Human Development and Stem Cells

Human embryonic development depends on stem cells. During the course of development, cells divide, migrate, and specialize. Early in development, a group of cells called the inner cell mass (ICM) forms. These cells are able to produce all the tissues of the body. Later in development, during gastrulation, the three germ layers form, and most cells become more restricted in the types of cells that they can produce.

Real-time PCR in Microbiology: From Diagnosis to Characterization

Ian M. MacKay "Real-time PCR in Microbiology: From Diagnosis to Characterization"

Publisher: Caister Academic Press 2007 | 454 Pages | ISBN: 1904455182 | PDF | 15.8 MB
Description or mention of instrumentation, sofhvare, or other products in this book does not imply endorsement by the author or publisher. The author and publisher do not assume responsibility for the validity of any products or procedures mentioned or described in this book or for the consequences of
their use. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the publisher. No claim to original U.S. Government works.

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Scripps Research Scientists Find E. Coli Enzyme Must Move to Function

Scripps Research Scientists Find E. Coli Enzyme Must Move to Function

Slight oscillations lasting just milliseconds have a huge impact on an enzyme's function, according to a new study by Scripps Research Institute scientists. Blocking these movements, without changing the enzyme's overall structure or any of its other properties, renders the enzyme defective in carrying out chemical reactions.

The study, published in the April 8, 2011 issue of the journal Science, adds to a growing body of evidence pointing to the importance of movement in the ability of enzymes and other types of proteins to do their job. The findings may also help scientists design more specific and effective drugs targeting enzymes.

"Ever since the first X-ray structures of proteins emerged, scientists have been talking about proteins as though their structures were fixed in space," said Peter Wright, chair of the Department of Molecular Biology and member of the Skaggs Institute for Chemical Biology at Scripps Research, who was senior author of the study. "But that is not how proteins work. They are like the machines we build. They have moving parts, and they need motion to work."

A Model Enzyme

The new study examined the enzyme dihydrofolate reductase (DHFR) from the common bacterium Escherichia coli, which the Wright group has been using as a model for understanding how enzymes catalyze (cause or accelerate) chemical reactions. Most strains of E. coli are harmless, but some can cause serious food poisoning.

Bacterial cells cannot live without DHFR, thus this enzyme is the target of many antibiotics. Human cells, and in particular rapidly dividing cells, also use DHFR; drugs that target human DHFR, such as methotrexate, are often used in cancer chemotherapy.

DHFR spurs the conversion of a compound called dihydrofolate (DHF) to a different form, tetrahydrofolate (THF), which is needed by cells for synthesis of DNA. In its chemical reaction, DHFR uses a helper or co-factor, called NADPH. It catalyzes the transfer of a hydride (a negative hydrogen ion) from NADPH to DHF to produce THF. Previous studies by Wright and others have shown that the loops surrounding the active site are flexible, and that one of the loops in particular, called the Met20 loop can adopt two different conformations during the catalytic cycle.

Until now, however, the significance of these motions remained obscure.

Linking Motion to Function

Wright, graduate student Gira Bhabha, and colleagues from both Scripps Research and Pennsylvania State University decided to investigate.

For the new study, the scientists turned to an imaging technique known as nuclear magnetic resonance (NMR) spectroscopy, in combination with X-ray crystallography. Unlike X-ray crystallography, a technique used to determine the structure of proteins in crystals, recently developed NMR methods allow scientists to visualize the motions of proteins in solution. The technique can capture protein motions "in a time scale that is relevant to biology, from microseconds to milliseconds to seconds," said Wright.

To determine the importance of the oscillations, the team set out to make a mutation in the DHFR enzyme that prevented the flexible Met20 loop from moving. To know which amino acids to change, the scientists compared the bacterial DHFR protein sequence to that of the human enzyme, since in the human enzyme, the Met20 loop is more rigid.

Using this approach, the scientists successfully produced a rigidified mutatant E. coli DHFR. When the scientists examined it using X-ray crystallography, they could see the mutant enzyme's structure was almost identical to the wild-type enzyme. However, NMR analysis revealed that the Met20 loop and other parts of the active site were no longer flexible in the mutant.

Significantly, the mutated E. coli enzyme transferred hydride at a rate that was 16-fold slower than that of the wild-type enzyme—a substantial loss in enzyme function.

"We demonstrated that locking down the motion in the active site prevents catalysis," said Wright.

