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Query: UNIPROT:P06889 (Mol)
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Several factors have been examined to improve in vitro fertilization and development of porcine oocytes. Cysteine is known to be beneficial for oocyte maturation and male pronuclear formation in pigs and glutathione is known to help prevent membrane disruption of sperm in other species, including human. It has also been reported that the presence of cumulus cells influences the outcome of in vitro fertilization in cattle. The objectives of the present study were to investigate the effects of several factors involved in porcine in vitro maturation (IVM) and fertilization (IVF) procedures on oocyte embryogenic competence. The following factors were examined: the effects of different concentrations (0, 0.285, 0.57, 1.14, 2.28 microM) and exposure duration (22 and 44 hr) of cysteine during IVM, glutathione inclusion and of cumulus presence during IVF, and the use of gradient Percoll (45%/90%) during sperm preparation. The presence of cysteine in maturation medium improved blastocyst development significantly regardless of the duration of exposure when compared to the control (11--16% vs. 4%, P < 0.01). However, no dose-responsive effect was observed at the concentrations tested. The use of gradient Percoll during sperm preparation significantly improved cleavage (85% vs. 57%, P < 0.01) and blastocyst development (24% vs. 6%, P < 0.01) over conventional sperm preparation. Significant improvement was also achieved by the addition of glutathione to Percoll gradient (30% vs. 20%, P < 0.05). In conclusion, cysteine and glutathione as well as Percoll and cumulus were beneficial to embryogenic competence of porcine oocytes in this study. Mol. Reprod. Dev. 59:330-335, 2001.
Mol Reprod Dev 2001 Jul
PMID:Cysteine, glutathione, and Percoll treatments improve porcine oocyte maturation and fertilization in vitro. 1142 19

Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.
Mol Biochem Parasitol 2001 Nov
PMID:Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense. 1170 74

Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence.
Mol Biochem Parasitol 2002 Jan
PMID:Cysteine proteinases from distinct cellular compartments are recruited to phagocytic vesicles by Entamoeba histolytica. 1175 83

Cysteine proteases play numerous indispensable roles in the biology of parasitic organisms. Aside from previously known general catabolic functions and protein processing, cysteine proteases may be key to parasite immunoevasion, excystment/encystment, exsheathing and cell and tissue invasion. Parasite cysteine proteases are unusually immunogenic and have been exploited as serodiagnostic markers and vaccine targets. Although host homologues exist, parasite cysteine proteases have distinct structural and biochemical properties including, pH optima and stability, the alteration in peptide loops or domain extensions, diverse substrate specificity and cellular location. The disparate nature of parasite cysteine protease compared to the host orthologous proteins has opened opportunities for chemotherapy. This review will highlight recent research on the 'papain-like' class of cysteine proteases, the most abundant family, and the newly discovered class of asparaginyl-endopeptidases. Cysteine protease classification will be re-examined in light of the diversity uncovered within parasitic organisms.
Mol Biochem Parasitol 2002 Mar
PMID:Cysteine proteases of parasitic organisms. 1184 1

Synthesis and secretion of certain epididymal proteins are regulated by lumicrine factors from the testis or from upstream regions of the excurrent ducts. Cysteine-rich secreted protein-1 (Crisp-1) is a major androgen regulated protein in the epididymal lumen fluid of the rat and other species. Previous research has demonstrated that disturbance of the luminal microenvironment through obstruction of the tract reduces Crisp-1 synthesis and secretion. The present study was undertaken to determine the influence of the luminal microenvironment on rat proximal caput epididymal Crisp-1 secretion into lumen fluid and on Crisp-1 gene expression in the same tubules. Western blot analysis demonstrated that Crisp-1 protein concentrations were reduced from control levels by perfusion with artificial caput fluid containing no testicular factors and were not increased by perfusion with fluids containing rete testis fluid proteins. Crisp-1gene expression was also reduced by perfusion with artificial caput fluid and not increased by perfusion with rete testis fluid proteins. Perfusion with artificial caput fluid containing 5alpha-dihydrotestosterone did increase one Crisp-1 transcript. This study demonstrates that intraluminal testicular proteins are not important co-regulators with androgens of Crisp-1gene expression or resulting Crisp-1 secretion into the rat proximal caput tubule lumen in vivo.
Mol Reprod Dev 2002 Apr
PMID:On the regulation of Crisp-1 mRNA expression and protein secretion by luminal factors presented in vivo by microperfusion of the rat proximal caput epididymidis. 1189 14

Protein-calorie malnutrition (PCM), as one of global health problems, arises during protein and/or energy deficit due to disease and nutritional inadequacy. Previously, we showed that PCM elicited oxidative stress with activation of the phase II detoxifying gene expression, which was reversed by cysteine supplementation. As part of the attempts to identify the cellular adaptive responses and the associated gene expression during PCM, the current study was initiated to analyze the genes differentially expressed in the rat during PCM. Among 1,916 bands amplified, 85 putative differentially amplified bands were enhanced by PCM in the liver, while the expression of 64 bands was suppressed. Northern and/or reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that PCM increased the expression of fibrinogen B beta chain, B cell translocation gene I (BTGI) and thyroid hormone responsive protein (THRP) mRNAs. The increase in the hepatic fibrinogen B beta chain mRNA was not prevented by cysteine supplementation, whereas cysteine decreased the enhancement in the rGSTA2 and microsomal epoxide hydrolase mRNA expression. Cysteine was also active in reversing the increase in BTG1 mRNA during PCM. This was supported by the increase in BTG1 mRNA in H4IIE cells exposed to sulfur amino acid-deprived medium. Northern blot analysis revealed that THRP, highly expressed in the brain in a tissue-specific manner, was induced by PCM and that cysteine supplementation abolished the THRP induction. Conversely, the level of hepatic albumin mRNA was markedly decreased by PCM, which was partially restored by cysteine supplementation. Differential display RT-PCR analysis allowed us to identify the genes that are responsive to oxidative stress during PCM and to characterize the differential role of cysteine on the expression of the fibrinogen B beta chain, BTG1 and THRP genes as a homeostatic adaptive response during protein deficiency.
Mol Cell Biochem 2002 Feb
PMID:Identification of genes enhanced by protein-calorie malnutrition by differential display polymerase chain reaction (expression of fibrinogen B beta chain, B cell translocation gene 1 and thyroid hormone responsive protein genes). 1195 59

