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New neurons are generated throughout life in distinct regions of the mammalian brain due to the proliferation and differentiation of neural stem cells (NSCs). Ubiquitin, a post-translational modification of cellular proteins, is an important factor in regulating neurogenesis. Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. Recent studies have provided growing evidence that deubiquitylases (DUBs) which reverse ubiquitylation process play critical roles in NSCs maintenance, differentiation and maturation. This review mainly focused on the relationship of DUBs and NSCs, and further summarized recent advances in our understanding of DUBs on regulating NSCs biological behaviors.

Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post-translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in-vitro assays. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. Here we demonstrate the use of a MspA nanopore for discriminating and quantifying Ub proteins. We further applied the MspA pore to measure the Ub-chain disassembly activity of UCH37, a proteasome associated deubiquitinase. The implementation of this MspA system into nanopore arrays could enable high throughput characterizations of unknown deubiquitinases as well as drug screening against disease related enzymes.

Author(s): Lam, Yi-Li | Advisor(s): Lew, John | Abstract: One of the hallmarks of Alzheimer’s disease is pathological tau aggregation. We previously reported that cinnamaldehyde, found in cinnamon, could inhibit tau aggregation in vitro, by reacting with one or both of the two cysteines in tau. In my thesis, I use spectrophotometric methods to show that both cysteines display hyperreactivity over model sulfhydryl compounds by as much as four fold. The hyperreactivity is specific to cysteines in tau as opposed to those in other sulfhydryl-containing cellular proteins. Cinnamaldehyde (CA) displays several characteristics that make it favorable as a possible inhibitor of aggregation in vivo: it is a water-soluble, brain-permeable molecule that is non-toxic and inexpensive. I now show in my thesis that the hyperreactivity of CA toward tau specifically, as described in this thesis, is an additional favorable property towards the goal of being a potential therapeutic.

A number of techniques now exist for decreasing the expression of cellular proteins without the need for genomic modification. One such technique involves engineered protein chimeras that combine the ubiquitination activity of E3 ubiquitin ligases with the binding affinity and substrate specificity of designer binding proteins (DBPs). These chimeras, called "ubiquibodies," are capable of selectively and controllably steering virtually any protein to the ubiquitin proteasome pathway (UPP) for degradation, making ubiquibodies a powerful addition to the protein knockout toolbox. A distinguishing feature of ubiquibodies is their modularity-simply swapping DBPs can generate a new ubiquibody with specificity for a different substrate protein. Moreover, by employing DBPs that recognize particular protein states (e.g., active versus inactive conformation, mutant versus wild-type, post-translational modification), it becomes possible to deplete certain protein subpopulations while sparing others. This protocol outlines the steps necessary to design and functionally evaluate ubiquibodies for customizable silencing of cellular proteins. © 2018 by John Wiley & Sons, Inc.

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an "overlap score," (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 "overlap factors," (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells.

The analysis of cellular subproteomes by 2DE is hampered by the difficulty of aligning gel images from samples that have very different protein composition. Here, we present a sensitive and cost-effective fluorescent labeling method for analyzing protein samples that is not dependent on their composition. The alignment is guided by inclusion of a complex mixture of proteins that is co-run with the sample. Maleimide-conjugated fluorescent dyes Dy-560 and Dy-635 are used to label the cysteine residues of the sample of interest and the alignment standard, respectively. The two differently labeled mixtures are then combined and separated on a 2D gel and, after selective fluorescence detection, an unsupervised image registration process is used to align the protein patters. In a pilot study, this protocol significantly improved the accuracy of alignment of nuclear proteins with total cellular proteins.

Within the last decade new technologies have been developed and implemented which employ light, often in the presence of a photosensitizer, to inactivate pathogens that reside in human blood products for the purpose of transfusion. These pathogen reduction technologies attempt to find the proper balance between pathogen kill and cell quality. Each system utilizes various chemistries that not only impact which pathogens they can inactivate and how, but also how the treatments affect the plasma and cellular proteins and to what degree. This paper aims to present the various chemical mechanisms for pathogen reduction in transfusion medicine that are currently practiced or in development.

