Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene A40R from vaccinia virus (VV) strain Western Reserve has been characterized. The open reading frame (ORF) was predicted to encode a 159 amino acid, 18152 Da protein with amino acid similarity to C-type animal lectins and to the VV A34R protein, a component of extracellular enveloped virus (EEV). Northern blotting and S1 nuclease mapping showed that gene A40R is transcribed early during infection from a position 12 nucleotides upstream of the ORF, producing a transcript of approximately 600 nucleotides. Rabbit anti-sera were raised against bacterial fusion proteins containing parts of the A40R protein. These were used to identify an 18 kDa primary translation product and N- and O-glycosylated forms of 28, 35 and 38 kDa. The A40R proteins were detected early during infection, formed higher molecular mass complexes under non-reducing conditions and were present on the cell surface but absent from virions. The proteins partitioned with integral membrane proteins in Triton X-114. Canine pancreatic microsomal membranes protected in vitro-translated A40R from proteinase K digestion, suggesting the A40R protein has type II membrane topology. A mutant virus with the A40R gene disrupted after amino acid 50, so as to remove the entire lectin-like domain, and a revertant virus were constructed. Disruption of the A40R gene did not affect virus plaque size, in vitro growth rate and titre, EEV formation, or virus virulence in a murine intranasal model.
J Gen Virol 1999 Aug
PMID:The vaccinia virus A4OR gene product is a nonstructural, type II membrane glycoprotein that is expressed at the cell surface. 1046 13

Mule deer fawns (Odocoileus hemionus) were inoculated orally with a brain homogenate prepared from mule deer with naturally occurring chronic wasting disease (CWD), a prion-induced transmissible spongiform encephalopathy. Fawns were necropsied and examined for PrPres, the abnormal prion protein isoform, at 10, 42, 53, 77, 78 and 80 days post-inoculation (p.i.) using an immunohistochemistry assay modified to enhance sensitivity. PrPres was detected in alimentary-tract-associated lymphoid tissues (one or more of the following: retropharyngeal lymph node, tonsil, Peyer's patch and ileocaecal lymph node) as early as 42 days p.i. and in all fawns examined thereafter (53 to 80 days p.i.). No PrPres staining was detected in lymphoid tissue of three control fawns receiving a control brain inoculum, nor was PrPres detectable in neural tissue of any fawn. PrPres-specific staining was markedly enhanced by sequential tissue treatment with formic acid, proteinase K and hydrated autoclaving prior to immunohistochemical staining with monoclonal antibody F89/160.1.5. These results indicate that CWD PrPres can be detected in lymphoid tissues draining the alimentary tract within a few weeks after oral exposure to infectious prions and may reflect the initial pathway of CWD infection in deer. The rapid infection of deer fawns following exposure by the most plausible natural route is consistent with the efficient horizontal transmission of CWD in nature and enables accelerated studies of transmission and pathogenesis in the native species.
J Gen Virol 1999 Oct
PMID:Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus). 1057 72

Different strains of transmissible spongiform encephalopathies in humans and rodent models are associated with the accumulation of PrP(Sc) of distinct molecular characteristics. These characteristics include glycosylation profiles, fragment sizes and long-term resistance of PrP(Sc) to proteinase K. The first objective of this study was to determine the applicability of these criteria to characterize and differentiate sheep scrapie PrP(Sc) and bovine spongiform encephalopathy (BSE) PrP(Sc). PrP(Sc) in sheep scrapie samples from Ireland had clearly distinct molecular characteristics to PrP(Sc) in cattle BSE samples using a monoclonal antibody (MAb P4) directed to position 89-104 of ovine PrP using either brain homogenates or semi-purified scrapie-associated fibrils. Similar glycoprofiles were found when analysing scrapie PrP(Sc) in six different CNS regions (thoracic spinal cord, thalamus, basal ganglia, mediobasal hypothalamus, medulla oblongata and cortex). While the long-term resistance results using a different monoclonal antibody (raised to ruminant PrP positions 145-163; MAb L42) were similar to the results obtained with MAb P4, different glycotyping results were obtained. Given the variation in glycosylation patterns using different antibodies, we conclude that standardization of methodology and antibodies is crucial to the applicability of molecular analysis of ruminant BSE and scrapie samples.
J Gen Virol 2000 Jun
PMID:Molecular analysis of Irish sheep scrapie cases. 1081 47

