Biomedical subjects
Publications and source records attributed to A Sali.
Three cases with foot drop following brain tumors in patients suffering from parasagittal pathology are reported. Foot drop was the first complaint in the patients in our series. We generally notice foot drop as a sign of lumbar disc herniation or peroneal nerve lesions but rarely foot drop may also occur with brain lesions.
Many developmentally regulated membrane proteins of lymphocytes are ecto-enzymes, with their active sites on the external surface of the cell. These enzymes commonly have peptidase, phosphodiesterase or nucleotidase activity. Their biological roles are just beginning to be discovered. Although their expression is usually associated with particular stages of lymphoid differentiation, the same gene products are often expressed on the surface of certain non-lymphoid cell types outside the immune system, indicating that their functions cannot be unique to lymphocytes, nor can they be ubiquitous. The plasma cell membrane protein PC-1 (phosphodiesterase I; EC 3.1.4.1/nucleotide pyrophosphatase; EC 3.6.1.9), which was one of the first serological markers for lymphocyte subsets to be discovered, is a typical example. Within the immune system, PC-1 is confined to plasma cells, which represent about 0.1% of lymphocytes. However, PC-1 is also expressed on cells of the distal convoluted tubule of the kidney, chondrocytes, osteoblasts, epididymis and hepatocytes. Recent work has shown that PC-1 is a member of a multigene family of ecto-phosphodiesterases that currently has two other members, PD-1 alpha (autotaxin) and PD-1 beta (B10). Within this family, the extracellular domains are highly conserved, especially around the active site. In contrast, the transmembrane and cytoplasmic domains are highly divergent. Individual members of the eco-phosphodiesterase family have distinct patterns of distribution in different cell types, and even within the same cell. For example, PC-1 is present only on the basolateral surface of hepatocytes, while B10 (PD-1 beta) is confined to the apical surface. Analysis of conservation and differences in the sequence of their cytoplasmic tails may illuminate intracellular targetting signals. Ecto-phosphodiesterases may play a part in diverse activities in different tissues, including recycling of nucleotides. They may also regulate the concentration of pharmacologically active extracellular compounds such as adenosine or its derivatives and cell motility. Some members may modulate local concentrations of pyrophosphate, and hence influence calcification in bone and cartilage.
Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used this in vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34-55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the alpha-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptase-specific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the alpha-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions.
Mouse mast cell protease (mMCP) 1, mMCP-2, mMCP-4, and mMCP-5 are members of a family of related serine proteases whose genes reside within an approximately 850 kilobase (kb) complex on chromosome 14 that does not readily undergo crossover events. While mapping the mMCP-1 gene, we isolated a novel gene that encodes a homologous serine protease designated mMCP-9. The mMCP-9 and mMCP-1 genes are only approximately 7 kb apart on the chromosome and are oriented back to back. The proximity of the mMCP-1 and mMCP-9 genes now suggests that the low recombination frequency of the complex is due to the closeness of some of its genes. The mMCP-9 transcript and protein were observed in the jejunal submucosa of Trichinella spiralis-infected BALB/c mice. However, in normal BALB/c mice, mMCP-9 transcript and protein were found only in those mast cells that reside in the uterus. Thus, the expression of mMCP-9 differs from that of all other chymases. The observation that BALB/c mouse bone marrow-derived mast cells developed with interleukin (IL) 10 and c-kit ligand contain mMCP-9 transcript, whereas those developed with IL-3 do not, indicates that the expression of this particular chymase is regulated by the cytokine microenvironment. Comparative protein structure modeling revealed that mMCP-9 is the only known granule protease with three positively charged regions on its surface. This property may allow mMCP-9 to form multimeric complexes with serglycin proteoglycans and other negatively charged proteins inside the granule. Although mMCP-9 exhibits a >50% overall amino acid sequence identity with its homologous chymases, it has a unique substrate-binding cleft. This finding suggests that each member of the chromosome 14 family of serine proteases evolved to degrade a distinct group of proteins.
The crystal structure of the delta' subunit of the clamp-loader complex of E. coli DNA polymerase III has been determined. Three consecutive domains in the structure are arranged in a C-shaped architecture. The N-terminal domain contains a nonfunctional nucleotide binding site. The catalytic component of the clamp-loader complex is the gamma subunit, which is homologous to delta'. A sequence-structure alignment suggests that nucleotides bind to gamma at an interdomain interface within the inner surface of the "C." The alignment is extended to other clamp-loader complexes and to the RuvB family of DNA helicases, and suggests that each of these is assembled from C-shaped components that can open and close the jaws of the "C" in response to ATP binding and hydrolysis.