Although cellular proteins conjugated to K48-linked Ub chains are targeted to proteasomes, proteins conjugated to K63-ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48- and K63-ubiquitinated substrates similarly. Therefore, we investigated why K63-ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63-ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48-ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48- or K63-ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal-lysosomal pathway.

Over the past 30 years joint replacement prostheses have been developed and refined to enhance durability and reproducibility. Total hip joint arthroplasty is being performed in an increasing number of younger patients; therefore orthopaedic surgeons seek implants with a longer life span. With regards to the progress of mechanical behaviour of the biomaterials used in an arthroplasty, little is known about the long-term biological effects of wear debris. Owing to the composition of the prostheses currently in use, systemic exposure to chromium (Cr), cobalt (Co), nickel (Ni) and aluminium (Al) alloys occurs as a result of the formation of metal wear nano-particles that are released both from metal-on-metal and polyethylene-on-metal bearings, resulting in a postoperative increase in metal ion levels at different organ sites. These particles circulate both locally and systemically, penetrate cell plasma membranes, bind to cellular proteins and enzymes and modulate cytokine expression. Their physiologic effects are poorly understood and their potential toxicity, hypersensitivity and carcinogenicity remain a cause for concern. In this article we will address the issue of whether these nanoscale degradation products are associated with adverse, clinically significant local or systemic toxicologic sequelae.

'RNA recognition motifs (RRMs)' are common domain-folds composed of 80-90 amino-acid residues in eukaryotes, and have been identified in many cellular proteins. At first they were known as RNA binding domains. Through discoveries over the past 20 years, however, the RRMs have been shown to exhibit versatile molecular recognition activities and to behave as molecular Lego building blocks to construct biological systems. Novel RNA/protein recognition modes by RRMs are being identified, and more information about the molecular recognition by RRMs is becoming available. These RNA/protein recognition modes are strongly correlated with their biological significance. In this review, we would like to survey the recent progress on these versatile molecular recognition modules.

We know where cellular proteins come from, but know very little about where they go. This is a fairly accurate generalization, since a great deal of emphasis has been placed on understanding the synthesis of proteins, the regulation of their synthesis, and if appropriate, their posttranslational modification, assembly into complexes, and insertion into membranes. In contrast, very little attention has been paid to the details of protein turnover—cellular proteins only have a limited lifetime and are somehow degraded to be replaced by new copies, but how? This generalization is certainly applicable to inositol 1,4,5-trisphosphate (IP3) receptors. In summary, it seems that under resting conditions, IP3 receptors are slowly disposed of in a nonspecific manner that may involve trafficking to lysosomes, whereas activated IP3 receptors are rapidly and selectively degraded by the ubiquitin-proteasome pathway. WIREs Membr Transp Signal 2012, 1:126–135. doi: 10.1002/wmts.4 For further resources related to this article, please visit the WIREs website.

Proper cellular proteostasis is essential to cellular fitness and viability. Exogenous stress conditions compromise proteostasis and cause aggregation of cellular proteins. We have developed a fluorogenic sensor (AgHalo) to quantify stress-induced proteostasis deficiency. The AgHalo sensor uses a destabilized HaloTag variant to represent aggregation-prone cellular proteins and is equipped with a series of fluorogenic probes that exhibit a fluorescence increase when the sensor forms either soluble oligomers or insoluble aggregates. Herein, we present protocols that describe how the AgHalo sensor can be employed to visualize and quantify proteome stress in live cells using a direct fluorescence read-out and visualization with a fluorescence microplate reader and a microscope. Additionally, protocols for using the AgHalo sensor in combination with fluorogenic probes and commercially available HaloTag probes to enable two-color imaging experiments are described. These protocols will enable use of the AgHalo sensor to visualize and quantify proteostasis in live cells, a task that is difficult to accomplish using previous, always-fluorescent methods. © 2018 by John Wiley & Sons, Inc.

