Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The major surface protease (MSP or GP63) of the Leishmania spp. protozoa facilitates parasite evasion of complement-mediated killing, phagocytosis by macrophages, and intracellular survival in macrophage phagolysosomes. Immunoblots of several Leishmania species have shown there are distinct MSP isoforms, but the biochemical bases for these differences are unknown. Northern blots show that transcripts of the three tandem gene classes encoding Leishmania chagasi MSP (MSPS, MSPL, MSPC) are differentially expressed during parasite growth in vitro. Cell-associated MSPs increase in abundance during growth, correlating directly with parasite virulence. We examined whether distinct products of these >18 MSP genes are either differentially expressed or differentially processed during parasite growth. Two-dimensional gel electrophoresis and immunoblots delineated more than 10 MSP isoforms in stationary phase L. chagasi, distributed between pIs of 5.2-6.1 and masses of 58-63 kDa. Post-translational modifications including N-glycosylation, GPI anchor addition and phosphorylation did not account for all differences among the isoforms. MALDI-TOF mass spectrometry demonstrated that at least some L. chagasi MSPs were the products of different MSP genes. One isoform was not available for surface biotinylation, suggesting it could be located internally. Parasites in logarithmic growth expressed only four MSP isoforms, and an attenuated strain of L. chagasi (L5) did not express one of the MSP classes (MSPS). These data demonstrate that the products of individual MSP genes are differentially expressed during Leishmania development. We hypothesize they may play different roles during parasite migration through its two hosts.
Mol Biochem Parasitol 2004 Jun
PMID:Multiple products of the Leishmania chagasi major surface protease (MSP or GP63) gene family. 1511 Apr 59

The elevated expression of 70 kDa heat shock protein (Hsp70) induces resistance to stress-induced apoptosis. We have screened a variety of natural products for their ability to enhance Hsp70 expression as anti-apoptotic agent. We found that glucuronic acid (GA) induced the synthesis of Hsp70 and that cells pretreated with GA were significantly tolerant to stress including heat shock and hydrogen peroxide. We also found that GA induces the production of reactive oxygen species (ROS), a process inhibited by NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI) and antioxidant N-acetylcysteine (NAC). GA-induced ROS production was also inhibited in RacN17 cell line overexpressing a dominant negative mutant of Rac1. Furthermore, GA treatment induces MAPKs activation (SAPK/JNK and p38) and Hsp70 expression in ROS dependent manner, suggesting that GA turns on the signaling pathway by generation of ROS through Rac1. We analyzed the profiles of newly synthesized proteins by GA with 2-dimensional gel electrophoresis and MALDI-TOF MS and found that two families of proteins were expressed by GA. One was similar to the protein family synthesized by heat shock (Hsp70, Hsp73, Hsp65, Hsp90, vimentin, tubulin, Ras homolog); and the other was a family of protein specific to GA (calreticulin, annexin III, thioredoxin peroxidase). These results suggest that GA-induced stress responses are mediated by ROS generation and are similar, in part, to heat shock-induced responses and GA can be possibly adopted for the protecting agent from cell death.
Mol Cell Biochem 2004 Apr
PMID:Glucuronic acid is a novel inducer of heat shock response. 1512 4

Capillary electrophoresis (CE) became a versatile technique for analysis of biological macromolecules. We have applied capillary zone electrophoresis (CZE) and SDS-gel CE for the characterization of recombinant proteins during development of major bioprocessing steps, including fermentation, hybridoma cell cultivation, chromatographic purification, and chemical transformation. Rapid SDS-gel CE was developed for the fast analysis of fermentation broth and hybridoma cell culture. The total analysis time was reduced to 4.5 min. We have developed system for fraction collection, which allows analyzing separated proteins by MALDI-TOF-MS. The main advantages of applied techniques were high resolution and selectivity, fast analysis, and high accuracy.
Methods Mol Biol 2004
PMID:Capillary electrophoresis in the analysis and monitoring of biotechnological processes. 1516 54

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.
Mol Reprod Dev 2004 Aug
PMID:Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase. 1523 38

Proteolytic activation of prophenoloxidase (proPO) is an integral part of the insect immune system against pathogen and parasite infection. This reaction is mediated by a proPO-activating proteinase (PAP) and its cofactor in the tobacco hornworm, Manduca sexta (Proc. Natl. Acad. Sci. USA 95 (1998) 12220; J. Biol. Chem. 278 (2003) 3552; Insect Biochem. Mol. Biol. 33 (2003) 1049). The cofactor consists of two serine proteinase homologs (SPHs), which associate with immulectin-2, a calcium-dependent lectin that binds to lipopolysaccharide (Insect Biochem. Mol. Biol. 33 (2003) 197). In order to understand the auxiliary effect of SPH-1 and SPH-2 in proPO activation, we started to investigate the molecular interactions among proPO, PAP-3, and the proteinase-like proteins. M. sexta SPH-1 and SPH-2 were purified from hemolymph of prepupae by hydroxylapatite, gel filtration, lectin-affinity, and ion exchange chromatography. They existed as non-covalent oligomers with an average molecular mass of about 790 kDa. MALDI-TOF mass fingerprint analysis revealed a new cleavage site in SPH-1 before Asp85. The PAP cofactor did not significantly alter Michaelis constant (KM) or kcat of PAP-3 towards a synthetic substrate, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide, but greatly enhanced proPO activation by PAP-3. The apparent KM for proPO was determined to be about 9.4 microg/ml, close to its estimated concentration in larval hemolymph. In the presence of excess proPO and a set amount of PAP-3, increasing levels of phenoloxidase (PO) activity were detected as more SPHs were added. Half of the maximum proPO activation occurred when the molar ratio of PAP-3 to SPH was 1:1.4. Gel filtration experiments suggested that proPO, PAP-3, and the cofactor formed a ternary complex.
Insect Biochem Mol Biol 2004 Aug
PMID:Prophenoloxidase (proPO) activation in Manduca sexta: an analysis of molecular interactions among proPO, proPO-activating proteinase-3, and a cofactor. 1526 78