Biogenesis of both filamentous phage and type-IV pili involves the assembly of many copies of a small, integral inner membrane protein (the phage major coat protein or pilin) into a helical, tubular array that passes through the outer membrane. The occurrence of related proteins required for assembly and export in both systems suggests that there may be similarities at the mechanistic level as well. This report summarizes the properties of filamentous phage and the proteins required for their assembly, with particular emphasis on features they may share with bacterial protein export and pilus biogenesis systems, and it presents evidence that supports the hypothesis that one of the phage proteins functions as an outer membrane export channel.
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The GreA and GreB proteins of Escherichia coli induce cleavage of the nascent transcript in ternary elongation complexes of RNA polymerase. Gre factors are presumed to have two biologically important and evolutionarily conserved functions: the suppression of elongation arrest and the enhancement of transcription fidelity. A three-dimensional structure of GreB was generated by homology modeling on the basis of the known crystal structure of GreA. Both factors display similar overall architecture and surface charge distribution, with characteristic C-terminal globular and N-terminal coiled-coil domains. One major difference between the two factors is the "basic patch" on the surface of the coiled-coil domain, which is much larger in GreB than in GreA. In both proteins, a site near the basic patch cross-links to the 3' terminus of RNA in the ternary transcription complex. GreA/GreB hybrid molecules were constructed by genetic engineering in which the N-terminal domain of one protein was fused to the C-terminal domain of the other. In the hybrid molecules, both the coiled-coil and the globular domains contribute to specific binding of Gre factors to RNA polymerase, whereas the antiarrest activity and the GreA or GreB specificity of transcript cleavage is determined by the N-terminal domain. These results implicate the basic patch of the N-terminal coiled-coil domain as an important functional element responsible for the interactions with nascent transcript and determining the size of the RNA fragment to be excised during the course of the cleavage reaction.
Authors make known the comprehensive results of a longitudinal epidemiological examination which commenced in the 10th District of Budapest with nearly 100,000 inhabitants in 1975. The observation period is 15-20 years. Its aim was the determination of the risk factors of lung cancer. They followed up the lot of 30,561 persons, 92% of the population over 40 years of age 14,191 patients took part in a control examination supplemented with respiratory test. They analysed the connection between the supposed external risk factors and parenchyma functional damages caused by them and the development of lung cancer by detailed mathematical statistical arithmetic. 571 patients fell ill with lung cancer. The cured lung tuberculosis also proved to be a leading risk factor besides smoking. From the not endangered group 43, the highly super highly endangered groups 458 people fell ill with lung cancer. There was no difference between men and women in the lung cancer risk of the risk free population. According to their supposition the connection between the parenchyma functional damages caused by external risk factors and the development of lung cancer is determined by the different immunological status of people. (During the 20 years observation period 4% of heavy smokers and 3% of cured lung tuberculous patients fell ill with lung cancer). They urge the organization of wide ranging international prospective examination for the testing of this supposition.
We evaluate homology-derived 3D models of dihydrofolate reductase (DFR1), phosphotransferase enzyme IIA domain (PTE2A3), and mouse/human UBC9 protein (UBC9(24)) which were submitted to the second Meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP). The DFR1 and PTE2A3 models, based on alignments without large errors, were slightly closer to their corresponding X-ray structures than the closest template structures. By contrast, the UBC9(24) model was slightly worse than the best template due to a misalignment of the N-terminal helix. Although the current models appear to be more accurate than the models submitted to the CASP meeting in 1994, the four major types of errors in side chain packing, position and conformation of aligned segments, position and conformation of inserted segments, and in alignment still occur to almost the same degree. The modest improvement probably originates from the careful manual selection of the templates and editing of the alignment, as well as from the iterative realignment and model building guided by various model evaluation techniques. This iterative approach to comparative modeling is likely to overcome at least some initial alignment errors, as demonstrated by the correct final alignment of the C terminus of DFR.