Modern cells present no signs of a putative prebiotic RNA world. However, RNA coding is not a sine qua non for the accumulation of catalytic polypeptides. Thus, cellular proteins spontaneously fold into active structures that are resistant to proteolysis. The law of mass action suggests that binding domains are stabilized by specific interactions with their substrates. Random polypeptide synthesis in a prebiotic world has the potential to initially produce only a very small fraction of polypeptides that can fold spontaneously into catalytic domains. However, that fraction can be enriched by proteolytic activities that destroy the unfolded polypeptides and regenerate amino acids that can be recycled into polypeptides. In this open system scenario the stable domains that accumulate and the chemical environment in which they are accumulated are linked through self coding of polypeptide structure. Such open polypeptide systems may have been the precursors to the cellular ribonucleoprotein (RNP) world that evolved subsequently.

Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.

Many cellular proteins exist as homo-oligomers. The mechanism of the assembly process of such proteins is still poorly understood. We have previously observed that Hsp16.3, a protein exhibiting chaperone-like activity, undergoes stepwise disassembly and nonstepwise reassembly. Here, the disassembly and reassembly of a nonchaperone protein RbsD, from Escherichia coli, was studied in vitro. The protein was found to mainly exist as decamers with a small portion of apparently larger oligomeric forms, both of which are able to refold/reassemble effectively in a spontaneous way after being completely unfolded. Disassembly RbsD intermediates including pentamers, tetramers, trimers, dimers, and monomers were detected by using urea-containing pore gradient polyacrylamide gel electrophoresis, while only pentamers were detected for its reassembly. The observation of stepwise disassembly and apparent nonstepwise reassembly for both a chaperone protein (Hsp16.3) and a nonchaperone protein (RbsD) strongly suggests that such a feature is most likely general for homo-oligomeric proteins.

The degradation of the majority of cellular proteins is mediated by the proteasomes. Ubiquitin-dependent proteasomal protein degradation is executed by a number of enzymes that interact to modify the substrates prior to their engagement with the 26S proteasomes. Alternatively, certain proteins are inherently unstable and undergo “default” degradation by the 20S proteasomes. Puzzlingly, proteins are by large subjected to both degradation pathways. Proteins with unstructured regions have been found to be substrates of the 20S proteasomes in vitro and, therefore, unstructured regions may serve as signals for protein degradation “by default” in the cell. The literature is loaded with examples where engagement of a protein into larger complexes increases protein stability, possibly by escaping degradation “by default”. Our model suggests that formation of protein complexes masks the unstructured regions, making them inaccessible to the 20S proteasomes. This model not only provides molecular explanations for a recent theoretical “cooperative stability” principle, but also provokes new predictions and explanations in the field of protein regulation and functionality. BioEssays 28: 844–849, 2006. © 2006 Wiley Periodicals, Inc.

Summary In this study, an unusual observation of Saccharomyces cerevisiae isolated from an infected root canal and from the blood of a patient undergoing endodontic therapy of a tooth with asymptomatic apical periodontitis is reported. Phenotypic (biochemical tests, antifungal susceptibility tests and SDS-PAGE of cellular proteins) and genetic (ribotyping) methods were used to characterize the strains. By using these methods it was found that the blood and root canal isolates were identical but differed from S. cerevisiae strains of other sources. It was therefore more than likely that the root canal was the source of the blood isolate and that it had been transferred unintentionally to the bloodstream during root canal treatment.

While much is known about protein folding in buffers, current methodology is limited in probing how protein folding in vivo is influenced by the cellular proteostasis network quantitatively, particularly with regard to partitioning client proteins between folded and functional, soluble and misfolded, and aggregated conformational states in the face of genetic and environmental stresses. Herein we develop protein-of-interest folding probes that specifically recognize the functional and folded fraction of the protein-of-interest, , and serve to quantify the amount of folded and functional protein in cell lysate. Rapid depletion of ATP levels during cell lysis minimizes folding equilibria shifts by creating sufficient cellular chaperone holdase activity. The folding probes strategy is exemplified with retroaldolase (RA), a de novo designed enzyme, and transthyretin, a tetrameric plasma protein. This strategy revealed that partitioning of RA and TTR mutants between the aforementioned conformational states was...

