The mechanism of glutamine-dependent amidotransferases

Glutamine-dependent amidotransferases have been known for more than 30 years. The mechanism by which these enzymes generate ammonia from the glutamine amide nitrogen and transfer it to seven different chemical classes of acceptors has been the subject of intense scrutiny for the last 5 years. The increasing number of biochemical and structural studies dealing with amidotransferases and with mechanistically related enzymes has disclosed the dichotomy of the mechanisms within these enzymes for achieving the glutamine amide bond cleavage. Some of them use a catalytic Cys/His/Glu triad similar to serine protease, whereas the aminoterminal cysteine of the others is believed to play the same function. The transfer of ammonia from the glutamine site to the acceptor site which must operate in a concerted manner has been demonstrated in two cases to involve channelling but is still matter of investigation.     

Identification of the bioactive peptide PEC-60 in brain

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60may play a role in the central nervous system disorders involving dopamine dysregulation. Received 6 December 2002; received after revision 10 December 2002; accepted 11 December 2002 RID="*" ID="*"Corresponding author.     

Differential display analysis of gene expression in invertebrates

Screening for differentially expressed genes is a straightforward approach to study the molecular basis for changes in gene expression. Differential display analysis has been used by investigators in diverse fields of research since it was developed. Differential display has also been the approach of choice to investigate changes in gene expression in response to various biological challenges in invertebrates. We review the application of differential display analysis of gene expression in invertebrates, and provide a specific example using this technique for novel gene discovery in the nematode Caenorhabditis elegans.     

CD100 is a leukocyte semaphorin

CD100 was originally described as an activation molecule on the surface of human T lymphocytes. Its triggering through distinct epitopes leads to different signals of costimulation with phorbol myristate acetate (PMA) or with CD3 and CD2. Interestingly, CD100 was shown to associate with different partner molecules in T cells. First, CD100 can associate with CD45, a key molecule with protein tyrosine phosphatase activity involved in T-cell transduction this association is physical and has functional consequences for both partners. Second, CD100 interacts in its cytoplasmic domain with a Ser/Thr kinase for which it represents a preferential substrate. Recently, CD100 was identified as a member of the semaphorin gene family. This family comprises approximately 20 structurally related proteins. The first semaphorins were identified in the developing nervous system. Function has been shown for only some of them and involves repulsion during growth cone guidance. Since CD100 was the first semaphorin identified in the immune system, this raises the possibility of the involvement of members of the semaphorin family in other physiological phenomena outside the nervous system. Received 1 March 1998; received after revision 8 June 1998; accepted 8 June 1998     

Water in enzyme reactions: biophysical aspects of hydration-dehydration processes

Water has been recognized as one of the major structuring factors in biological macromolecules. Indeed, water clusters influence many aspects of biological function, and the water-protein interaction has long been recognized as a major determinant of chain folding, conformational stability, internal dynamics, binding specificity and catalysis. I discuss here several themes arising from recent progress in understanding structural aspects of ‘direct’ and ‘indirect’ ligands in terms of enzyme-substrate interactions, and the role of water bridges in enzyme catalysis. The review also attempts to illuminate issues relating to efficiency, through solvent interactions associated with enzymic specificity, and versatility. Over the years, carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) has played a significant role in the continuing delineation of principles underlying the role of water in enzyme reactions. As a result of its pronounced catalytic power and robust constitution CA was transformed into a veritable ‘laboratory’ in which active site mechanisms were rigorously tested and explored.     

Strategies for the inhibition of serine proteases

Serine proteases have been shown to play a multifarious role in health and disease. As a result, there has been considerable interest in the design and development of synthetic inhibitors of these enzymes. In view of their diverse roles in biological processing events, one of the great challenges in such endeavours has been the need to produce compounds with exquisite selectivity. Inhibitor design has been broadly guided by the use of either peptide- or heterocyclic-based compounds, designed to exploit the known substrate specificity characteristics of individual enzymes. This review describes the thinking and strategies employed in such efforts. Received 8 August 2000; received after revision 16 November 2000; accepted 17 November 2000     

The role of thrombospondin-1 in apoptosis

The thrombospondins are a family of extracellular proteins that participate in cell-to-cell and cell-to-matrix communication. They regulate cellular phenotype during tissue genesis and repair. Five family members, each representing a separate gene product, probably exist in most vertebrate species. Like most extracellular proteins, the thrombospondins are composed of several structural domains that are responsible for the numerous biological functions that have been described for this protein family. Considerable progress has been made towards understanding the function of thrombospondins. The role of thrombospondin in the process of apoptosis or programmed cell death has recently come into focus. In this review we will concentrate on the role of thrombospondin-1 in the broad field of apoptotis research. Received 5 December 2001; received after revision 28 March 2002; accepted 28 March 2002     

Chromosome 17-linked dementias

Chromosome 17-linked dementias have been defined by linkage analysis. The most common of these syndromes has been estimated to be the cause of between 2 and 20% of all dementia and has alternately been called frontotemporal dementia, Pick's disease (without Pick bodies) and dementia lacking distinctive features [1 – 3]. The identification of the mutation responsible for these conditions in a group of clinically and pathologically heterogeneous disorders may allow us to gain broad insight into the processes of neurodegeneration.     

