Cross-linked, degradable starch microspheres as carriers of paramagnetic contrast agents for magnetic resonance imaging: synthesis, degradation, and relaxation properties.

Biodegradable particles were produced by the cross-linking of starch with epichlorohydrin. Diethylenetriaminepenta-acetic acid (DTPA) was covalently linked to the particles by using DTPA bisanhydride. The small, gadolinium-labelled particles were 40-260% more efficient in vitro proton relaxation agents than the corresponding unbound chelate gadolinium-DTPA. The relaxation properties were dependent on the metal chelate, the particle size, the metal content, and the degree of substitution (d.s.). For the small gadolinium-DTPA particles, an increased d.s. decreased the rate of degradation by alpha-amylase.     

A dominant interfering mutation (CYR3) of the Saccharomyces cerevisiae RAS2 gene.

The dominant cyclic AMP-requiring mutation CYR3 had been previously reported as a mutation in the regulatory subunit of cyclic AMP-dependent protein kinase. However, recharacterization revealed that the CYR3 mutation was a nonconditional dominant lethal mutation and was a missense allele of RAS2 which results from the substitution of aspartic acid for glycine at amino acid 22.     

Enzymatic synthesis and isolation of thymidine diphosphate-6-deoxy-D-xylo-4-hexulose and thymidine diphosphate-L-rhamnose. Production using cloned gene products and separation by HPLC.

A two-step enzymatic synthesis of dTDP-L-rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rfb gene cluster of Salmonella enterica LT2. The intermediate dTDP-6-deoxy-D-xylo-4-hexulose was isolated after a 1-h reaction, using only dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89%). In a two-step reaction using dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase in the first step, and with NADPH, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase in the second hour of incubation, the dTDP-D-glucose was fully converted to dTDP-L-rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP-L-rhamnose, after protein precipitation, anion-exchange chromatography and desalting by gel chromatography, was 62%, corresponding to more than 150 mg, starting from 250 mg of dTDP-D-glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months. Both dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP-6-deoxy-D-xylo-4-hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier. Furthermore, an HPLC technique is presented for determining the net activity of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase, based on separation of dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.     

The disaccharide L-alpha-D-heptose1-->7-L-alpha-D-heptose1-->of the inner core domain of Salmonella lipopolysaccharide is accessible to antibody and is the epitope of a broadly reactive monoclonal antibody.

We generated a panel of mAb containing at least one specificity against each of the known chemotypes of the Salmonella LPS core domain and used them to investigate the accessibility of core determinants in smooth LPS. Most of the mAb were reactive with at the most three chemotypes of the core as determined by enzyme immunoassay and failed to bind smooth LPS or any of the complete cores of E. coli. One mAb, MASC1-MM3 (MM3), reacted with six different Salmonella core chemotypes, the R2 core of Escherichia coli and a variety of smooth LPS. This mAb reacted equally well with live and heat-killed bacteria. It bound to 123 of 126 clinical isolates of Salmonella and 11 of 73 E. coli strains in a dot-immunoblot assay. Typical ladder-like patterns of bands were observed after immunoblotting of this mAb against electrophoretically resolved smooth LPS from the five major serogroups of Salmonella species (A, B, C1, D1, and E). MM3 had no reactivity with BSA conjugates of O-Ag polysaccharides from the above serogroups confirming specificity for a core epitope. Polysaccharides derived from or synthetic saccharides representative of the various chemotypes of Salmonella LPS core were tested as competitive inhibitors of the binding of MM3 to LPS. The results led to a conclusion that MM3 recognizes the structure, L-alpha-D-Heptose1-->7-L-alpha-D-Heptose1-->disaccharide present as a branch in the Ra, Rb1, Rb2, Rb3 and Rc but lacking in the Rd1, Rd2, and Re chemotypes of the Salmonella LPS core. This disaccharide seems free and accessible on the basis of the previously calculated conformations of the Salmonella (Ra) and E. coli complete cores (R1, R2, R3, R4, and K12). It therefore defines or contains an epitope within the inner core subdomain of Salmonella LPS that is accessible to antibody in long-chained LPS and in intact bacteria with complete LPS.     

Selenium status of patients with Balkan endemic nephropathy

The selenium (Se) contents in common cereals in endemic and nonendemic areas in Serbia are very low. Plasma Se levels of both patients and healthy subjects, were also low, reflecting low Se intakes. Patients with Balkan endemic nephropathy (BEN) had significantly lower (p<0.05) plasma Se levels than healthy individuals, both from regions close to endemic areas, and from Belgrade. Mean plasma Se of BEN patients was slightly but insignificantly higher in samples taken immediately after dialysis than in those taken before, suggesting that very little of the Se present in plasma is dialyzable. Plasma SeGSH-Px activities before and after hemodialysis in both BEN and Nonendemic chronic renal failure (NCRF) patients were not significantly different, but BEN patients had lower enzyme activities than those with NCRF and healthy controls. In BEN patients, a significant correlation between plasma Se and SeGSH-Px activity was found. NCFR patients were with diagnoses: TBC of kidneys, chronic glomerulonephritis, chronic pyelonephritis, and polycystic kidneys.     