The conversion of the cellular isoform of the prion protein (PrP(C)) to the abnormal disease-associated isoform (PrP(Sc)) has been simulated in cell-free conversion reactions in which PrP(Sc)-enriched preparations induce the conformational transition of PrP(C) into protease-resistant PrP (PrP-res). We explored the utility of recombinant hamster (Ha)PrP(C) purified from baculovirus-infected insect cells (bacHaPrP(C)) as a replacement for mammalian-derived HaPrP(C) in the conversion reactions. Protease-resistant recombinant HaPrP was generated after incubation of (35)S-bacHaPrP(C) with PrP(Sc)-enriched preparations. Moreover strain-specific PrP-res was also reproduced using insect-cell derived HaPrP(C) and PrP(Sc) from two different strains of hamster-adapted transmissible mink encephalopathy, designated hyper (HY) and drowsy (DY). Two strain-mediated properties were tested: (i) molecular mass of the protease-digested products and (ii) relative resistance to proteinase K (PK) digestion. Similar to in vivo generation of PrP(HY) and PrP(DY), the converted products selectively reproduced both characteristics, with the DY conversion product being smaller in size and less resistant to PK digestion than the HY product. These data demonstrate that non-mammalian sources of recombinant HaPrP can be converted into PK-resistant form and that strain-mediated properties can be transmitted into the newly formed PrP-res.
J Gen Virol 2000 Oct
PMID:Strain-specific propagation of PrP(Sc) properties into baculovirus-expressed hamster PrP(C). 1099 47

A sequence of 1425 nt was established that included the complete coat protein (CP) gene of Lettuce big-vein virus (LBVV). The LBVV CP gene encodes a 397 amino acid protein with a predicted M(r) of 44486. Antisera raised against synthetic peptides corresponding to N-terminal or C-terminal parts of the LBVV CP reacted in Western blot analysis with a protein with an M(r) of about 48000. RNA extracted from purified particles of LBVV by using proteinase K, SDS and phenol migrated in gels as two single-stranded RNA species of approximately 7.3 kb (ss-1) and 6.6 kb (ss-2). After denaturation by heat and annealing at room temperature, the RNA migrated as four species, ss-1, ss-2 and two additional double-stranded RNAs (ds-1 and ds-2). The Northern blot hybridization analysis using riboprobes from a full-length clone of the LBVV CP gene indicated that ss-2 has a negative-sense nature and contains the LBVV CP gene. Moreover, ds-2 is a double-stranded form of ss-2. Database searches showed that the LBVV CP most resembled the nucleocapsid proteins of rhabdoviruses. These results indicate that it would be appropriate to classify LBVV as a negative-sense single-stranded RNA virus rather than as a double-stranded RNA virus.
J Gen Virol 2001 Jun
PMID:Nucleotide sequence of the coat protein gene of Lettuce big-vein virus. 1136 98

The distribution of disease-associated prion protein (PrP) was investigated in eight animals (20-24 months of age) from a flock of Suffolk sheep that had experienced frequent cases of natural scrapie over a period of several years. Tissue from the central nervous system (CNS), alimentary tract, peripheral nervous system and lymphoreticular system was examined by histopathology and immunohistochemistry. The lymphoid tissues were subjected further to histoblot and immunofluorescence examination. The four clinically affected PrP(ARQ/ARQ) sheep had widespread accumulations of disease-associated PrP in the CNS, lymphoreticular system and peripheral ganglia. In the two PrP(ARQ/ARQ) sheep that did not show clinical signs of scrapie, only limited vacuolation and PrP accumulation were detected in the brain, but the results from the lymphoreticular system and peripheral nervous system were comparable with the clinically affected animals. The remaining PrP(ARR/ARR) and PrP(ARR/ARQ) sheep did not show proteinase K-resistant PrP accumulations in the lymphoid tissues examined and immunohistochemistry did not reveal the presence of disease-associated PrP. In lymphoid tissues of the PrP(ARQ/ARQ) sheep, the dominant localization of disease-associated PrP was in lymphoid nodules and double immunofluorescence labelling for PrP and CD21 provided further support for the role of follicular dendritic cells in scrapie in sheep. A striking finding in the present study was the large accumulations of disease-associated PrP in the lymphoid nodules of the alimentary tract at the late sub-clinical and clinical stage of the infection. The study also identified disease-associated PrP in extra-nodular sites of lymphoid tissues, such as the marginal zone of the spleen, and these observations were used to argue that cells of the mononuclear phagocyte system of sheep may be involved in the uptake, transport, elimination and shedding of the scrapie agent.
J Gen Virol 2002 Feb
PMID:Distribution and accumulation of PrP in gut-associated and peripheral lymphoid tissue of scrapie-affected Suffolk sheep. 1180 42