Cysteine proteinase inhibitors (cystatins) confer resistance to plant-parasitic nematodes when expressed in transgenic plants. The survival and growth of nymphs of the peach-potato aphid, Myzus persicae, were adversely affected when cystatins were added to artificial diets. When aphids were clip-caged onto transgenic plants expressing chicken egg white cystatin (CEWc) there was no adverse effect on aphid fitness. Field populations of aphids on transgenic Desiree potatoes, expressing CEWc or a modified version of oryzacystatin I, were not significantly different from populations on control Desiree plants. The effect of other nematode management options on aphid numbers was also studied. A conventionally bred cultivar, with partial nematode resistance, supported higher populations of aphids than the transgenic lines at the beginning of the sampling period. Peak aphid densities on the untreated control and untreated transgenic lines were 7 and 5.2 aphids per plant. Aldicarb, commonly used to control nematodes on potatoes, reduced the value to less than 0.2 aphids per plant. The results demonstrate that levels of expression in the plant tissue actually consumed are important in determining the risk of cystatins to nontarget invertebrates. The study also highlights the importance of including currently used management options in any assessment of the impact of transgenic plants on nontarget organisms.
Mol Ecol 2002 Apr
PMID:Transgenic potatoes with enhanced levels of nematode resistance do not have altered susceptibility to nontarget aphids. 1197 67

A gene coding for a protein containing two Scavenger Receptor Cysteine-Rich (SRCR) motifs, four Limulus factor C, Coch-5b2 and Lgl1 (LCCL) motifs; and one Polycystin-1, Lipoxygenase and Alpha Toxin (PLAT) motif was cloned from Plasmodium chabaudi and homologues identified in the P. falciparum and P. yoelii genome data bases. At least one of these sequence motifs (SRCR) has adhesive properties in other proteins, therefore, we propose to name this protein PSLAP for Plasmodium SRCR, LCCL Adhesive-like Protein. Southern blotting and chromosome analysis showed that pslap is a single copy gene on chromosome 14 in P. falciparum 3D7. pslap mRNA is strongly expressed in P. falciparum gametocytes, but was undetectable on Northern blots of RNA from the asexual blood stages. Polyclonal antibodies raised to different parts of PSLAP detected a protein expressed in late gametocytes, but not in the early stages of gametocytogenesis or asexual blood stages of P. falciparum. We suggest that PSLAP functions in the mosquito, for example, in modulation of the invertebrate host immune response or in protection against complement factors in the blood meal.
Mol Biochem Parasitol 2002 Apr 30
PMID:PSLAP, a protein with multiple adhesive motifs, is expressed in Plasmodium falciparum gametocytes. 1198 59

Iron regulatory protein 1 (IRP1), a major posttranscriptional regulator of cellular iron and energy metabolism, is controlled by an iron-sulfur cluster switch. Cysteine-437 is critical for coordinating the cluster, and its replacement yields mutants that do not respond to iron perturbations and constitutively bind to cognate mRNA iron-responsive elements (IREs). The expression of IRP1(C437S) in cells has been associated with aberrations in iron homeostasis and toxicity. We have established clones of human lung (H1299) and breast (MCF7) cancer cells that express high levels of IRP1(C437S) in a tetracycline-inducible manner. As expected, IRP1(C437S) stabilizes transferrin receptor mRNA and inhibits translation of ferritin mRNA in both cell types by binding to their respective IREs. However, H1299 transfectants grown at high densities are able to overcome the IRP1(C437S)-mediated inhibition in ferritin synthesis. The mechanism involves neither alteration in ferritin mRNA levels nor utilization of alternative transcription start sites to eliminate the IRE or relocate it in less inhibitory downstream positions. The derepression of ferritin mRNA translation occurs under conditions where global protein synthesis appears to be impaired, as judged by a significant enrichment in the expression of the underphosphorylated form of the translational regulator 4E-BP1. Collectively, these data document an example where ferritin mRNA translation evades control of the IRE-IRP system. The physiological implications of this response are reflected in protection against iron-mediated toxicity, oxidative stress, and apoptosis.
Mol Cell Biol 2002 Jul
PMID:Conditional derepression of ferritin synthesis in cells expressing a constitutive IRP1 mutant. 1205 72

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic alpha-helices (H1-H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C-apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C-apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene "excimer" fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene-A8C-apoLp-III/DMPC or pyrene-A138C-apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene-A8C/A138C-apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle.
J Mol Biol 2002 Aug 09
PMID:Lipid-triggered conformational switch of apolipophorin III helix bundle to an extended helix organization. 1214 79


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