Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The incorporation of radioactivity into various cells in the sequence of spermatogenesis was measured by preparing highly purified spermatozoan nuclei from the cauda epididymidis of mice at daily intervals after injection of (3H)thymidine. The stages of differentiation of these sperm at the time of thymidine administration were calculated from the kinetics of spermatogenesis. The procedure for purification of sperm nuclei included sonication, mechanical shearing, and treatment with trypsin, DNase, Triton X-100, 2M NaC1, and sodium dodecyl sulfate. DNA was isolated from these nuclei by treatment with dithiothreitol and pronase, followed by phenol extraction and ethanol precipitation. The levels of radioactivity in the epididymal sperm head preparations were low (less than 13 dpm/mouse) for 27 days after injection, and then rose dramatically to over 4 times 104 dpm/mouse. Further experiments demonstrated that the 11 dpm of 3H radioactivity contained in sperm heads at 21 or 26 days after injection of (3H)TdR was significantly above background and contamination levels from other cells or other sources. Most of the radioactivity was in the sperm DNA and represented incorporation of tritium from (3H)TdR into the nuclear DNA of meiotic cells at 0.002 percent of the rate of incorporation into S-phase cells. Little, if any, (3H)TdR was incorporation into the DNA of spermatids. The levels of DNA synthesis during the meiotic prophase in the mouse appear to be much lower than those reported for other organisms.
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PMID:Meiotic DNA synthesis during mouse spermatogenesis. 110 31

A method is described for isolating Clara cells from the mouse lung that does not require the technique of elutriation. Mouse lungs totally perfused of blood are instilled with crystalline trypsin (0.25%) and incubated for the optimum time of 15 min. The lung tissue is chopped, mechanically agitated, and sequentially filtered to obtain a primary digest of 3 to 5 x 10(6) cells. Clara cells, identified routinely by histochemical localization of NADPH diaphorase, using the stain nitrotetrazolium blue (NBT), accounts for between 20 to 40% of the cells in the primary digest. Layering the cells of the primary digest on a discontinuous Percoll gradient followed by centrifugation gives rise to a major band of cells, 52% that are Clara cells (0.77 +/- 0.28 x 10(6)/mouse). A second method was devised to purify the Clara cells by simply centrifuging (32g, 6 min, 10 degrees C) the primary digest and discarding the supernatant that contained only a few NBT positive cells. When this process was repeated three times, the final pellet contained 68% Clara cells realizing 0.55 +/- 0.16 x 10(6) cells/mouse. The cells have typical Clara cell morphology as confirmed by electron microscopy and have a high level of P-450 enzymes (7-ethoxycoumarin deethylase and coumarin hydroxylase). Furthermore, the primary digests and the purified isolates contain less than 1% alveolar Type II cells, although such cells, identified by the histochemical localization of alkaline phosphatase, can be obtained by a second, more extensive digestion procedure. The simple procedure described for the isolation of mouse Clara cells could be further advanced if methods could be devised to prevent the loss of NADPH diaphorase activity during enzymatic digestion and cell centrifugation.
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PMID:Isolation of Clara cells from the mouse lung. 220 Jun 69

Degradation of internalized insulin was studied after binding at 25 degrees C and 37 degrees C to isolated hepatocytes. The cells were washed to avoid extracellular insulin contamination. Degradation of both, intracellular and extracellular 125I-insulin, was measured with TCA and insulin antibody. In these conditions binding at 25 degrees C and 37 degrees C was equal but both intra and extracellular degradation were greater at 37 degrees C than at 25 degrees C. At both temperatures, intracellular degradation was greater than extracellular degradation with accumulation of degraded and non-degraded intracellular insulin. To study in what state hepatocytes release internalized insulin into the medium, 125I-insulin association was performed at an intermediate temperature (30 degrees C). Extracellular insulin contamination (whether associated or not) was avoided by three methods: 1) washing; 2) treatment with insulin degrading enzyme(s) and washing; 3) treatment with insulin degrading enzyme(s) then with trypsin and washing. Kinetics of radioactivity released from the cells was identical in the three conditions and the radioactivity was released throughout the experiments. Complete degradation of the released insulin was observed by gel filtration when the previous binding was 0.4 ng insulin/10(6) cells. When the dose of associated insulin increased (25 ng/10(6) cells) 3.5% of non-degraded insulin was liberated and when the dose was 14,300 ng/10(6) cells, the insulin released was 44.3%. In one experiment during the first 30 min, the insulin released was 52.88% and in the last 45 min 39.59%. To study the biologic behavior of the insulin released from cells, a group of mice were injected with this insulin (8.4 mU/mouse) and blood glucose was measured. The released insulin behaved as intact insulin as far as blood glucose responses were concerned. We may conclude that liver cells have the ability to internalize insulin and release biologically active insulin after accumulation.
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PMID:Internalization and release of insulin from hepatocytes. 267 18