Comparative modelling of protein 3D structure can now be applied with reasonable accuracy to ten times more protein sequences than the number of experimentally determined protein structures. A protein sequence that has at least 40% identity to a known structure can be modelled automatically with an accuracy approaching that of a low resolution X-ray structure or a medium resolution NMR structure. Currently, the errors in comparative models include mistakes in the packing of sidechains, in the conformation and shifts of the core segments and loops, and, most importantly, in an incorrect alignment of the modelled sequence with related known structures. Nevertheless, the number of applications in which comparative modelling has been proven to be useful has grown rapidly.
The science of psychoneuroimmunology looks at the role of the mind in the cause of disease. A number of studies have demonstrated that stress increases the risk of viral infection. Stress and depression can depress immunity whereas stress reduction can enhance immunity. Preliminary data suggest that cancer prognosis can be improved by enhanced immunity as a result of stress reduction. An understanding of psychoneuroimmunology should contribute to significant changes in the management of many diseases, especially cancer and infection.
beta2-Glycoprotein I (beta2GPI) is a phospholipid-binding serum protein with anticoagulant properties. It plays a vital role in the binding of anti-cardiolipin Abs purified from patients with autoimmune disease when assayed in a cardiolipin (CL) ELISA. Based on a three-dimensional model of beta2GPI, electrostatic calculations, and earlier peptide studies, a highly positively charged amino acid sequence, Lys282-Asn-Lys-Glu-Lys-Lys287, located in the fifth domain of beta2GPI, has been predicted to be the phospholipid binding site. We tested this hypothesis by site-directed mutagenesis of residues in the predicted phospholipid binding site and by assessing the mutants for phospholipid binding and anti-beta2GPI activity. A single amino acid change from Lys286 to Glu significantly decreased the binding of beta2GPI to CL. Double and triple mutants 2k (from Lys286, 287 to Glu286, 287), 2ka (from Lys284, 287 to Glu284, 287), and 3k (from Lys284, 286, 287 to Glu284, 286, 287) possessed no binding of Ab to beta2GPI in a CL ELISA, as well as no inhibitory activity on the binding of iodinated native beta2GPI to CL. These results indicate that the residues Lys284, Lys286, and Lys287 in the fifth domain of beta2GPI are critical for its binding to anionic phospholipids and its subsequent capture for binding of anti-beta2GPI Abs.
Brain lipid-binding protein (BLBP) is a member of the fatty acid-binding protein (FABP) family. Although BLBP expression in the developing central nervous system is complex, a close correlation between its expression and radial glial differentiation has been observed. Furthermore, antibodies to BLBP can block glial cell differentiation in mixed primary cell cultures. Here we describe the ligand binding properties of BLBP. The binding affinities of BLBP for oleic acid (Kd approximately 0.44 microM) and arachidonic acid (Kd approximately 0.25 microM) are similar to those reported for other FABPs, but BLBP does not bind to palmitic acid or arachidinic acid. These and other experiments establish that BLBP has a strong preference for binding long chain polyunsaturated fatty acids. A probable in vivo ligand for BLBP is docosahexaenoic acid (DHA), since its binding affinity (Kd approximately 10 nM) is the highest yet reported for an FABP/ligand interaction, exceeding even the affinity of retinoic acid for its binding proteins. Furthermore, the requirement of DHA for nervous system development and the coincident expression of BLBP during these developmental stages suggest that the physiologic role of BLBP may involve DHA utilization. Finally, we present a structural model of BLBP/DHA interaction that provides insight into both the structural characteristics important for ligand binding and the effects of specific mutations upon BLBP/ligand interactions.
The mouse mast cell protease granule tryptases designated mMCP-6 and mMCP-7 are encoded by highly homologous genes that reside on chromosome 17. Because these proteases are released when mast cells are activated, we sought a basis for distinctive functions by examining their fates in mice undergoing passive systemic anaphylaxis. 10 min-1 h after antigen (Ag) was administered to immunoglobulin (Ig)E-sensitized mice, numerous protease/proteoglycan macromolecular complexes appeared in the extracellular matrix adjacent to most tongue and heart mast cells of normal BALB/c mice and most spleen and liver mast cells of V3 mastocytosis mice. These complexes could be intensively stained by anti-mMCP-6 Ig but not by anti-mMCP-7 Ig. Shortly after Ag challenge of V3 mastocytosis mice, large amounts of properly folded, enzymatically active mMCP-7 were detected in the plasma. This plasma-localized tryptase was approximately 150 kD in its multimeric state and approximately 32 kD in its monomeric state, possessed an NH2 terminus identical to that of mature mMCP-7, and was not covalently bound to any protease inhibitor. Comparative protein modeling and electrostatic calculations disclosed that mMCP-6 contains a prominent Lys/Arg-rich domain on its surface, distant from the active site. The absence of this domain in mMCP-7 provides an explanation for its selective dissociation from the exocytosed macromolecular complex. The retention of exocytosed mMCP-6 in the extracellular matrix around activated tissue mast cells suggests a local action. In contrast, the rapid dissipation of mMCP-7 from granule cores and its inability to be inactivated by circulating protease inhibitors suggests that this tryptase cleaves proteins located at more distal sites.
