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)

The polypeptide composition of neurofibrillary tangles (NFTs) and senile plaques (SPs) has been characterized extensively within the Alzheimer's disease (AD) brain. Because few data exist on the nonproteinaceous components of these lesions, we sought to determine if NFTs, neuropil threads (NTs), and SPs contain RNA species. To accomplish this, acridine orange (AO) histofluorescence was employed, alone or in combination with thioflavine S (TS) staining and immunohistochemistry to identify RNAs in paraffin-embedded tissue sections of hippocampus and entorhinal cortex. Postmortem brain samples came from 32 subjects including AD and elderly Down's syndrome (DS) patients, age-matched normal controls, and non-AD diseased controls. AO stained the cytoplasm of normal hippocampal and entorhinal neurons in all of the cases, while NFTs, NTs, and SPs were AO-positive in the same regions of AD and DS brains. Cytoplasmic AO histofluorescence was abolished with RNase, but not DNase or proteinase K, indicating the relative specificity of AO for RNA species. Quantitative analysis of double-labeled sections demonstrated that approximately 80% of TS-positive NFTs also were AO-positive, whereas approximately 55% of TS-stained SPs contained AO labeling. These novel observations demonstrate the presence of RNAs in NFTs, NTs, and SPs.
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PMID:Sequestration of RNA in Alzheimer's disease neurofibrillary tangles and senile plaques. 902 69

A new method for isolation of prawn baculovirus and subsequent extraction of viral DNA was developed. No density gradient centrifugation, ultracentrifugation or phenol-chloroform extraction steps were involved. Phenylmethylsulfonyl fluoride (PMSF) was used to prevent proteinase degradation, DNase and RNase were used to degrade prawn DNA and RNA respectively. The nucleocapsid was a bacilliform virion, about 58 62 nm in width and 300-350 nm in length as observed by transmission electron microscopy. Intact viral DNA was obtained by lysing nucleocapsids with guanidine hydrochloride and degrading protein with proteinase K. As the viral DNA was digested with restriction endonuclease and separated by electrophoresis, restriction fragments were clearly shown on the agarose gel. The size of the DNA was estimated approximately to be 290 kb. The virus which appeared to be a prawn baculovirus was named prawn white spot baculovirus (PWSBV) due to the white spots which appeared on the inside surface of the crust of infected prawns.
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PMID:A simple and efficient method for purification of prawn baculovirus DNA. 927 11

Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for beta-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.
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PMID:Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7. 1022 67

Hydroxymethylacylfulvene (HMAF, MGI 114) is a novel antitumor drug and a potent pro-apoptotic agent that has the potential to alkylate cellular nucleophiles. The objective of these studies was to characterize drug uptake and cellular targets for drug binding in human leukemia CEM cells. The uptake of [14C]HMAF had two components: a rapid phase (0-10 min) and a slow phase. At 10 microM drug (37 degrees), the rapid and slower phase amounted to 0.86 and 0.13 pmol/min/10(6)cells, respectively. HMAF uptake was inhibited 82% by low temperature (4 degrees) at 4 hr. Cell-associated HMAF localized to nuclear (50%), cytoplasmic (37%), and membrane fractions (10%). Continued drug uptake appeared to be driven by covalent binding to cellular macromolecules. Approximately 1/4 and 2/3 of cell-associated HMAF formed covalent adducts after 10 min and 4 hr, respectively, as found by perchloric acid precipitation. Drug adducts were not readily reversible; 77% of the covalently bound radiolabel was retained by the cells 20 hr after drug treatment. Combinations of DNase, RNase, and proteinase K with perchloric acid precipitation showed that approximately 60, 30, and 10% of the covalently bound drug was associated with the protein, DNA, and RNA fractions, respectively. Incubation of 100 microM [14C]HMAF (24 hr) with purified DNA, serum albumin, thioredoxin, and thioredoxin reductase resulted in 6, 22, 14, and 11 pmol [14C]HMAF/microg DNA or protein, respectively. Results indicate that multiple targets for HMAF binding may contribute to the pro-apoptotic and antiproliferative action of the drug.
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PMID:Drug uptake and cellular targets of hydroxymethylacylfulvene (HMAF). 1042 61

