The enzyme that cleaves apolipoprotein A-II upon in vitro incubation of human plasma high-density lipoprotein-3 with blood polymorphonuclear cells is an elastase

The proteolytic activity directed against apolipoprotein A-II (apo-A-II) which is released from human blood polymorphonuclear cells (PMN) when they are incubated with human plasma high-density lipoprotein-3 (HDL3) was studied to assess the properties and site specificity of the enzyme. When 125I-apo-A-II-labeled HDL3 was incubated with the PMN protease at 37 degrees C, a complete cleavage of apo-A-II was observed which paralleled the formation of bands of approximately 11,000 and 7,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 7,000-dalton component had the following N-terminal sequence: NH2-Thr-Asp-Tyr-Gly-Lys-Asp-Leu-Met-Glu-Lys. This corresponds to residues 19 through 28 of the intact apo-A-II monomer. Methoxysuccinyl (MeO-Suc)-Ala-Ala-Pro-Val-chloromethylketone-(CH2Cl) caused a 90% inhibition of apo-A-II hydrolysis at the highest concentration tested (6 X 10(-4)M). Besides apo-A-II, the PMN enzyme also hydrolyzed a synthetic substrate, MeO-Suc-Ala-Ala-Pro-Val-4-nitroanilide and its 4-methylcoumaryl-7-amide analogue. The protease appeared to have a mass of 28,000 daltons as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]diisopropylfluorophosphate-labeled PMN enzyme. That the PMN enzyme which cleaves apo-A-II is an elastase was derived from the following criteria: 1) cleavage at the Val-X bond in apo-A-II and in the two synthetic substrates studied; 2) prevention of the cleavage by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a known specific elastase inhibitor; and 3) a mass comparable to that reported for a pure PMN elastase. These studies establish that apolipoproteins can be suitable substrates for enzymes of the elastase family.     

Biologic-response-modifier-induced indoleamine 2,3-dioxygenase activity in human peripheral blood mononuclear cell cultures

Degradation of tryptophan to kynurenine, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been augmented in human epithelial cell lines treated with human interferon-gamma (HuIFN-gamma). Several human biologic response modifiers, including HuIFN-gamma, HuIFN-beta, HuIFN-alpha, interleukin 2 (HuIL-2), and tumor necrosis factor alpha, have now been assessed for their ability to enhance tryptophan degradation in human peripheral blood mononuclear cell (PMC) cultures. PMC were isolated from normal donors, cultivated in RPMI 1640 medium containing [3H]tryptophan, and treated with individual biologic response modifiers. At various intervals, culture supernatants were removed, fractionated by reversed-phase high performance liquid chromatography, and radioactivity in resultant fractions was determined. Significantly increased amounts of tryptophan catabolites were observed after treatment with HuIFN-gamma, HuIFN-beta, HuIFN-alpha, and HuIL-2, but not human tumor necrosis factor alpha. Often, greater than 30% of available tryptophan was degraded by treated PMC cultures. Although antibodies to HuIFN-alpha, HuIFN-beta, and HuIFN-gamma specifically neutralized the induction of IDO activity in PMC by their respective HuIFN, only anti-HuIFN-gamma antibody also neutralized HuIL-2-induced IDO activity. Furthermore, T24 bladder carcinoma cells, in which IDO was induced by HuIFN-gamma but not by the other biologic response modifiers, were induced to degrade tryptophan by supernatants of HuIL-2-stimulated PMC cultures, but not by HuIFN-beta-stimulated PMC culture supernatants. Thus, whereas HuIL-2 indirectly induced IDO in PMC cultures by stimulating production of HuIFN-gamma, all cases of interferons appeared to induce IDO directly in PMC cultures.     