The folding mechanism of a 125-bead heteropolymer model for proteins is investigated with Monte Carlo simulations on a cubic lattice. Sequences that do and do not fold in a reasonable time are compared. The overall folding behavior is found to be more complex than that of models for smaller proteins. Folding begins with a rapid collapse followed by a slow search through the semi-compact globule for a sequence-dependent stable core with about 30 out of 176 native contacts which serves as the transition state for folding to a near-native structure. Efficient search for the core is dependent on structural features of the native state. Sequences that fold have large amounts of stable, cooperative structure that is accessible through short-range initiation sites, such as those in anti-parallel sheets connected by turns. Before folding is completed, the system can encounter a second bottleneck, involving the condensation and rearrangement of surface residues. Overly stable local structure of the surface residues slows this stage of the folding process. The relation of the results from the 125-mer model studies to the folding of real proteins is discussed.
We describe here the cloning and sequencing of a new auxiliary gene identified by Tn551 insertional mutagenesis of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL. The insertionally inactivated mutant RUSA315 had intact mecA and normal amounts of PBP2A, but drastically reduced antibiotic resistance (drop in methicillin MIC from 1600 to 1.5 micrograms ml-1), a unique heterogeneous phenotype, and a compositional change in the cell wall characterized by the complete disappearance of the unsubstituted disaccharide pentapeptide from the peptidoglycan. Cloning in E. coli followed by sequencing located the Tn551 insert omega 720 in an open reading frame of 451 codons, provisionally called femR315, defining a polypeptide with a deduced amino acid sequence that showed over 26% sequence identity and 57% overall sequence similarity with the phosphoglucomutase (PGM) gene of E. coli. The Tn551 insertion site of a previously described mutant 12F (femD) also lies in the same gene as femR315. The wild-type form of femR315 subcloned in a shuttle vector fully restored expression of high level (parental) methicillin resistance in mutant RUSA315. The exact biochemical function of femR315 is not known. However, enzymes similar to PGM catalyze the isomerization of hexose and hexosamine phosphates leading to the formation of glucosamine-1-P, which is an obligate precursor in the biosynthesis of UDP-N-acetylglucosamine (UDP-NAGA). We propose that the suppression of methicillin resistance in RUSA315 is related to some functional or quantitative abnormality of UDP-NAGA metabolism.
Mouse mast cell protease 7 (mMCP-7) is a tryptase stored in the secretory granules of mast cells. At the granule pH of 5.5, mMCP-7 is fully active and is bound to heparin-containing serglycin proteoglycans. to understand the interaction of mMCP-7 with heparin inside and outside the mast cell, this trytase was first studied by comparative protein modeling. The "pro" form of mMCP-7 was then expressed in insect cells and studied by site-directed mutagenesis. Although mMCP-7 lacks known linear sequences of amino acis that interact with heparin, the three-dimensional model of mMCP-7 revealed an area on the surface of the folded protein away from the substrate-binding site that exhibits a strong positive electrostatic potential at the acidic pH of the granule. In agreement with this calculation, recombinant pro-mMCP-7 bound to a heparin-affinity column at pH 5.5 and readily dissociated from the column at pH > 6.5. Site-directed mutagenesis confirmed the prediction that the conversion of His residues 8,68, and 70 in the positively charged region into Glu prevents the binding of pro-mMCP-7 to heparin. Because the binding requires positively charged His residues, native mMCP-7 is able to dissociate from the protease/proteoglycan macromolecular complex when the complex is exocytosed from bone marrow-derived mast cells into a neutral pH environment. Many hematopoietic effector cells store positively charged proteins in granules that contain serglycin proteoglycans. The heparin/mMCP-7 interaction, which depends on the tertiary structure of the tryptase, may be representative of a general control mechanism by which hematopoietic cells maximize storage of properly folded, enzymatically active proteins in their granules.