences.

Science.gov (United States)

Alberti, Michael O; Drake, Thomas A; Song, Lu

2015-12-01

Immunoglobulin paraproteins can interfere with multiple chemistry assays. We want to investigate the mechanisms of immunoglobulin interference. Serum samples containing paraproteins from the index patient and eight additional patients were used to investigate the interference with the creatinine and total protein assays on the Beckman Coulter AU5400/2700 analyzer, and to determine the effects of pH and ionic strength on the precipitation of different immunoglobulins in these patient samples. The paraprotein interference with the creatinine and total protein assays was caused by the precipitation of IgM paraprotein in the index patient's samples under alkaline assay conditions. At extremely high pH (12-13) and extremely low pH (1-2) and low ionic strength, paraprotein formed large aggregates in samples from the index patient but not from other patients. The pH and ionic strength are the key factors that contribute to protein aggregation and precipitation which interfere with the creatinine and total protein measurements on AU5400/2700. The different amino acid sequence of each monoclonal paraprotein will determine the pH and ionic strength at which the paraprotein will precipitate.

Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th-century pandemics.

Science.gov (United States)

Pasricha, Gunisha; Mishra, Akhilesh C; Chakrabarti, Alok K

2013-07-01

PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. Analysis showed that 96·4% of the H5N1 influenza viruses harbored full-length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th-century pandemic influenza viruses contained full-length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human- and avian host-specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host-specific evolution of the virus. However, studies are required to correlate this sequence variability with the virulence and pathogenicity. © 2012 John Wiley & Sons Ltd.

[ABIN1 is not involved in imatinib upregulating A20 to inhibit the activation of NF-κB pathway in Jurkat T cells].

Science.gov (United States)

Chen, Qian; Wang, Senlin; Lin, Chen; Chen, Shaohua; Zhao, Xiaoling; Li, Yangqiu

2017-05-01

Objective To investigate the effect of imatinib (IM) on the expressions of A20-binding inhibitor of NF-κB1 (ABIN1) and A20 in Jurkat T cells. Methods Jurkat T cells were treated with 25, 50 and 100 nmol/L IM for 24 hours. The mRNA and protein levels of ABIN1, A20 and NF-κB were detected by real-time quantitative PCR and Western blotting. Results IM significantly inhibited both mRNA and protein levels of ABIN1 and NF-κB, but raised the mRNA and protein levels of A20; while phorbol 12-myristate 13-acetate/ionomycin increased the expression levels of ABIN1 and A20 mRNA and protein. Conclusion IM could upregulate A20 protein to inhibit the activation of NF-κB pathway in Jurkat T cells, which was independent of the ABIN1 protein.

Improving the expression of recombinant proteins in E. coli BL21 (DE3) under acetate stress: an alkaline pH shift approach.

Science.gov (United States)

Wang, Hengwei; Wang, Fengqing; Wang, Wei; Yao, Xueling; Wei, Dongzhi; Cheng, Hairong; Deng, Zixin

2014-01-01

Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5-8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0 ± 0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

Compartment-specific pH monitoring in Bacillus subtilis using fluorescent sensor proteins: a tool to analyze the antibacterial effect of weak organic acids.

NARCIS (Netherlands)

van Beilen, J.W.A.; Brul, S.

2013-01-01

The internal pH (pHi) of a living cell is one of its most important physiological parameters. To monitor the pH inside Bacillus subtilis during various stages of its life cycle, we constructed an improved version (IpHluorin) of the ratiometric, pH-sensitive fluorescent protein pHluorin by extending

Nanosensors for pH measurements in plants

DEFF Research Database (Denmark)

Ytting, Cecilie Karkov; Fuglsang, Anja Thoe

Traditionally in vivo pH measurements in plants are carried out using different fluorescent probes such as BCECF and SNARFs by injecting plant cells or incubating tissues with the probes. This approach, however, carries several problems, the most important one being the handling of the plant cells...... or tissues. Stress and wounding of the plant can possibly influence the outcome of the pH measurements. Other problems are the difficulties of getting the probes more than a few cell layers into the plant tissue and to get an even distribution of the probe. Increasing numbers of different variants...... of the green fluorescent protein, GFP, have become available, some of which are pH sensitive. This offers the opportunity to simply express the pH sensitive protein in different plant tissues, making it possible to conduct in vivo pH measurements in a non-invasive manner. One sensor being characterized...

Plaque pH changes following consumption of two types of plain and bulky bread

Directory of Open Access Journals (Sweden)

Shiva Mortazavi

2011-01-01

Full Text Available Background: Consistency, backing process and content differences could influence cariogenic potential of foods. The aim was to compare plaque pH changes following consumption of two types of bread with different physical characteristics. Methods : In this clinical trial, interproximal plaque pH of 10 volunteers with high risk of dental caries (saliva Streptococcus mutans > 10 5 , high dental caries experience, and average DMFT =6.10 ± 1.56 was measured. Plain traditionally backed "Sangak bread" and soft bulky "Baguette bread" and %10 sucrose solution were tested in a cross over designed experiment. Baseline plaque pH was recorded and followed by 1, 5, 10, 15, 20, and 30 minutes intervals. Data was analyzed using ANOVA and Tukey test (α = 0.05. Results: Sucrose solution caused the most pronounced pH and ∆pH drop from 7.15 ± 0.33 at baseline to 6.78 ± 0.29. Means plaque pH of 10% sucrose solution and Baguette were not statistically different at 1, 20 and 30 minutes (P > 0.05. Mean plaque pH of Sangak and Baguette showed significant differences at 0, 1, 20 and30 minutes (P < 0.05. Sucrose solution caused a dramatic plaque pH drop during first 10 minutes and then within 30 minutes returned to baseline pH. For two bread samples within first 10 minutes, pH increased and then started to decrease during tenth to fifteenth minutes. Conclusion: During all experiment phases, the mean pH of Baguette with less consistency and carbohydrate content and higher rate of starch gelatination was lower compared to Sangak.

Engineering a pH responsive pore forming protein.

Science.gov (United States)

Kisovec, Matic; Rezelj, Saša; Knap, Primož; Cajnko, Miša Mojca; Caserman, Simon; Flašker, Ajda; Žnidaršič, Nada; Repič, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor

2017-02-08

Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

Engineering a pH responsive pore forming protein

Science.gov (United States)

Kisovec, Matic; Rezelj, Saša; Knap, Primož; Cajnko, Miša Mojca; Caserman, Simon; Flašker, Ajda; Žnidaršič, Nada; Repič, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor

2017-02-01

Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

International Nuclear Information System (INIS)

Zhang, Nawei; Zhang, Zhenyu; Feng, Shan; Wang, Qingtao; Malamud, Daniel; Deng, Haiteng

2013-01-01

Highlights: ► A high-throughput method for profiling and quantification of the differentially expressed proteins in saliva samples was developed. ► Identified that DMBT1, S100A7, S100A8, S100A9 and alpha defensin were up-regulated in saliva from HIV-1 seropositive patients. ► Established analytical strategies are translatable to the clinical setting. -- Abstract: In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity