The yeast SEC17 gene product is functionally equivalent to mammalian alpha-SNAP protein.

The SEC17 gene of Saccharomyces cerevisiae is required for vesicular transport between the endoplasmic reticulum and the Golgi apparatus. Here we report that the product of the SEC17 gene has the exact biochemical properties expected for a yeast homologue of the mammalian transport factor, alpha-SNAP. The DNA sequence of SEC17 codes for a protein of predicted molecular mass of 33 kDa. Immunoblotting indicates that Sec17p fractionates as a peripheral membrane protein and is mostly soluble when overexpressed, suggesting the presence of a saturable membrane receptor for Sec17p. Sec17p was purified from yeast cytosol using a SNAP-dependent in vitro mammalian Golgi transport assay. Kinetic analysis using this assay shows Sec17p acts temporally close to the fusion of transport vesicles with the medial Golgi compartment. In yeast extracts, Sec17p binds to Sec18p with a 1:1 stoichiometry. The interaction between Sec17p and Sec18p requires an activity provided by yeast membranes, and this putative membrane receptor activity is not extracted by high salt treatment of membranes.     

Chronopharmacokinetics and Cardiovascular Effects of Nifedipine

Orcadian phase dependency in pharmacokinetics and hemodynamic effects on blood pressure and heart rate of different galenic formulations of nifedipine (immediate-release, sustained-release, and i.v. solution) were studied in healthy subjects or in hypertensive patients. Pharmacokinetics of immediate-release but not sustained-release and i.v. nifedipine were dependent on time of day: immediate-release nifedipine had higher C_max (peak concentration) and shorter t_max (time-to-peak concentration) after morning than evening application, and bioavailibility in the evening was reduced by about 40%. Orcadian rhythm in estimated hepatic blood flow as determined by indocyanine green kinetics may contribute to these chronokinetics. A circadian time dependency was also found in nifedipine-induced effects on blood pressure and heart rate as monitored by 24-h ambulatory blood pressure measurements. In conclusion, the dose response relationship of oral nifedipine is influenced by the circadian organization of the cardiovascular system as well as by the galenic drug formulation.     

Defects in fruiting body development caused by Tn5 lac insertions in Myxococcus xanthus.

Mutations caused by insertions of Tn5 lac that block development are rare. At least six of the eight mutations examined appeared to be regulatory. Three of these were found to disrupt social motility, suggesting a particular importance for this function. One other occurred in a known cell-cell interaction gene, bsgA, and the remaining two were located in genes operative early in the developmental program.     

Physical and chemical scavenging of singlet molecular oxygen by tocopherols

Singlet molecular oxygen (1O2) arising from the thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate was used to assess the effectiveness of alpha-, beta-, gamma-, and delta-tocopherol in the physical quenching as well as the chemical reaction of 1O2. The relative physical quenching efficiencies of the tocopherol homologs were found to decrease in the order of alpha greater than or equal to beta greater than gamma greater than delta-tocopherol. The ability of physical quenching depends on a free hydroxyl group in position 6 of the chromane ring. Chemical reactivity of the tocopherol homologs with 1O2 was low, accounting for 0.1-1.5% of physical quenching with beta-tocopherol showing particularly low reactivity, resulting in the sequence alpha greater than gamma greater than delta greater than beta-tocopherol. Tocopheryl quinones were products of all tocopherol homologs, and in addition a quinone epoxide was a major product from gamma-tocopherol. This quinone epoxide was not cleaved by rat liver microsomal epoxide hydrolase; however, it reacted further with 1O2. It is concluded that methylation in position 5 of the chromane ring enhances physical quenching of 1O2, whereas chemical reactivity is favored by a methylated position 7. In view of the fact that beta-tocopherol is as effective as alpha-tocopherol in physical quenching of 1O2 but shows very low chemical reactivity, this tocopherol homolog might be particularly suitable for biological conditions in which an accumulation of oxidation products might weaken the antioxidant defense.     

Avian alcohol dehydrogenase. Characterization of the quail enzyme, functional interpretations, and relationships to the different classes of mammalian alcohol dehydrogenase

The primary structure of the major quail liver alcohol dehydrogenase was determined. It is a long-chain, zinc-containing alcohol dehydrogenase of the type occurring also in mammals and hence allows judgement of the gene duplications giving rise to the classes of the human alcohol dehydrogenase system. The avian form is most closely related to the class I mammalian enzyme (72-75% residue identity), least related to class II (60% identity), and intermediately related to class III (64-65% identity). This pattern distinguishes the mammalian enzyme classes and separates classes I and II in particular. In addition to the generally larger similarities with class I, the avian enzyme exhibits certain residue patterns otherwise typical of the other classes, including an extra Trp residue, present in both class II and III but not in class I, with a corresponding increase in the UV absorbance. The avian enzyme further shows that a Gly residue at position 260 previously considered strictly conserved in alcohol dehydrogenases can be exchanged with Lys. However, zinc-binding residues, coenzyme-binding residues, and to a large extent substrate-binding residues are unchanged in the avian enzyme, suggesting its functional properties to be related to those of the class I mammalian alcohol dehydrogenases. In contrast, the areas of subunit interactions in the dimers differ substantially. These results show that (a) the vertebrate enzyme classes are of distant origin, (b) the submammalian enzyme exhibits partly mixed properties in relation to the classes, and (c) the three mammalian enzyme classes are not as equidistantly related as initially apparent but suggest origins from two sublevels.     

Synthesis and structural stability of helichrome as an artificial hemeproteins

A detailed procedure is described for the synthesis of helichrome, which is the first successful example of polypeptide-based artificial hemeprotein. The segment synthesis-condensation approach used for the assembly of small proteins has proven to be extremely useful for protein mimetics as well. The final deprotection was performed using the TMSOTf-thioanisole method instead of the less-convenient hydrogen fluoride method. The unfolding transition of the alpha-helical conformation of helichrome induced by guanidine hydrochloride was studied to understand the stability and dynamics of the folded structure. The resulting parameters (C0.5 = 5.2 M and delta GH2O = -4.4 kcal mol-1) characterizing helichrome denaturation were comparable to that of native globular proteins.     

Determination of plasma free fatty acids, free cholesterol, cholesteryl esters, and triacylglycerols directly from total lipid extract by capillary gas chromatography

An accurate capillary gas chromatographic method using different internal standards for determining free fatty acids, cholesterol, cholesteryl esters, and triacylglycerols in plasma and other biological sources is described. It is designed to give information about species composition and, consequently, more detailed information about changes in lipid metabolism of patients suffering from metabolic disorders. After plasma extraction the lipids, except phospholipids, are directly examined without any further derivatization. For free fatty acid determination the programmed temperature vaporizer (PTV) injector was heated from 40 degrees C (sample introduction) to 190 degrees C. In a second gas chromatographic run the PTV-injector system was heated from 60 degrees C (sample introduction) to 400 degrees C, enabling the determination of free cholesterol, cholesteryl esters, and triacylglycerol species, differing in the number of carbon atoms. Evaluation of the values obtained resulted in coefficients of variation (%) of 1.0-2.8, 2.0, 1.29-2.24, and 2.8, for free fatty acid standards, plasma free fatty acids, cholesterol and cholesteryl ester standards, and plasma total cholesterol, respectively. Free fatty acids, cholesterol, and cholesteryl esters were not influenced by storage of plasma at -24 degrees C up to 4 days prior to extraction. The results of the gas chromatographic method and the enzymatic methods correlated well. Determination by gas chromatography yielded higher total cholesterol and lower triacylglycerol values than those values obtained by enzymatic methods.     

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 micrograms/ml MMC, (3)HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of (3)HdT incorporation than did those sorted from the first half of S. Fanconi's anemia cells exhibited a marked accumulation in the G(2) + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with .0.5 micrograms/ml MMC, the G(2) + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.02. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a G(2) + M increment than did FA cells, even after exposure to 0.1 micrograms/ml MMC. Examination of cells sorted from the G(2) + M peak revealed that MMC-treated FA cells were blocked prior to mitosis. To determine whether the response of FA cells was specific for bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G(2) + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.     

Oxygen uptake of carp,Cyprinus carpio,swimming in normoxic and hypoxic water

Synopsis Oxygen uptake (V_{o 2}) was measured in carp of approximately 40 cm length swimming at controlled variable oxygen tensions (P_{o 2}). At P_{o 2}> 120 mm Hg V_{o 2} increased with an increase in swimming speed from 5.6 to 11.3 cm · sec^–1. Extrapolation of V_{o 2} to zero activity at P_{o 2} = 140 mm Hg revealed a standard O₂ uptake of 36.7 ml O₂ · kg^–1 · h^–1 at 20° C. At the lowest swimming speed (5.6 cm · s^–1) the oxygen uptake increased when the water P_{o 2} was reduced. A near doubling in V_{o 2} was seen at P_{o 2} = 70 mm Hg compared to 140 mm Hg. At higher swimming speeds in hypoxic water V_{o 2} decreased relative to the values at low swimming speeds. As a result the slope of the lines expressing log V_{o 2} as a function of swimming speed decreased from positive to negative values with decreasing P_{o 2} of the water. pH of blood from the caudal vein drawn before and at termination of swimming at P_{o 2} = 70 mm Hg and 100 mm Hg did not show any decrease in relation to rest values at P_{o 2} = 140 mm Hg. Blood lactate concentration did not increase during swimming at these tensions.