While previous work had indicated that enzymes can exist in different shapes and forms and that changes in enzyme shape enable enzymes to bind to their substrates and co-factors or release the products, "This is the first demonstration that motions play a role in the actual chemistry of a reaction," said Wright.

Clamping Down on the Active Site

The scientists reason that when the E. coli DHFR carries out its chemical reaction, motions in the active site assist in pushing NADPH and DHF closer to one another. This proximity makes the transfer of the hydride from NAPDH to DHF more efficient. If the active site can't move, the molecules are not sufficiently close to one another for the chemical reaction to occur. "We think that the mutations prevent the enzyme from clamping down on the hydride donor and acceptor, so they can no longer get as close to each other as is necessary for efficient catalysis," explained Bhabha.

Taking motion into account when designing drugs to either inhibit or increase enzyme function could result in more effective or more specific drugs. For example, because the motions in the bacterial DHFR differ from those in the human enzyme, this difference might be exploited to design drugs that are specific for the bacterial enzyme. "It might help reduce the serious side effects of drugs that target DHFR," said Wright.

"The idea is to harness these motions in drug design," added Bhabha. "It's a difficult and challenging problem, but it could have huge impact."

In addition to Wright and Bhabha, co-authors for the paper "A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis," include Damian C. Ekiert, Ian A. Wilson, and H. Jane Dyson at Scripps Research, and Jeeyeon Lee, Jongsik Gam, and Stephen J. Benkovic at Pennsylvania State University.

The research was supported by the National Institutes of Health and the Skaggs Institute for Chemical Biology.

About The Scripps Research Institute

The Scripps Research Institute is one of the world's largest independent, non-profit biomedical research organizations. Scripps Research is internationally recognized for its discoveries in immunology, molecular and cellular biology, chemistry, neuroscience, and vaccine development, as well as for its insights into autoimmune, cardiovascular, and infectious disease. Headquartered in La Jolla, California, the institute also includes a campus in Jupiter, Florida, where scientists focus on drug discovery and technology development in addition to basic biomedical science. Scripps Research currently employs about 3,000 scientists, staff, postdoctoral fellows, and graduate students on its two campuses. The institute's graduate program, which awards Ph.D. degrees in biology and chemistry, is ranked among the top ten such programs in the nation. For more information, see www.scripps.edu .

Advanced Molecular Biology Free Ebook Download

Advanced Molecular Biology
Publisher: Garland/BIOS Scientific Publishers | ISBN: 185996141X | edition 2000 | PDF | 512 pages | 104,3 mb

Advanced Molecular Biology emphasises the unifying principles and mechanisms of molecular biology, with frequent use of tables and boxes to summarise experimental data and gene and protein functions. Extensive cross-referencing between chapters is used to reinforce and broaden the understanding of core concepts. This is the ideal source of comprehensive, authoritative and up-to-date information for all those whose work is in the field of molecular biology.

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Apoptosis in Neurobiology Free Ebook Download

 

Apoptosis in Neurobiology (Frontiers in Neuroscience)
Publisher: CRC | ISBN: 0849333520 | edition 1998 | File type: PDF | 271 pages | 11,5 mb

The rapid growth of the study of apoptosis-mechanism-driven, regulated cell death-has created an urgent need for reliable documentation of the different approaches to and methods of studying the various aspects of the field. Apoptosis in Neurobiology is an important resource for researchers in this emerging frontier of biomedical study. This volume allows the uninitiated neuroscientist intellectual and practical access to the study of apoptosis, with special consideration to the nervous system. The first section concentrates on conceptual approaches to the study of apoptosis in neurobiology and its significance to the nervous system. The second section provides a user-friendly approach to methods and techniques in the study of apoptosis as applied to neurobiology. 

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Methods in Molecular Biology Volume 5: Animal Cell Culture Free Ebook Download

Methods in Molecular Biology Volume 5: Animal Cell Culture
Publisher: Humana Press | ISBN: 0896031500 | edition 1990 | PDF | 704 pages | 52,8 mb

Animal Cell Culture, the latest volume in Humana's highly successful Methods in Molecular Biology series, provides detailed practical techniques for the culture of a broad spectrum of basic cell cell types. Chapters offer hands-on methods for creating mammalian fibroblastic cell cultures and maintaining culture conditions for epithelial, neuronal, and hematopoietic cells among others. Attention is given to the diversity of culture media and extracellular matrices needed to maintain the differentiated functions of the cultured cells.

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