Angiotensin converting enzyme (ACE) plays an important role in the regulation of renal blood pressure by the hydrolysis of the inactive precursor peptide angiotensin I to the potent vasopressor angiotensin II. Renal ACE is a surface membrane protein of both endothelium and tubular epithelium. Enzymatically active ACE was isolated from renal homogenates by chromatography using an affinity column constructed by linking an ACE inhibitor, lisinopril, to Affi-Gel 15. Analysis of eluates from this column showed that ACE activity was increased greater than 500-fold. SDS-polyacrylamide gel electrophoresis demonstrated a single band of molecular weight 144 kD (mouse) and 149 kD (bovine). N-terminal amino acid sequence analysis revealed: (formula; see text) Though bovine ACE has one additional N-terminal amino acid, these two partial sequences are highly homologous (16 of 20 positions are identical). Mouse ACE was digested with trypsin and the peptides were isolated by reverse phase HPLC. Analysis of the amino acid sequences showed that these tryptic peptides were unique to ACE. Thus, we were able to isolate ACE from bovine and mouse kidneys and show that they had substantial structural homology. They were also quite similar to that from rabbit lung.
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PMID:Partial protein sequence of mouse and bovine kidney angiotensin converting enzyme. 283 38

Ciliary neurotrophic factor (CNTF) is a protein supporting the in vitro survival of a characteristic spectrum of embryonic chicken and rat peripheral neurons. High-speed supernatants of extracts from two neuroblastoma (NB) cell lines--the mouse C 1300 N2a and the human IMR 32--mimic the effects of CNTF on identical target neurons. Promotion of survival is dose-dependent with an ED50 of 80 micrograms (IMR 32) and 140 micrograms (C 1300 N2a) of protein per ml and saturable at plateau values for surviving neurons identical to those achieved with purified CNTF. Small amounts of a CNTF-like material are also detectable in medium conditioned by NB cells. The activity is destroyed by heat and trypsin and not blocked by antibodies to (mouse) nerve growth factor. Unlike the neurite-promoting and neuronal-survival modulating agent laminin, it cannot be depleted on poly(L-alpha-ornithine)-coated plastic surfaces. NB IMR 32 cell extracts were electrophoresed using NaDodSO4/PAGE and transferred to nitrocellulose. Ciliary ganglion neurons seeded on the blotting paper in culture medium lacking CNTF ("cell blot") exclusively survive on two distinct bands with apparent molecular masses of 24 and 48 kDa. Twenty-four kilodaltons is the molecular mass of a CNTF purified from rat sciatic nerve. These results suggest that NB cells may contain a CNTF-like protein and provide further evidence that neurons may store neurotrophic factors. Purified (chicken) CNTF failed to affect proliferation and neurite growth of NB cells. The biological relevance of CNTF for NB cells, therefore, remains to be elucidated.
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PMID:Neuroblastoma cells contain a trophic factor sharing biological and molecular properties with ciliary neurotrophic factor. 347 25

A liver DNA synthesis promoter activity was detected in human plasma from subjects with hepatitis. The assay procedure consisted of intraperitoneal injection into mice of aliquots of plasma, previously chromatographed on Sephadex G-25. After 24 hr, [3H]thymidine was injected and its incorporation into liver DNA measured. The increase in [3H]thymidine uptake of injected mice was not detected in those administered plasma from normal subjects (basal [3H]thymidine incorporation was that corresponding to saline-injected mouse values). At a maximal effective dose (0.3 mg protein per mouse), plasma from subjects with hepatitis increased the mitotic index of mouse liver hepatocytes; at the same dose, plasma from normal subjects had no effect. This DNA synthesis promoter activity appears to be a protein, as it is sensitive to trypsin digestion and heat.
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PMID:Liver DNA synthesis promoter activity detected in human plasma from subjects with hepatitis. 373

A protein was isolated from plasma of partially (70%) hepatectomized rats that, injected in mice, increases the uptake of [3H]thymidine by liver DNA by 200-300% over that by injected control saline. The purification procedure consists essentially of three chromatography steps, employing Sephadex G-75, DEAE-cellulose and hydroxyapatite. The hepatic promoter (HP) preparation shows a single band in SDS/polyacrylamide (15%)-gel electrophoresis (silver stained), with an Mr of 64 000; its activity is suppressed by trypsin or pepsin and is unaffected by deoxyribonuclease or ribonuclease. On injection into mice (150 ng/mouse), it increases the mitotic index of the liver. It shows organ-specificity, acting on liver but not on spleen, kidney, lung or brain. In primary liver cultures, it produces an increase in uptake of [3H]thymidine into DNA in the range 1-10 ng/ml. In this system in vitro, it increases the uptake of 22Na+ immediately after addition.
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PMID:Purification of a liver DNA-synthesis promoter from plasma of partially hepatectomized rats. 374 89