Despite much progress over the last six decades, we are still far from understanding the mechanism of protein folding in vitro and, most importantly, in the cell. This lack of information poses tremendous challenges to progress in many areas of fundamental biology, biotechnology, protein design, drug production and neurodegenerative-disease research. Most soluble cellular proteins fold co- and immediately post-translationally and the ribosome serves as an obligatory host for this process. A compelling unanswered question is whether the ribosome plays any active role in protein folding in the cellular environment. This lecture will discuss how the ribosomal surface and its peculiar electrostatic properties, together with the vectorial nature of protein synthesis, modulate the balance between protein folding and aggregation during the crucial stages of protein birth.

Two-dimensional polyacrylamide gel electrophoresis 1 (2-D PAGE 1) is currently the method of choice for separating complex mixtures of cellular proteins. Despite its usefulness, 2-D PAGE is not being applied to its full potential because of difficulties with both the method and analysis of the results. One of the key problems is the difficulty and slowness of image analysis, especially registration (image alignment), which is laborious and the results unsatisfactory. We have developed a novel system for analysis of 2-D PAGE images, called Z3, that performs the analysis faster and more precisely. The Z3 system employs novel approaches to image registration, image display, computation of differential expression, and the design and analysis of 2-D gel experiments. This paper describes in detail the registration algorithm, and briefly discusses the merits of complementary pseudocolor display. The registration algorithm is novel in that for the first time raw-image-based registration technology is applied to 2-D gel analysis.

In order to evaluate the role of protein synthesis in apoptosis, (13)C-NMR has been used to study the levels of protein synthesis in three different human leukemic cell lines in the presence and absence of dexamethasone-induced apoptosis. Measurements were done on one dexamethasone-sensitive (CEM-C7-14) and two different dexamethasone-resistant variants (CEM-4R4 and CEM-ICR27-4). The incorporation of (13)C-labeled amino acids into cellular proteins, which reflects the level of new protein synthesis, was monitored by (13)C-NMR spectroscopy. In the absence of dexamethasone, the level of protein synthesis was found to be significantly different among the three cell lines. Dexamethasone caused a significant reduction ( congruent with 60-87%) in the level of protein synthesis in dexamethasone-sensitive CEM-C7-14 cells, while having no significant effect on protein synthesis in dexamethasone-resistant CEM-4R4 cells. Dexamethasone treatment caused a significant enhancement of the level of protein synthesis in the CEM-ICR27-4 cells. Synthesis of proteins was found to occur during apoptosis, albeit at a low level, suggesting a role for the synthesis of specific proteins in the mechanism of apoptosis.

Two-dimensional (2-D) polyacrylamide gel electrophoresis has much to contribute to experimental analysis of the proteomes of microbial organisms, since this method separates most cellular proteins and allows synthesis rates to be determined quantitatively. Databases generated using 2-D gels can grow to be very large from even just a few experiments, since each sample provides the data for a field (or column) in the database for several hundreds to even thousands of records (or rows), each of which represents a single polypeptide species. The value of such databases for generating an encyclopedia of how each of the cell's proteins behave in different conditions (protein phenotypes) has been recognized for some time. The potential exists, however, to glean even more valuable information from such databases. Because the measurements of each protein are made in the context of all other proteins, a comprehensive glimpse of the cell's physiological state is theoretically achievable with each 2-D gel. By examining enough conditions (and 2-D gels), expression patterns of subsets of proteins (proteomic signatures) can be found that correlate with the cell's state. This type of information can provide a unique contribution to proteomic analysis, and should be a major focus of such analyses.