Pale and homogeneous-looking nuclei of degenerative acinar cells selectively seen in an early regression stage of the human lactating breast were periodic acid-Schiff (PAS)-reactive. In our preceding paper, this peculiar morphologic feature was designated as 'magentosis'. The present paper was aimed at histochemically clarifying the nature of the 'magentotic' nuclei. The diffuse PAS reactivity was not influenced by pretreatments with alpha-amylase, DNase, RNase, proteinase K, nor by hydrochloric acid or heating. The nuclei were negative for acid mucosubstances and secretory glycoproteins, and were unreactive with a variety of lectins. In contrast, the presence of single-stranded DNA stretches or breaks was proven. The 'magentotic' nuclei in non-heated paraffin sections were hybridized with a heat-denatured DNA probe for human DNA consensus sequences and were focally immunoreactive with an antibody to single-stranded DNA. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method turned to be positive after digestion by mung bean nuclease, a single-stranded DNA-specific enzyme. The 'magentotic' nuclei were further clearly labeled by the in situ nick translation method. The nucleoli were devoid of reactivity for both the PAS and single-stranded DNA signals. We propose that 'magentosis' represents a unique mode of cell death, distinct from apoptosis and necrosis or oncosis. The PAS reactivity in the 'magentotic' nuclei may be correlated with the occurrence of single-stranded stretches or breaks in the DNA chain.
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PMID:'Magentosis' in human lactating breast: a mode of cell death accumulating single-stranded DNA stretches or breaks. 1084 59

Lipopolysaccharide (LPS) of Burkholderia cepacia was purified by the conventional phenol-water extraction method (preparation BcLPS-1), followed by enzymatic treatments with DNase, RNase, trypsin, and proteinase K (preparation BcLPS-2), and finally by deoxycholate-phenol-water extraction (preparation BcLPS-3). Cells of LPS-hyporesponsive C3H/HeJ mice were activated by both the BcLPS-1 and the BcLPS-2 preparations but barely activated by BcLPS-3. When LPS-responsive C3H/HeN mice were used as targets, endotoxic activities such as lethal toxicity to galactosamine-sensitized mice, mitogenicity to spleen cells, and activation of macrophages to induce tumor necrosis factor alpha and interleukin-6 (IL-6) were strongly exhibited even by highly purified BcLPS-3 at levels comparable to those of the highly active enterobacterial LPS of Salmonella enterica serovar Abortus-equi (SaeLPS), used as the control. The ability of BcLPS-3 to activate murine macrophages for induction of IL-1beta was, however, much weaker than that of SaeLPS. Both accumulation of pro-IL-1beta protein and expression of IL-1beta mRNA in macrophages by stimulation with BcLPS-3 were much weaker than by stimulation with SaeLPS. These results indicate that LPS of B. cepacia has the potential to play a role as a pathogenic factor with strong activity comparable to that of usual enterobacterial LPS, but unlike the latter, this LPS has a relative lack of ability in the activation of murine macrophages to induce IL-1beta. The lack of IL-1beta-inducing ability appears to be caused by incomplete signal transduction somewhere in the upstream step(s) of IL-1beta gene transcription.
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PMID:Lipopolysaccharide of Burkholderia cepacia and its unique character to stimulate murine macrophages with relative lack of interleukin-1beta-inducing ability. 1134 28

Endothelins (ETs) are a family of potent vasoconstrictor and comitogenic polypeptides consisting of 21-amino acids. Using in situ hybridization, ET-1 mRNA has previously been localized to neuronal cell bodies in fourteen human brain regions. However, because in situ hybridization has a limited detection sensitivity of 20 mRNA copies per cell, ET-1 mRNA may be present in previously undetected areas. Hence, our objective was to localize ET-1 mRNA in specific human brain regions and astrocytic tumours using the more sensitive in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR). Human brain autopsy tissue and surgical cerebral tumour tissue were treated with proteinase K and DNase, followed by RT-PCR using primers specific for ET-1 mRNA and digoxygenin-labelled dUTP in the PCR mixture. The DIG-dUTP was localized with an immunodetection system. We demonstrate ET-1 mRNA labelling in twenty two of the twenty four brain regions studied including those regions in which ET-1 mRNA has been observed by in situ hybridization. In addition, the localization of ET-1 mRNA observed in astrocytomas suggests a role for ET-1 in tumour pathogenesis. In situ RT-PCR has proven to be highly sensitive in its ability to detect low mRNA expression at the cellular level. Our results confirm a role for ET-1 in the human nervous system.
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PMID:Cellular distribution of endothelin-1 mRNA in human brain by in situ RT-PCR. 1176 33