The complete nucleotide sequence of the extracellular glucoamylase gene STA1 from the yeast Saccharomyces diastaticus has been determined. A single open reading frame codes for a 778-amino-acid protein which contains 13 potential N-glycosylation sites. In the 5'- and 3'-flanking regions of the gene, there are striking sequence homologies to the corresponding regions of ADH1 for alcohol dehydrogenase and MAT alpha 2 for mating type control in the yeast Saccharomyces cerevisiae. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. The presumptive signal sequence showed a significant homology to that of Bacillus subtilis alpha-amylase precursor. The next segment, of ca. 320 amino acids, contains a threonine-rich tract in which direct repeat sequences of 35 amino acids exist, and is bordered by a pair of basic amino acid residues (Lys-Lys) which may be a proteolytic processing signal. The carboxy-terminal half of the precursor is a presumptive glucoamylase which contains several peptide segments showing a high degree of homology with alpha-amylases from widely diverse organisms including a procaryote (B. subtilis) and eucaryotes (Aspergillus oryzae and mouse). Analysis of both the nucleotide sequence of the STA1 gene and the amino acid composition of the purified glucoamylase suggested that the putative precursor is processed to yield subunits H and Y of mature enzyme by both trypsin-like and chymotrypsin-like cleavages.
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PMID:Nucleotide sequence of the extracellular glucoamylase gene STA1 in the yeast Saccharomyces diastaticus. 391 17

A cell line of a benign tumor, trichilemmoma, was established in vitro and has been maintained in culture for 1.5 years with more than 30 passages. Plating efficiency was less than 0.1%, and population doubling time was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) e was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) e was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) as observed in vivo. Gas chromatographic analysis revealed that extracted glycogen was composed of glucose alone. Chromosome analyses with trypsin-Giemsa banding showed an abnormal karyotype with hypodiploid modal numbers of 44 and 45. There were four marker chromosomes observed in 100% of cells in 100 metaphase cells examined. Cells did not grow on fibroblast monolayers or in soft agar in vitro but did induce tumors in athymic nude mice (12 of 15) after the s.c. injection of tissue-cultured cells (2.5 x 10(6) to 4.5 x 10(7) cells/mouse). The histological characteristics of the tumors in nude mice were similar to those of the original tumor. This is the first time, to our knowledge, that a benign human tumor cell line has been established in vitro which can induce tumors in nude mice.
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PMID:Morphological, biological, and biochemical characteristics of a benign human trichilemmoma cell line in vivo and in vitro. 616 63

The two most basic beta-bungarotoxins (beta 3- and beta 4-toxins) and another, less neurotoxic beta-bungarotoxin (beta 5-toxin) were purified from Bungarus multicinctus venom, by a combination of CM-Sephadex C-25 column chromatography and Sephadex G-75 gel filtration. The three toxins consisted of two dissimilar polypeptides (A chain, 120 amino acid residues; B chain, 60 residues). The LD50 values of the beta 3- and beta 4-toxins were 0.066 micrograms and 0.072 micrograms/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. beta 5-Toxin was weaker in neurotoxicity (LD50, 0.13 micrograms/g of mouse) than the others, and its phospholipase activity was 47.6 units/mg of toxin. Each toxin was separated into RCM-A and RCM-B chains after reduction and S-carboxymethylation. The RCM-polypeptides were maleylated and digested with TPCK-trypsin. The tryptic peptides were sequenced with manual Edman degradation or the dansyl-Edman method. The final alignment of the tryptic peptides from the respective RCM-polypeptides was deduced on the basis of the amino acid sequences of the A and B chains of beta 1-bungarotoxin (beta 1-toxin). The amino acid sequences of the A chains of the beta 3- and beta 4-toxins were identical but differed from those of the A chains of the beta 1- and beta 2-toxins by 4 amino acid substitutions in the COOH-terminal portions (residues 109-120) and substitution at position 87. The amino acid sequences of the B chains of the beta 3- and beta 4-toxins differed from each other, but they were identical with those of the B chains of the beta 1- and beta 2-toxins, respectively. The amino acid sequence of the A chain of beta 5-toxin differed from that of the A chain of beta 1-toxin by consecutive substitutions in residues 55-60 and substitutions at positions 23, 87, and 89. The amino acid sequence of the B chain of beta 5-toxin was identical with those of the B chains of beta 1- and beta 3-toxin. From our results on the effects of the amino acid displacements found in the A chains on the neurotoxicity, it was concluded that the COOH-terminal portion in the A chains was not essential to their neurotoxicity, whereas the region of residues 55-60 in the A chains appeared to participate in the constitution of the neurotoxically active site of the beta-toxins.
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PMID:Amino acid sequences of three beta-bungarotoxins (beta 3-, beta 4-, and beta 5- bungarotoxins) from Bungarus multicinctus venom. Amino acid substitutions in the A chains. 709 5


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