Plasma Free Amino Aoids in Workers Working under Different Stress Conditions: Tang-Chun Wu et al. Institute of Occupational Medicine, Tongji Medical University-Many harmful occupational factors induce the stress response and result in both the induction of heat stress proteins and the inhibition of the synthesis of other cellular proteins. Since amino acid levels are intimately linked to protein synthesis, we examined the free amino acid levels in the plasma of workers subjected to prolonged exposure to heat, carbon monoxide (CO) or both. The results showed that the levels of most amino acids tended to increase in the plasma of workers in stressful environments, and that free methionine and tryptophan increased significantly (p

Human immunodeficiency virus type-1 (HIV-1) virions contain both virus-encoded and cellular proteins. Recent advances in the detection, isolation, and functional characterisation of host proteins incorporated in the virion have begun to provide for new perspectives on the interactions between virus and cell. The acquisition of host proteins by HIV-1 may also influence viral pathology in vivo. This article reviews detection and analysis of host proteins found in HIV-1 particles and presents some potential roles that these proteins might play in the biology of this important virus. Copyright 1997 John Wiley & Sons, Ltd.

Chemical modification of backbone structures has been an important strategy in designing oligonucleotides capable of improved antisense effects. However, altered backbone chemistry may also affect the binding of oligonucleotides to key cellular proteins, and thus may impact on the overall biological action of antisense agents. In this study we have examined the binding of oligonucleotides having four different backbone chemistries to single-strand binding protein (SSB), a protein having a key role in DNA repair and replication. The oligomers tested had the same sequence, while the internucleoside linkages were phosphodiester (PO), phosphorothioate (PS), phosphorodithioate (PS2), or methylphosphonate (MP). We found that both PS and PS2 oligomers bound to SSB with higher affinity than PO oligonucleotides, while MP oligonucleotides did not bind appreciably at the concentrations tested. Oligonucleotide length was also an important factor in binding to SSB, but sequence was less critical. These observations indicate that backbone chemistry is an important factor in interactions between oligonucleotides and critical cellular proteins, and thus may be a key determinant of the biological effects of antisense oligonucleotides. © 1997 John Wiley & Sons, Ltd.

Picornaviral proteinases are responsible for maturation cleavages of the viral polyprotein, but also catalyze the degradation of cellular targets. Using graphical visualization techniques and neural network algorithms, we have investigated the sequence specificity of the two proteinases 2Apro and 3Cpro. The cleavage of VP0 (giving rise to VP2 and VP4), which is carried out by a so-far unknown proteinase, was also examined. In combination with a novel surface exposure prediction algorithm, our neural network approach successfully distinguishes known cleavage sites from noncleavage sites and yields a more consistent definition of features common to these sites. The method is able to predict experimentally determined cleavage sites in cellular proteins. We present a list of mammalian and other proteins that are predicted to be possible targets for the viral proteinases. Whether these proteins are indeed cleaved awaits experimental verification. Additionally, we report several errors detected in the protein databases. A computer server for prediction of cleavage sites by picornaviral proteinases is publicly available at the e-mail address NetPicoRNA@cbs.dtu.dk or via WWW at http:@www.cbs.dtu.dk/services/NetPicoRNA/.

Gas chromatographic analysis of cellular fatty acids (CFA), biochemical reactions, electrophoresis of soluble cellular proteins, as well as immunodiffusion were used to discriminate between 16 fresh spirochete isolates from periodontal and endodontic infections. CFA patterns were compared by the Hewlett Packard MIDI library to all available reference strains, and results of the biochemical tests were compared to VPI's records for treponemal strains. The electrophoretograms of soluble proteins of the fresh isolates were compared to those of previously well described strains of Treponema denticola, T. pectinovorum, T. vincentii, T. socranskii subsp. socranskii, T. socranskii subsp. paredis, T. socranskii subsp. buccale, and T. socranskii 04. Immunodiffusion was carried out by using adsorbed polyclonal rabbit antibodies to representative strains of the species mentioned. These methods separated most clinical strains from approved species strains, suggesting that new species had been isolated.

