Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.     

Changes in Esterification of the Uronic Acid Groups of Cell Wall Polysaccharides during Elongation of Maize Coleoptiles

Cell walls of grasses have two major polysaccharides that contain uronic acids, the hemicellulosic glucuronoarabinoxylans and the galactosyluronic acid-rich pectins. A technique whereby esterified uronic acid carboxyl groups are reduced selectively to yield their respective 6,6-dideuterio neutral sugars was used to determine the extent of esterification and changes in esterification of these two uronic acids during elongation of maize (Zea mays L.) coleoptiles. The glucosyluronic acids of glucuronoarabinoxylans did not appear to be esterified at any time during coleoptile elongation. The galactosyluronic acids of embryonal coleoptiles were about 65% esterified, but this proportion increased to nearly 80% during the rapid elongation phase before returning to about 60% at the end of elongation. Methyl esters accounted for about two-thirds of the total esterified galacturonic acid in cell walls of unexpanded coleoptiles. The proportion of methyl esters decreased throughout elongation and did not account for the increase in the proportion of esterified galactosyluronic acid units during growth. The results indicate that the galactosyluronic acid units of grass pectic polysaccharides may be converted to other kinds of esters or form ester-like chemical interactions during expansion of the cell wall. Accumulation of novel esters or ester-like interactions is coincident with covalent attachment of polymers containing galactosyluronic acid units to the cell wall.     

Purification and some properties of a phospholipase A2 from bovine platelets

An intracellular form of phospholipase A2 was purified about 47,500-fold to near homogeneity from bovine platelets 100,000 x g supernatant by sequential use of column chromatographies on Heparin-Sepharose, DEAE-Sephacel, Butyl-Toyopearl, Sephacryl S-300, DEAE-5PW HPLC, TSK G 3000 SW HPLC and Mono Q FPLC. The final preparation showed a single band on SDS-polyacrylamide gel, and its molecular mass was estimated to be approximately 100,000 daltons. The purified PLA2 showed maximal activity at alkaline pH(pH 9.0-10.0) and considerable activity at 0.3-1.0 microM calcium concentration. It hydrolyzed phosphatidylcholine containing arachidonate at sn-2 position with high selectivity in comparison to linoleate.     

Insulin-sensitive myelin basic protein phosphorylation on tyrosine residues.

Rat brain plasma membranes were solubilized in detergent and a glycoprotein-enriched fraction was obtained by lectin affinity chromatography. This glycoprotein fraction contained insulin receptors, as well as protein kinases capable of phosphorylating some exogenously added substrates such as MAP2 (microtubule associated protein 2) and MBP (myelin basic protein), but not ribosomal protein S6. Phosphoamino acid analysis of MAP2 and MBP showed that phosphotyrosine residues, as well as phosphoserine/phosphotheronine residues, were present in both proteins under basal conditions. Whereas the addition of insulin to the rat brain membrane glycoprotein fraction in vitro had no effect on MAP2 phosphorylation, MBP phosphorylation was stimulated 2.7-fold in response to insulin. This phenomenon was dose-dependent, with half-maximal stimulation of MBP phosphorylation observed with 2 nM insulin. Phosphoamino acid analysis of MBP indicated that insulin stimulated the phosphorylation of tyrosine residues nearly three-fold, whereas the phosphorylation of serine or threonine residues was not increased. These results identify MBP as a substrate for the rat brain insulin receptor tyrosine-specific protein kinase in vitro.     

An endonuclease activity in human polymorphonuclear neutrophils that removes 8-hydroxyguanine residues from DNA+

An endonuclease that specifically removes 8-hydroxyguanine (oh8Gua) from DNA has been isolated from Escherichia coli. As the amount of oh8Gua produced in DNA of X-ray-irradiated mice is known to decrease with time after irradiation, an attempt was made to find a similar activity in human polymorphonuclear neutrophils (PMNs) using a synthetic dsDNA containing oh8Gua as a substrate. The PMN enzyme was isolated free of other DNases, and found to cleave the substrate DNA simultaneously at 2 sites, the phosphodiester bonds 5' and 3' to oh8Gua, producing free hydroxyl and phosphate groups, respectively. The enzyme showed almost no activity on DNAs containing other kinds of modified base tested or mismatched DNA. Thus human cells also contain an endonuclease that specifically removes oh8Gua residues from DNA.     