Conversion of the cellular isoform of the prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)) is thought to be the causative event in prion diseases. Biochemically, PrP(Sc) differs from PrP(C) in its partial resistance to proteinase K (PK). The amino acid sequence AGAAAAGA, comprising residues 112-119 of the murine PrP(C), has been shown to be amyloidogenic and evolutionarily conserved. To assess the effect of mutations at and around this hydrophobic sequence on protease resistance, the sequence was replaced either by alanines or by glycines and, in a third mutant, a large part surrounding this region was removed. The PrP mutant carrying substitutions of glycines for alanines showed PK resistance and aberrant proteolytic processing. Tetracycline-induced expression of this mutant indicated that resistance to protease is acquired concurrent with the synthesis of the protein. These findings indicate that mutations in the central hydrophobic region lead to immediate alterations in PrP structure and processing.
J Gen Virol 2002 May
PMID:Mutant prion protein acquires resistance to protease in mouse neuroblastoma cells. 1196 Dec 79

The antigenic determinant of a monoclonal antibody (MAb) (API9-2) having specific reactivity with the fungi grouped into the genus Fusarium was analyzed. The culture supernatant of the fungi showed antigenicity against MAb API9-2, proving that the antigen exists as an exoantigen. The heat-resistant, proteinase K-resistant and periodate oxidation-labile features of the antigenic determinant indicated its carbohydrate nature. Also, lectin affinity tests and thin-layer chromatography analysis suggested that the monosaccharide making up the antigenic determinant was mainly mannose. Considering previous reports that the antigen exists on the surface of mycelia (by immunofluorescence assay) and is a - 55 kDa molecule (by Western blotting analysis), it was concluded that the antigenic determinant of MAb API9-2 on F. oxysporum is a mannan component existing on the surface of mycelia.
J Gen Appl Microbiol 1998 Feb
PMID:Characterization of the antigenic determinant on Fusarium oxysporum recognized by a genus-specific monoclonal antibody. 1250 Dec 92

Bulk DNA isolated from the ectomycorrhizal basidiomycete Hebeloma circinans was treated with proteinase K and submitted to agarose gel electrophoresis. In addition to high molecular weight genomic DNA, three minor bands were detected. The band with the highest electrophoretic mobility (2.2 kbp) corresponds to double-stranded RNA. The two other bands, termed pHC1 and pHC2, were shown to be dsDNA molecules of 10.3 and 9.1 kbp, respectively. Treatment of the pHC elements with 3'- and 5'-specific exonucleases revealed a linear structure and proved that the 5' ends are protected from digestion; for pHC2, linearity was confirmed by restriction mapping. A 3.2 kbp HindIII fragment of pHC2 was cloned and sequenced; it contains two open reading frames encoding putative viral B type DNA and RNA polymerases. Thus, the fungus harbors a typical linear plasmid, up to now, rarely described for basidiomycetes and hitherto unknown for mycorrhizal species.
J Gen Appl Microbiol 1997 Oct
PMID:The ectomycorrhizal basidiomycete Hebeloma circinans harbors a linear plasmid encoding a DNA- and RNA polymerase. 1250 14

Prion diseases are associated with the conversion of the normal cellular prion protein, PrP(C), to the abnormal disease-associated protein, PrP(Sc). This conversion can be mimicked in vitro using PrP(Sc) isolated from the brains of scrapie-infected animals to induce conversion of recombinant PrP(C) into a proteinase K-resistant isoform, PrP(res). Traditionally, the 'cell-free' conversion assay has used, as substrate, recombinant PrP(C) purified from mammalian tissue culture cells or, more recently, from baculovirus-infected insect cells. The cell-free conversion assay has been modified by replacing the tissue culture-derived PrP(C) with recombinant PrP purified from bacteria. Bacterial expression and chromatographic purification give high yields of recombinant radiolabelled untagged protein, eliminates artefacts that may be due to cellular factors or antibody fragments normally present in labelled PrP preparations and allows accurate and rapid variation of protein sequence using standard molecular biological techniques. In addition, these cell-free conversion assays were carried out under more physiological conditions, giving more relevance to the assay as a model for conversion. To validate its use in this assay, this bacterial recombinant PrP has been shown to have the conversion properties of mammalian PrP(C): (i) it converts to a proteinase K-resistant isoform in the presence of PrP(Sc); (ii) the efficiency of this conversion by PrP(Sc) of different strains and species parallels that found in vivo; and (iii) its cell-free conversion is inhibited by Congo Red analogues in a structure-dependent manner similar to that seen in in vivo and in vitro cell assays.
J Gen Virol 2003 Apr
PMID:In vitro cell-free conversion of bacterial recombinant PrP to PrPres as a model for conversion. 1265 5


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