Encouraging results obtained for modulation of gene expression by antisense oligonucleotides and their analogues have kindled hopes for a new generation of therapeutics against viral infections, cancer, and many other diseases. Among such analogues, oligo(nucleoside phosphorothioate)s (Oligo-S) have generally shown the highest efficacy in inhibiting the biosynthesis of “unwanted” proteins. The first clinical trials of antisense agents are now in progress using Oligo-S against genital warts and acute myeloid leukemia, and tests of Oligo-S against AIDS should follow soon. Nevertheless, their mechanism of action, internalization, cellular trafficking, subcellular localization, and interaction with cellular proteins is still poorly understood. It is assumed a priori that application involves rapid and efficient molecular recognition of target RNA by Oligo-S; however, the effects of the chirality of Oligo-S have so far been unappreciated, because Oligo-S has not yet been synthesized with stereocontrol. Indeed, the diastereomeric composition of Oligo-S has never been determined, primarily because of the lack of appropriate analytical methods. Since each of the diastereomers is a stereochemically unique chemical entity, questions arise as to which diastereomer is responsible for an observed biological response, including positive (curative) or possibly negative (toxic) side effects. In this review we intend provide a perhaps somewhat speculative assessment of the problems associated with the stereo-controlled synthesis of Oligo-S and to discuss the state-of-the-art in this field including strategies that may lead to Oligo-S of predetermined chirality. This article is not intended to discourage researchers from further studies of dia-steromeric mixtures of Oligo-S as potential pharmaceuticals. Throughout the history of medicinal chemistry numerous useful medicines were discovered, developed, and employed without the detailed knowledge of their structure. Indeed, the composition of the vaccines discovered by Pasteur is a subject of vigorous study still today.

The identification of small molecule ligands for cellular proteins of biologically importance has not only provided new insights in biology, but has also provided organic chemists with the opportunity to create new synthetic molecules which efficiently regulate specific protein-protein interactions and biological processes. Small-molecule microarrays are recently developed as platforms to screen such small molecule ligands for proteins of interest. The small-molecule microarray technology is highly dependent on chemistry to immobilize small molecules on a variety of solid surfaces, and synthetic organic chemistry will continue to play a significant role in this field. This review covers the recent advances in the field with particular emphasis on the immobilization chemistry, as well as technical advances in the platform optimization and the successful applications of this platform.

A large fraction of cellular proteins are oligomeric. Protein oligomerization may often be an advantageous feature from the perspective of protein evolution and has probably evolved by a variety of mechanisms. The study of protein oligomerization may provide insights into the early protein environment and the evolution of modern proteins. Oligomeric mini-proteins, short peptides with discrete protein-like structures, may serve as valuable models for understanding features of protein oligomerization.

Genomic integrity is maintained by a network of cellular activities that assesses the status of the genome at a given point in time and that provides signals to proceed with or to halt cell cycle progression. Recent studies have identified cellular proteins that are the targets for the viral oncoproteins involved in these processes. We demonstrate that the expression of human papilloma virus type 16 E6 and E7 oncoproteins in normal mortal cells disrupts the integration of the network of signals that maintain genomic integrity. © 1995 Wiley-Liss, Inc.

Procedures detailing the production of monoclonal antibody supernatants, including the production of larger amounts (liters), are presented here. The protocol for large-scale production of hybridomas or cells (e.g., for isolation of cellular proteins) involves a similar procedure. A method for producing and obtaining ascites fluid containing the monoclonal antibody is also presented. The unit ends with a set of recommendations developed by the Committee on Methods of Producing Monoclonal Antibodies (Institute of Laboratory Animal Research, National Research Council). The recommendations are designed to foster the judicious use of animals for the production of monoclonal antibodies and to strongly encourage the use of in vitro methods whenever possible.

