Inhibitors of sterol synthesis. Chemical syntheses and spectral properties of 26-oxygenated derivatives of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

26-Oxygenated derivatives of delta 8(14)-15-ketosterols have been synthesized from (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one (IX) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Partial hydrolysis of IX gave a mixture, from which the 3 beta,26-diol II and the 26-acetate (XI) and 3 beta-acetate (X) monoesters were isolated. Mitsunobu reaction of XI followed by hydrolysis gave (25R)-3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (VI). Oxidation of XI with pyridinium chlorochromate followed by hydrolysis of the acetate gave (25R)-26-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione (VII). Oxidation of X with Jones reagent followed by hydrolysis of the acetate gave (25R)-3 beta-hydroxy-15-keto-5 alpha-cholest-8(14)-en-26-oic acid (IVa). Jones oxidation of II gave (25R)-3,15-diketo-5 alpha-cholest-8(14)-en-26-oic acid (VII). 1H and 13C nuclear magnetic resonance assignments and analyses of mass spectral fragmentation data are presented for each of the new compounds and their derivatives. The 3,15-diketone VII was found to be highly active in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells, with a potency comparable to that of I. In contrast, 3 alpha,26-diol VI was less potent than I or VII. The two carboxylic acid analogs IVa and VIII were considerably less potent than VI in lowering the levels of HMG-CoA reductase activity.     

Similar Intracellular Location and Stimulus Reactivity,but Differential Mobility of Tailless (Vicia faba) and Tailed Forisomes (Phaseolus vulgaris) in Intact Sieve Tubes

Sieve elements of legumes contain forisomes—fusiform protein bodies that are responsible for sieve-tube occlusion in response to damage or wound signals. Earlier work described the existence of tailless and tailed forisomes. This study intended to quantify and compare location and position of tailless (in Vicia faba) and tailed (in Phaseolus vulgaris) forisomes inside sieve elements and to assess their reactivity and potential mobility in response to a remote stimulus. Location (distribution within sieve elements) and position (forisome tip contacts) of more than altogether 2000 forisomes were screened in 500 intact plants by laser scanning confocal microscopy in the transmission mode. Furthermore, we studied the dispersion of forisomes at different locations in different positions and their positional behaviour in response to distant heat shocks. Forisome distribution turned out to be species-specific, whereas forisome positions at various locations were largely similar in bushbean (Phaseolus) and broadbean (Vicia). In general, the tailless forisomes had higher dispersion rates in response to heat shocks than the tailed forisomes and forisomes at the downstream (basal) end dispersed more frequently than those at the upstream end (apical). In contrast to the tailless forisomes that only oscillate in response to heat shocks, downstream-located tailed forisomes can cover considerable distances within sieve elements. This displacement was prevented by gentle rubbing of the leaf (priming) before the heat shock. Movement of these forisomes was also prohibited by Latrunculin A, an inhibitor of actin polymerization. The apparently active mobility of tailed forisomes gives credence to the idea that at least the latter forisomes are not free-floating, but connected to other sieve-element structures.     

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.     

牛膝多糖抑制大肠埃希菌细胞粘附的实验研究

目的 :研究牛膝多糖能否影响大肠埃希菌对细胞的粘附。方法 :使用 Hela细胞进行了粘附试验及粘附抑制试验。结果 :发现牛膝多糖浓度为 0 .8mg/ml时 ,对细菌的细胞粘附抑制最为明显 ,粘附率由(2 5 7.0± 5 .2 )个细菌 /细胞 降低到 (63 .6± 3 .6)个细菌 /细胞 。结论 :牛膝多糖对大肠埃希菌的细胞粘附具有抑制作用 ,提示该多糖具有调节肠道微生态的潜在应用价值     

Quantitative determination of hemoglobin and cytochemical staining for peroxidase using 3,3',5,5'-tetramethylbenzidine dihydrochloride, a safe substitute for benzidine.

The quantitative determination of hemoglobin employing 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB-d) is described. This agent can also be used for staining myeloperoxidase-containing granules in granulocytes. TMB-d is at least as sensitive a reagent as benzidine but, unlike benzidine and diaminobenzidine, it is not a carcinogen.     

Variability in response to UV-B among species and strains of Metarhizium isolated from sites at latitudes from 61 degrees N to 54 degrees S.