Hormone-induced subcellular redistribution of trimeric G proteins

Trimeric guanine nucleotide-binding proteins (G proteins) function as the key regulatory elements in a number of transmembrane signaling cascades where they convey information from agonist-activated receptors to effector molecules. The subcellular localization of G proteins is directly related to their functional role, i.e., the dominant portion of the cellular pool of G proteins resides in the plasma membrane. An intimate association of G protein subunits with the plasma membrane has been well known for a long time. However, results of a number of independent studies published in the past decade have indicated clearly that exposure of intact target cells to agonists results in subcellular redistribution of the cognate G proteins from plasma membranes to the light-vesicular membrane fractions, in internalization from the cell surface into the cell interior and in transfer from the membrane to the soluble cell fraction (high-speed supernatant), i.e., solubilization. Solubilization of G protein α subunits as a consequence of stimulation of G protein-coupled receptors (GPCRs) with agonists has also been observed in isolated membrane preparations. The membrane-cytosol shift of G proteins was detected even after direct activation of these proteins by non-hydrolyzable analogues of GTP or by cholera toxin-induced ADP-ribosylation. In addition, prolonged stimulation of GPCRs with agonists has been shown to lead to down-regulation of the relevant G proteins. Together, these data suggest that G proteins might potentially participate in a highly complex set of events, which are generally termed desensitization of the hormone response. Internalization, subcellular redistribution, solubilization, and down-regulation of trimeric G proteins may thus provide an additional means (i.e., beside receptor-based mechanisms) to dampen the hormone or neurotransmitter response after sustained (long-term) exposure. Received 31 August 2001; received after revision 31 October 2001; accepted 7 November 2001     

Structural studies of a phosphatidyl serine-amorphous calcium phosphate complex

A phosphatidyl serine-amorphous calcium phosphate complex has been synthesized as a model of the matrix vesicle system that is associated with the induction of mineral deposition in bone, cartilage and dentine. The complex has been studied using a novel technique of subtractive extended X-ray absorption fine structure (EXAFS). This enables spectra of the components of the molecules to be subtracted from the complex so as to identify the sites of interaction. The results suggest there is a movement in the nitrogen atom of the phosphatidyl serine which approaches the calcium atom in the mineral phase. This interpretation would link the membrane structure of the vesicle to the structure of the mineral in a way that could explain some of its roles in biomineralization. Received 14 November 1997; accepted 23 December 1997     

Three-dimensional structure of annexins

Annexins constitute a family of structurally related calcium- and phospholipid-binding proteins whose molecular structure has been investigated in detail in the crystalline and membrane-bound form. Their polypeptide chain is folded into four or eight α-helical domains of similar structure with a central hydrophilic pore. Bound to phospholipid membranes, the four-domain arrangement of the annexin molecule is conserved. A peripheral binding mode has been well documented by electron microscopy and a variety of other techniques.     

SecB,one small chaperone in the complex milieu of the cell

SecB is only one of a plethora of cytosolic chaperones in E. coli whose common property is that they bind nonnative proteins. It plays a crucial role during protein export via the general secretory pathway by modulating the partitioning of precursors between folding or aggregation and delivery to the membrane-bound translocation apparatus. In this latter role SecB demonstrates specific binding to a unique partner, SecA. SecB has the potential to participate in functions outside of export acting as a general nonspecific chaperone to provide buffering capacity of the nonnative state of proteins in the cytosolic pool. We discuss the interactions of SecB with its many binding partners in light of its recently determined structure, emphasizing both kinetic and thermodynamic parameters. RID="*" ID="*"Corresponding author.     