Studies of the behavior of many ligand-gated ion channels have long provided evidence that more than one molecule of ligand was able to bind to each receptor complex, a phenomenon termed cooperativity. The conclusion that some receptors possessed more than one binding site for ligands thus invoked another phenomenon that was originally described in the field of enzymology, that is, the concept of allosteric binding sites. The biological activity of enzymes can be modified, either in a positive or negative fashion, by the binding of ligands to sites that are topographically distinct from the substrate-binding site. These accessory binding sites are termed allosteric sites, in contrast to the substrate-binding (active) site (the isosteric site). Thus, allosteric interactions arise because the binding of a ligand to the allosteric site induces a conformational change in the protein that modulates the binding of the substrate to the isosteric site, and vice versa. The biological activity of the enzyme arises from the subsequent (modified) properties of the substrate-binding site, and not through a direct effect of the allosteric modulator itself. This overview unit discusses definitions of various terms used in the field of receptor allosterism, and explains in clear terms how allosteric transitions and interactions can affect receptor activation. In addition, the functions of allosteric modulators are discussed along with explanations of how these modulators can be attractive tools for drug development.

Immunochemical detection of xenobiotics covalently bound to cellular proteins can provide information about toxic mechanism and is more specific than the alternative radiochemical studies. Both immunoblotting and immunohistochemical methods are used to pinpoint the target protein(s) and to identify the tissue targets. Also included in this unit are protocols for synthesizing artificial antigens, immunizing suitable host species, and using noncompetitive and competitive ELISA assays to characterize the antibodies produced.

Genetic polymorphism of human lymphocyte proteins was studied by two-dimensional electrophoresis in the members of 12 families and in additional 20 unrelated individuals from Southern Germany. Cell lysates of phytohemagglutinin-stimulated, [3H-]leucine labeled peripheral lymphocytes were subjected to two-dimensional electrophoresis. Among the approximate 300 labeled proteins on two-dimensional fluorograms we found six polymorphic proteins. Five proteins, named p23 (Mr 23 000), p30 (Mr 30 000), p40 (Mr 40 000), p60 (Mr 60 000) and p66 (Mr 66 000), existed as two charge variants, an acidic (a) and a basic (b) variant. Three different electrophoretic phenotypes, two homozygous (a and b) and one heterozygous (ab), could be observed for the proteins p23, p40 and p66. The proteins p30 and p60 occurred in our population sample only in two phenotypes, a common homozygous (a) and a rare heterozygous (ab) phenotype. Preliminary results are presented for the sixth polymorphic protein p75 (Mr 75 000), which occurred in at least three or even more different electrophoretic variants, resulting in six or more phenotypes. The genetic basis of the protein variations was confirmed by the quantitative gene-dosage dependence, the identity of the protein patterns in three monozygotic twin pairs and the transmission of the variants as Mendelian traits within the 12 families examined. Gene frequencies of the rare alleles of the five proteins which occurred in two charge variants ranged between 0.012 and 0.477 in a sample of 44 unrelated individuals from Southern Germany. Thus, the term genetic polymorphism is justified.

Human peripheral blood lymphocytes were isolated from healthy donors and cultivated in the absence and presence of phytohemagglutinin (PHA) or pokeweed mitogen (PWM). Cell lysates of unlabelled lymphocytes or following stimulation of [H]leucine metabolically labelled lymphocytes were subjected to two-dimensional electrophoresis. Differences in the protein patterns are described which are related to the method applied for visualization of the proteins (silver staining or fluorography), to the culture conditions (unstimulated, PHA or PWM stimulated lymphocytes) and to individual variations. Comparative analysis of the resulting patterns on silver stained gels and fluorograms revealed three qualitative and several quantitative differences. Comparing the two-dimensional patterns of unstimulated and mitogenstimulated lymphocytes with silver staining, we recognized the quantitative increase of 6 protein spots after stimulation. Whereas protein patterns of PHA and PWM stimulated cells showed only minor quantitative differences when compared in silver stained gels, one qualitative and two quantitative differences between the two mitogens could be observed in fluorograms. Individual variability was generally low: within the approximately 30 unrelated individuals examined, thus far, the presence of two polymorphic proteins was demonstrated.