Microcarrier culture of bowes melanoma cells in serum-free medium with Human plasma fraction IV-4+ V

Bowes melanoma cells were cultivated successfully in a serum-free medium which was constructed by the concept of maximum retention of proteins from fractionated human plasma having growth stimulatory activities. The cells could be cultivated in the serum-free medium without any adaptation period. The major serum-free component of the medium was the fraction IV-4 + V of the Cohn fractionation process of human plasma. Approximately six times increase of tissue-type plasminogen activator (t-PA) activity as compared with that in serum-free medium even though the cell growth was much slower. In addition, the growth stimulatory activities of thrombin and fibronectin were investigated during the cultivation of Bowes melanoma cells in this serum-free medium. These proteins contributed significantly to the enhanced growth of cells by reducing doubling time to 25 and 35 h as compared with 55 h in the serum-free medium without them. Especially, fibronectin supported cells to propagate near to the maximum cell density achieved in the medium with 10% FBS.     

Effects of periodic backflushing on ultrafiltration performance.

Periodic backflushing was introduced to a membrane separation process to improve the performance. Hemoglobin (M.W. = 62,500) and dextran (M.W. = 10,000) were used as model compounds. Filtration performance of an ultrafiltration membrane system (Amicon hollow fiber membrane, H1P30-43, molecular weight cutoff = 30,000) was measured in terms of apparent permeability and retention coefficient of dextran to determine the effects of backflushing frequency and duration of one cycle. An optimum frequency around 0.2 min-1 existed to give a maximum permeability while the retention of dextran decreased with increasing frequencies. The improvement in permeability by periodic backflush was more than doubled. The retention of dextran decreased as backflushing duration was increased in one cycle. With the duration of 33.75 s, the retention of dextran was less than 50% and dextran output was 1.14 g/h, which was 1.3 times the value without backflushing. Also, periodic backflush made possible the long-term filtration of yeast cells for more than 20 h.     

Identification of new urinary metabolites of trimeprazine in rats by gas chromatography—mass spectrometry

The metabolites of trimeprazine were identified in urine of rats by gas chromatography—mass spectrometry. After the oral administration of trimeprazine, the urinary metabolites were extracted with diethyl ether before or after hydrolysis with β-glucuronidase. The identified metabolites were N-demethyltrimeprazine, 3-hydroxytrimeprazine, N-demethyl-3-hydroxytrimeprazine and trimeprazine sulphoxide.     

Molecular cloning of cDNAs derived from a novel human intestinal mucin gene

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.     

Comparison of the DNA-alkylating properties and mutagenic responses of a series of S-(2-haloethyl)-substituted cysteine and glutathione derivatives

The mutagenicity of 1,2-dibromoethane is highly dependent upon its conjugation to glutathione by the enzyme glutathione S-transferase. The conjugates thus formed can react with DNA and yield almost exclusively N7-guanyl adducts. We have synthesized the S-haloethyl conjugates of cysteine and glutathione, as well as selected methyl ester and N-acetyl derivatives, and compared them for ability to produce N7-guanyl adducts with calf thymus DNA. The cysteine compounds were found to be more reactive toward calf thymus DNA and yielded higher adduct levels than did the glutathione compounds. Adduct levels tended to be suppressed when there was a net charge on the compound and were not affected by substitution of bromine for chlorine, as expected for a mechanism known to involve an intermediate episulfonium ion. Sequence-selective alkylation of fragments of pBR322 DNA was investigated. The compounds produced qualitatively similar patterns of alkylation, with higher levels of alkylation at runs of guanines. The compounds were also tested for their ability to act as direct mutagens in Salmonella typhimurium TA98 and TA100. None of the compounds caused mutations in the TA98 frameshift mutagenesis assay. In the strain TA100, where mutation of a specific guanine by base-pair substitution produces reversion, all compounds were found to produce mutations, but the levels of mutagenicity did not correlate at all with the levels of DNA alkylation. The ratio of mutations to adducts varied at least 14-fold among the various N7-guanyl adducts examined.(ABSTRACT TRUNCATED AT 250 WORDS)