INTERACTIONS BETWEEN KURIL SEALS AND SALMON TRAP NET FISHERY IN THE COASTAL WATERS OF SOUTHEASTERN HOKKAIDO

An annual average of 163 Kuril seals was found dead in two years in salmon trap nets along the coastal waters of the Nemuro Peninsula and adjacent areas. The seal-caused damage to the total salmon catch at the salmon trap nets was concentrated in some of them, particularly No. 27, where seals killed or injured 5.1% of the catch in 1982, and 1.8% in 1983. Based on the proportion of Kuril seals among the dead seals in the trap nets, it was estimated that Kuril seals damaged 4.7% of the total salmon catch at No. 27 in 1982, and 1.7% in 1983. Not all seals that entered the trap net drowned; some killed or damaged salmon, and then escaped.     

The functional domain of adenylate cyclase associated with entry into meiosis in Saccharomyces cerevisiae.

Diploid yeast cells that carry a part of the CYR1 gene deficient in a region coding for the N-terminal domain of adenylate cyclase were growth arrested and accumulated unbudded cells after inoculation into complete medium or nitrogen-free medium, but produced many cells which had one or more buds after incubation in sporulation medium. The cells incubated in sporulation medium had abnormal spindles which were free from the spindle pole bodies, larger in size, or frequently distributed in cytoplasm. The levels of cyclic AMP in these cells did not decrease to the wild-type level after transfer to the sporulation medium and remained at a constant level. The results suggest that the N-terminal domain of adenylate cyclase is associated with the regulatory function for sporulation. The environmental signals for sporulation may be transferred to the adenylate cyclase system through a factor that negatively interacts with the N-terminal domain of this enzyme.     

Characterization of rabbit liver P450IIE1 synthesized in transformed yeast cells

cDNA for chimeric protein, P450(3P4), consisting of the amino-terminal 43 residues (the membrane-anchor region) of rabbit P450IIC14 and the remaining 447 residues of rabbit P450IIE1 was constructed, then cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. P450(3P4) thus synthesized in the transformed yeast cells was partially purified, and its spectral and catalytic properties were examined. In the oxidized state P450(3P4) exhibited a high-spin type absorption spectrum even in the absence of a substrate. The reduced CO complex of the P450 showed a Soret absorption maximum at 452 nm. P450(3P4) catalyzed aniline p-hydroxylation, N-nitrosodimethylamine demethylation, benzphetamine N-demethylation, and laurate and caprate (omega-1)-hydroxylation in the reconstituted system containing the P450 and NADPH-P450 reductase. These results indicate that P450(3P4) preparation obtained from the transformed yeast cells has spectral and catalytic characteristics identical with those of P450IIE1 purified from rabbit liver microsomes, confirming the substrate specificity reported of P450IIE1.     

Characterization of cyclic AMP-requiring yeast mutants altered in the regulatory subunit of protein kinase

The CYR3 mutant of yeast, Saccharomyces cerevisiae, partially accumulated unbudded cells and required cAMP for the best growth at 35 degrees C. The CYR3 mutation was partially dominant over the wild type counterpart and suppressed by the bcy1 mutation which is responsible for the deficiency of the regulatory subunit of cAMP-dependent protein kinase. The molecular weights of cAMP-dependent protein kinase and its catalytic and regulatory subunits were 160,000, 30,000, and 50,000, respectively. No significant differences in the molecular weights of cAMP-dependent protein kinase and the subunits were found between the wild type and CYR3 mutant strains. However, the cAMP-dependent protein kinase activity of CYR3 cells showed significantly higher Ka values for activation by cAMP at 35 degrees C than those of wild type and a clear difference in the electrophoretic mobility of the regulatory subunit was found between the wild type and CYR3 enzymes. The CYR3 mutation was suppressed by the IAC mutation which caused the production of a significantly high level of cAMP. The results indicate that the CYR3 phenotype was produced by a structural mutation in the CYR3 gene coding for the regulatory subunit of cAMP-dependent protein kinase in yeast.     

Characterization of Enkephalin Release from Rat Striatum

Abstract: Using antisera specific for methionine- and leucine-enkephalin, we studied the characteristics of the release of these peptides from rat striatal slices. Only 2–3% of the total tissue stores of enkephalin could be released by potassium depolarization; similar percentages were released from globus pallidus, thalamus, and nucleus accumbens. Enkephalin release from hippocampus could not be detected. The striatal release of both enkephalins was affected similarly by changes in potassium and calcium levels in the superfusion medium. Lithium has no effect on either basal or potassium-stimulated release; tyr-arg did not affect basal release of either peptide. Striatal enkephalin levels were stable during the short-term incubation periods used in these experiments.