This review is concerned with rates of N flux in the living animal rather than with mechanisms of protein synthesis and breakdown at the cellular level. Methods of measuring protein turnover in the whole body are discussed, with special emphasis on studies in man, and results obtained by different methods have been compared. Aspects of whole body protein turnover which are of physiological interest include its relation to body size, growth and development, energy metabolism and food intake. There are substantial increases in protein turnover in injury, and changes that occur in exercise are beginning to be explored. From the physiological point of view these results point the need for future research along two main lines. The first is that of regulation: a wide variety of hormones stimulate or repress protein synthesis and breakdown, with varying actions in different tissues. These effects, however, do not in themselves explain the mechanism by which a balance between synthesis and breakdown is maintained. Secondly, the fact that all cellular proteins are in a dynamic state poses questions about the relation between structure and function in tissues such as muscle and brain, which physiologists have hardly begun to tackle.

Exposure of chick myotube cultures to a temperature (45 degrees C) higher than their normal growing temperature (37 degrees C) caused extensive synthesis of three major polypeptides of Mr = 25 000, 65 000 and 81 000 referred to as 'heat-shock polypeptides' (hsps). When these cells were allowed to recover from heat-shock treatment at 37 degrees C for 6-8 h, the rate of accumulation of isotope into the 65 000-Mr and 81 000-Mr hsps declined to levels comparable to those in control cultures maintained at 37 degrees C. However, incorporation of isotope in the 25 000-Mr hsp continued at an elevated rate for a longer period than the 65 000-Mr and 81 000-Mr hsps. When heat-shocked cells were allowed to recover at 37 degrees C in the presence of actinomycin D to block new mRNA synthesis, the hsp synthesis as measured by the incorporation of radioactive isotope in these polypeptides continued at levels comparable to those in heat-shocked cells prior to recovery. The block of recovery by actinomycin D was due to the presence of a greater amount of functional hsp mRNAs in the polysomes as compared to untreated controls. The role of competition between the mRNAs for hsps and normal cellular proteins for the translation machinery in regulating protein synthesis during the recovery from heat shock has been discussed.

We have investigated the qualitative and quantitative differences in the two-dimensional (2-D) gel fluorographic patterns of biosynthetically pulse-labeled cellular proteins from the malignant cells of patients with various leukemias (acute myelogenous, acute lymphoblastic, chronic lymphocytic, and hairy cell). Significant similarities and differences between the major types of leukemias were found. An initial small data base was constructed using our GELLAB computer analysis system for 2-D gels in order to define methods for data base definition, normalization, and visual verification of results. Search strategies to find significant spot differences, both qualitative and quantitative, between currently accepted classes of leukemias were explored. Potential methods of expressing in a summary statistic the overall similarities and differences between gels were also investigated. A series of proteins whose relative synthetic rates may serve as potential markers of states of differentiation in human leukemias were identified in this initial data base and are reported here.

Summary Evidence is presented that in the malignant cell of rat liver tumours a highly specific cellular protein has either been lost or has been modified; this is judged by the cell's control the growth of tissues and organs and, in ordinary circumstances, limit the proliferation of tissue cells. The significance of these observations in human neoplasms is as yet uncertain. However, this is being studied and if the staining technique should prove applicable it will be not only a valuable method of investigating tumours but possibly also important in differentiating tumour-like masses from true neo-plasms.

The oxidized tannin-azo (OTA) method was used to study the location of tannophilic protein in hepatic cells. In this method tannic acid was first attached to the tissue proteins and then oxidized with periodic acid; the oxidized tannin, which was firmly bound to the cellular proteins, was subsequently converted to a highly coloured azo dye by coupling with diazotized o-dianisidine. Preliminary deamination with nitrous acid markedly diminished the capacity of hepatic cells to stain with OTA; this indicates that -NH3 groups in the tissue proteins are the principal loci revealed by the OTA method. Sections of liver, after tanning and oxidation, were also treated with 6-amino-3-dimethylaminophenol; this technique produced intense colouration of the hepatic cells probably owing to the formation of an oxazine dye. The colouration of oxidized tannin by 6-amino-3-dimethylaminophenol confirms the view that bound tannin is oxidized by periodic acid to a 1–2-quinone.