The effects of irradiances of 920 and 1200 mW m(-2) (biologically effective weighted irradiance) were examined in 2 Metarhizium album strains, 26 M. anisopliae strains, 1 M. flavoviride strain, and 1 M. taii strain isolated from sites located at latitudes from 61 degrees N to 54 degrees S. Conidia were exposed to UV-B from 1 to 6 h and subsequently examined for relative percentage culturability. Total dosage received at the end of the exposure periods ranged from 3.3 to 19.9 kJ m(-2) for the lower irradiance and from 4.3 to 25.9 kJ m(-2) for the higher irradiance. Both the irradiance values and the doses are environmentally realistic and can be observed even in temperate regions. The relationships between latitude of origin and UV-B tolerance were compared for the two levels of irradiance for the data from 1 and 2 h exposure. Exposure to both irradiances drastically reduced the relative percentage culturability of all strains. Tolerance to UV-B varied widely among strains and high variation was observed for both irradiances after all periods of exposure. After 1 h of exposure, a difference between the two irradiance levels was detectable, and this difference was magnified at longer irradiations. A significant quadratic relationship of decreasing UV-B tolerance with increasing latitude was observed after exposure of 1 and 2 h. The shape of the relationship did not differ for the two levels of irradiance. Also, we studied the effect of 1200 mW m(-2) irradiance on conidial germination time in 1 M. album strain, 7 M. anisopliae strains, and 1 M. taii strain. Exposure to UV-B delayed the germination of surviving conidia of all strains. In general, the delay in germination was directly proportional to the dose.     

Scanning electron microscopy of the heart of the climbing perch

The air-breathing climbing perch Anabas testudineus has two ventral aortas, one directs blood through well developed anterior gill arches into the suprabranchial chambers and back to the heart and the other sends blood through rudimentary shunt-like posterior arches and onto the systemic circulation. The sinus venosus is a thin-walled structure and lacks myocardial trabeculae. The atrium is similar to that of other teleosts and it is traversed by numerous myocardial trabeculae. There are no sinoatrial valves, whereas the atrioventricular aperture is guarded by one pair of large wing-shaped and two small cap-shaped valves. The ventricle is composed only of spongy myocardium and has numerous branching lacunae extending to the epicardium. The thick-walled bulbus arteriosus is lined with longitudinal ridges and this and the ventricular trabeculae may minimize mixing of respiratory and systemic flow while blood is passing through the heart. However, with the exception of the absence of sinoatrial valves and the ridged bulbar lumen, the heart of the climbing perch is essentially similar to that of most other non air-breathing teleosts.     

Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells

Pertussis toxin (PT) comprises an active subunit (S1), which ADP-ribosylates the alpha subunit of several mammalian G proteins, and the B oligomer (S2–S5), which binds glycoconjugate receptors on cells. In a previous report, expression of S1 in Cos cells resulted in no observable cytotoxicity, and it was hypothesized that either S1 failed to locate its target proteins or the B oligomer was also necessary for cytotoxicity. To address this, we stably transfected S1 with and without a signal peptide into mammalian cells. Immunofluorescence analysis confirmed the function of the signal peptide. Surprisingly, we found that S1 was active in both transfectants, as determined by clustering of transfected Chinese hamster ovary (CHO) cells and ADP-ribosylation of G proteins. Constructs with a cysteine-to-serine change at residue 201 or a truncated S1 (residues 1–181) were also active when transfected into cells. Constructs with an inactive mutant S1 had no activity, confirming that the observed results were due to the activity of the toxin subunit. We conclude that S1 is active when expressed in mammalian cells without the B oligomer, that secretion into the endoplasmic reticulum does not prevent this activity and that the C-terminal portion of S1 is not required for its activity in cells.     

The Drosophila VEGF receptor homolog is expressed in hemocytes.

Several signalling pathways have been defined by studies of genes originally characterised in Drosophila. However, some mammalian signalling systems have so far escaped discovery in the fly. Here, we describe the identification and characterisation of fly homologs for the mammalian vascular endothelial growth factor/platelet derived growth factor (VEGF/PDGF) and the VEGF receptor. The Drosophila factor (DmVEGF-1) gene has two splice variants and is expressed during all stages, the signal distribution during embryogenesis being ubiquitous. The receptor (DmVEGFR) gene has several splice variants; the variations affecting only the extracellular domain. The most prominent form is expressed in cells of the embryonic haematopoietic cell lineage, starting in the mesodermal area of the head around stage 10 of embryogenesis. Expression persists in hemocytes as embryonic development proceeds and the cells migrate posteriorly. In a fly strain carrying a deletion uncovering the DmVEGFR gene, hemocytes are still present, but their migration is hampered and the hemocytes remain mainly in the anterior end close to their origin. These data suggest that the VEGF/PDGF signalling system may regulate the migration of the Drosophila embryonic haemocyte precursor cells.