From the Drosophila virilis late puff region 31C, we microcloned two neighbouring genes, Kil-1 and Kil-2, that encode putative Kunitz serine protease inhibitor like proteins. The Kil-1 gene is expressed exclusively in prepupal salivary glands. Using a size mutant of the KIL-1 protein and MALDI-TOF analysis, we demonstrate that during pupation this protein is released from the prepupal salivary glands into the pupation fluid covering the surface of the pupa. 3-D-structure predictions are consistent with the known crystal structure of the human Kunitz type protease inhibitor 2KNT. This is the first experimental proof for the extracorporal presence of a distinct Drosophila prepupal salivary gland protein. Possible functions of KIL-1 in the context of the control of proteolytic activities in the pupation fluid are discussed.
Insect Biochem Mol Biol 2004 Aug
PMID:A Kunitz type protease inhibitor related protein is synthesized in Drosophila prepupal salivary glands and released into the moulting fluid during pupation. 1526 89

Tobacco plants secrete a limited array of proteins (nectarins) into their floral nectar. N-terminal sequencing of the Nectarin II ( NEC2; 35kD) and the Nectarin III ( NEC3; 40kD) proteins revealed that they both share identity with dioscorin, the major soluble protein of yam tubers. These sequences also revealed that NEC2 is a breakdown product of NEC3. Using these N-terminal peptide sequences, degenerate oligonucleotides were designed that permitted the isolation of a partial NEC3 cDNA. This cDNA was then used to probe a nectary specific cDNA library and a full-length NEC3 cDNA clone was isolated. Complete sequence analysis confirmed the identity of NEC3 as a dioscorin-like protein. MALDI-TOF mass spectrometric fingerprinting of tryptic peptides derived from the purified NEC3 confirmed that this protein was encoded by the isolated cDNA. NEC3 was shown to possess both carbonic anhydrase and monodehydroascorbate reductase activities. RT-PCR based expression analyses demonstrated that NEC3 transcript is expressed throughout nectary development as well as in other floral organs. A proposed function in the maintenance of pH and oxidative balance in nectar is discussed.
Plant Mol Biol 2004 Feb
PMID:Tobacco Nectarin III is a bifunctional enzyme with monodehydroascorbate reductase and carbonic anhydrase activities. 1528 96

When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C(2)C(12) cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C(2)C(12) myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C(2)C(12) myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C(2)C(12) myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.
Mol Cell Proteomics 2004 Nov
PMID:Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program. 1528 12

(4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-beta-d-galactopyranoside was synthesized and successfully tested on beta-galactosidases from Xanthomonas manihotis (Wong-Madden, S. T., and Landry, D. Glycobiology (1995) 5, 19-28 and Taron, C. H., Benner, J. S., Hornstra, L. J., and Guthrie, E. P. (1995) Glycobiology 5, 603-610), Escherichia coli (Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Nature 369, 761-766), and Bacillus circulans (Fujimoto, H., Miyasato, M., Ito, Y., Sasaki, T., and Ajisaka, K. (1988) Glycoconj. J. 15, 155-160) for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from beta-galactosidases. It was demonstrated by using X. manihotis beta-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42 (Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., and Wakagi, T. (2002) J. Mol. Biol. 322, 79-91). A catalytic glutamic acid (Glu-537) of E. coli beta-galactosidase was proved to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B. circulans beta-galactosidase, indicating that Glu-259 is one of the nucleophiles in the active site. The present method can be readily extended to other glycosidases and should greatly aid the high throughput proteomics of many glycoside hydrolases showing both retaining- and inverting-type mechanisms.
 ...
PMID:Mechanism-based fluorescent labeling of beta-galactosidases. An efficient method in proteomics for glycoside hydrolases. 1530 75

A fully automated synthesizer was constructed and designed to perform high speed miniaturized syntheses of compound libraries using the SPOT technique. Utilizing magnetically controlled drop-on-demand ink jet nozzles, an r/phi array format of 2500 spots can be simultaneously dispensed from up to 24 separate reagent valves onto a rotating disc as the solid phase in less than three minutes. In addition, a complete wash station is on board allowing for fully programmable combinatorial syntheses without manual attention. A new carbon black/polypropylene composite solid phase disc was developed and tested for its functionalisation/loading, spot detection, durability and MALDI-TOF target capabilities. The carbon black/polypropylene composite was then successfully employed jointly as the solid phase in the syntheses of short peptide and PNA oligomers and as the target probe holder for MALDI-TOF measurement without transfer of the material. Several protocols for PNA syntheses were also investigated and an optimised PNA methodology for the carbon black/polypropylene composite is reported.
Mol Divers 2004
PMID:A new compact disc format of high density array synthesis applied to peptide nucleic acids and in situ MALDI analysis. 1538 13


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