The structure and function of Escherichia coli penicillin-binding protein 3

Escherichia coli penicillin-binding protein PBP3 is a key element in cell septation. It is presumed to catalyse a transpeptidation reaction during biosynthesis of the septum peptidoglycan but, in vitro, its enzymatic activity has only been demonstrated with thiolester analogues of the natural peptide substrate. It has no detectable transglycosylase activity with lipid II as substrate. This tripartite protein is constructed of an N-terminal membrane anchor-containing module that is essential for cell septation, a non-penicillin-binding (n-PB) module of unknown function and a C-terminal penicillin-binding (PB) module exhibiting all the characteristic motifs of penicilloyl serine transferases. The n-PB module, which is required for the folding and stability of the PB module, may provide recognition sites for other cell division proteins. Initiation of septum formation is not PBP3-dependent but rests on the appearance of the FtsZ ring, and is thus penicillin-insensitive. The control of PBP3 activity during the cell cycle is briefly discussed.     

T-cell signal transduction and the role of protein kinase C

The T lymphocyte has a vital part to play in maintaining the host response to bacterial and viral infection and also appears to play a key pathological role in autoimmune diseases such as rheumatoid arthritis. In this review, we summarize the signalling pathways which trigger antigen-driven T-cell proliferation and examine the evidence which suggests that protein kinase C (PKC) is fundamental to this process. Finally, we discuss the therapeutic potential that PKC inhibitors may have in the treatment of autoimmune disease. Received 31 March 1998; received after revision 19 May 1998; accepted 19 May 1998     

Protein farnesylation in mammalian cells: effects of farnesyltransferase inhibitors on cancer cells

Protein farnesylation, catalyzed by protein farnesyltransferase, plays important roles in the membrane association and protein-protein interaction of a number of eukaryotic proteins. Recent development of farnesyltransferase inhibitors (FTIs) has led to further insight into the biological significance of farnesylation in cancer cells. A number of reports point to the dramatic effects FTIs exert on cancer cells. In addition to inhibiting anchorage-independent growth, FTIs cause changes in the cell cycle either at the G1/S or at the G2/M phase. Furthermore, induction of apoptosis by FTIs has been reported. FTIs also affects the actin cytoskeleton and cell morphology. This review summarizes these reports and discusses implications for farnesylated proteins responsible for these FTI effects. Received 17 April 2001; received after revision 28 May 2001; accepted 28 May 2001     

Introduction: molecular and biomechanical basis of osteoarthritis

Osteoarthritis has developed into the most common chronic disease in the highly industrialized nations. Moreover, because of the prevalence of the disease in the elderly, this trend occurs worldwide as a consequence of increasing longevity due to the overall improvement in living conditions and health status. In contrast, research on osteoarthritis is still financially marginalized within biomedical research, so that the molecular and biophysical bases for disease initiation and progression are largely unmapped. The following sequence of five reviews highlights a remarkable change in that body of knowledge taking place at the beginning of the World Health Organization (WHO) 'Bone and Joint Decade 2001-2010'. The data and ideas presented in these articles reflect to some extent the guidelines set up by the WHO and by the National Institutes of Health of the USA and therefore allow a glimpse into the directions that research in osteoarthritis will take in the future.     

Recent advances in the genetics of schizophrenia

The genetic etiology of schizophrenia, a common and debilitating psychiatric disorder, is supported by a wealth of data. Review of the current findings suggests that considerable progress has been made in recent years, with a number of chromosomal regions consistently implicated by linkage analysis. Three groups have shown linkage to 1q21-22 using similar models, with HLOD scores of 6.5, 3.2, and 2.4. Other replicated loci include 13q32 that has been implicated by two independent groups with significant HLOD scores (4.42) or NPL values (4.18), and 5p14.1-13.1, 5q21-33, 8p21-22, and 10p11-15, each of which have been reported as suggestive by at least three separate groups. Different studies have also replicated evidence for a modest number of candidate genes that were not ascertained through linkage. Of these, the greatest support exists for the DRD3 (3q13.3), HTR2A (13q14.2), and CHRNA7 (15q13-q14) genes. The refinement of phenotypes, the use of endophenotypes, reduction of heterogeneity, and extensive genetic mapping have all contributed to this progress. The rapid expansion of information from the human genome project will likely further accelerate this progress and assist in the discovery of susceptibility genes for schizophrenia. A greater understanding of disease mechanisms and the application of pharmacogenetics should also lead to improvements in therapeutic interventions. Received 11 May 2001; received after revision: 20 July 2001; accepted 18 September 2001     

Structural view of cadherin-mediated cell-cell adhesion

Following the multiplication of biochemical, biophysical and structural studies describing cadherin molecules and their interactions, several ideas have emerged to explain the mechanisms of cadherin-mediated cell adhesion. Although different models were proposed for cadherin interactions, a consensus has come forth considering lateral dimerization of cadherins as being a central component of the cell-cell adhesion process. This review summarizes the recent development in structural studies of cadherins. Received 14 September 1998; received after revision 14 November 1998; accepted 16 November 1998