Campylobacter jejuni 81116 has been extensively investigated in studies on genes associated with the synthesis of Campylobacter lipopoly/lipooligosaccharides (LPS/LOS). Despite these investigations, data on the chemical structure of polysaccharides from C. jejuni 81116 have been absent. The present study was undertaken to fill that void. Biomass was grown in large quantities on agar medium, harvested and extracted by hot phenol-water extraction. Subsequently, extracts were treated by DNase, RNase and proteinase K to remove contaminants. After mild acid treatment, followed by preparative gel-permeation and anion-exchange chromatography, fractions were isolated and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, 1H,(13)C HMQC and HMBC experiments. These advanced investigations revealed the occurrence of two different polysaccharides in the approximate ratio of 3:1, each having a tetrasaccharide repeating unit. Polysaccharide A contained glucose, glucuronic acid and mannose, and is O-acetylated. Polysaccharide B contained glucose, galactose and N-acetylglucosamine. Importantly, polysaccharide A is acidic, whereas polysaccharide B is neutral. [carbohydrate structure: see text]
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PMID:Structures of two polysaccharides of Campylobacter jejuni 81116. 1243 86

Brucella is a Gram-negative facultative bacterium that persists intracellularly in macrophages. However, the intracellular survival mechanisms used by Brucella are not fully understood. Isolation of Brucella RNA from infected macrophages has been challenging, and the inability to isolate sufficient Brucella RNA from infected macrophages has contributed to the failure in understanding bacterial transcriptional events. We describe the isolation of sufficient Brucella abortus RNA from its infective host cell environment using osmotic lysis and RNase and DNase digestion. This method takes advantage of the B. abortus cell envelope that protects bacterial RNA and DNA. The cell envelope of B. abortus was digested using SDS/proteinase K (PK) that, importantly, inhibits any residual RNase after digesting macrophage RNA permitting the extraction of B. abortus RNA. In our experiments, 4.5 microg of RNA was routinely isolated from 1 ml bacterial culture and 2-9 microg of bacterial RNA from infected macrophages without detectable host cell RNA or DNA contamination. The method is rapid and uses inexpensive, commonly available reagents. Total bacterial RNA was isolated in quantities sufficient for RT-PCR and microarray analysis.
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PMID:Isolation of Brucella abortus total RNA from B. abortus-infected murine RAW macrophages. 1564 40

During mammalian follicle development, a fluid-filled antrum develops in the avascular centre of the follicle. We investigated the hypothesis that follicular fluid contains osmotically-active molecules, sufficiently large so as to not freely escape the follicular fluid. Such molecules could generate an osmotic differential and thus recruit fluid from the surrounding vascularised stroma into the antrum. Follicular fluid was collected from bovine follicles classified histologically as healthy (n = 4 pools) or atretic (n = 4 pools). Dialysis of the follicular fluid at 300 kDa or 500 kDa resulted in a reduction in colloid osmotic pressure of 35% and 60%, respectively, in fluid from healthy follicles and 29% and 80% from atretic follicles. Digestion of follicular fluid with Streptomyces hyaluronidase, chondroitinase ABC or DNase 1 followed by dialysis resulted in reductions in osmotic pressure of 43%, 53% and 43% respectively for fluids from healthy follicles and 34%, 20% and 31% for atretic follicles. Digestion with collagenase I, proteinase K, heparanase 1 or keratanase had no significant effect on the osmotic pressure of follicular fluid of healthy follicles. Ion exchange and size exclusion, Western blotting and ELISA identified the proteoglycans versican and inter-alpha trypsin inhibitor and the glycosaminoglycan hyaluronan in follicular fluid. We conclude that these molecules or aggregates of them are of sufficient size to contribute to the osmotic potential of follicular fluid and thus recruit fluid into the follicular antrum. DNA may also contribute but it is probably not a component that is regulated for this role.
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PMID:Formation of ovarian follicular fluid may be due to the osmotic potential of large glycosaminoglycans and proteoglycans. 1681 38


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