下丘脑外侧区注射TRH对大鼠胃酸分泌的影响

本文采用连续收集胃腔灌流法,观察下丘脑外侧区(LHA)注射促甲状腺激素释放激素(TRH)对大鼠胃酸分泌的影响,并分析TRH在LHA促进胃酸分泌的作用机制。结果表明:(1)LHA注射TRH(1μg)明显地刺激胃酸分泌;(2)预先向LHA注射酚妥拉明(10μg)、美多心安(5μg)及胃泌素抗体1μl(1:640)并不影响TRH的泌酸作用,如预先向LHA注射阿托品(5μg)则可消除TRH的泌酸效应;(3)垂体摘除及肾上腺切除均不影响TRH的泌酸作用;(4)隔下迷走神经切断后,LHA注入TRH的泌酸效应仍然出现,但持续时间显著缩短;腹腔交感神经节摘除后,TRH仍能促进胃酸分泌,但分泌量少而平稳。以上结果提示:LHA是TRH中枢泌酸效应的有关结构之一,其中枢机制是通过胆碱能M受体中介的,腹腔交感神经节和膈下迷走神经是TRH泌酸效应的传出途径。前者引起的泌酸反应出现较早且引起泌酸高峰,但持续时间短;后者则引起低平的持续分泌。     

Opioidergic control of gonadotropin secretion in the female rabbit: divergent effects of morphine on secretion of follicle-stimulating hormone and luteinizing hormone

The nature of secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was followed in female rabbits on a daily basis from age 36 to 60 days by sequential 5-min blood sampling over 1- to 2-h periods each day. Both LH and FSH were found to be secreted in a pulsatile manner. The mean LH pulse amplitude over the 25 days was 0.95 +/- 0.32 ng/mL and for FSH it was 10.15 +/- 1.11 ng/mL. Mean plasma LH levels were significantly increased from 1.46 +/- 0.08 ng/mL in 36 to 42-day-old rabbits to 1.89 +/- 0.12 ng/mL in 43 to 50-day-old rabbits and remained elevated from 50 to 60 days. FSH levels during the same periods also rose significantly from 14.93 +/- 0.79 to 19.57 +/- 2.05 ng/mL. To examine the influence of endogenous opioid peptides on the release of LH and FSH in 36 to 60-day-old female rabbits, morphine sulfate at 0.2, 0.5, 2.0, and 5.0 mg/kg was administered subcutaneously after 30 min baseline sampling, and blood was taken for another 60-120 min. Morphine at all doses and at all ages inhibited the amplitude and frequency of LH pulses but had no effect on FSH secretion. To determine whether the effects of morphine on LH secretion could be reversed with naloxone, females aged 82-114 days were used. Naloxone administered 1 h after morphine reversed the inhibitory effects of morphine, whereas the simultaneous administration of naloxone with morphine had variable effects but seemed to delay the LH increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Callus formation and plantlet regeneration from protoplasts derived from suspension cultures of wheat (Triticum aestivum L.)

Protoplasts were isolated from anther-derived suspension cultures of commercial wheat (Triticum aestivum L. cv. Chris). The protoplasts were released enzymatically and isolated by centrifugation on a sucrose cushion. The isolated protoplasts were initially cultured in a liquid medium in the dark. Numerous microcalli were produced under these conditions, some of which differentiated into globular embryos. Upon transfer to a solid medium and exposure to 16h/8h light/dark cycle, the protocalli proliferated and many of the somatic embryos matured. Complete plantlets were obtained and maintained in sterile culture.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - MES 2-[N-morpholino] ethanesulfonic acid     

Histochemical localization of IGF-I and IGF-II mRNA in the rat between birth and adulthood

We describe the postnatal ontogeny and localization of insulin-like growth factors I and II (IGF-I and -II) in the rat. We have used oligodeoxyribonucleotide probes for in situ hybridization (hybridization histochemistry) and for Northern blotting. IGF-II mRNA is strongly expressed in liver, skeletal muscle, perichondrium, leptomeninges and choroid plexus of the newborn. Demonstrable levels fall dramatically in the liver at 18-20 days postnatally but persist for longer periods in muscle and remain undiminished throughout life in the pia/choroid plexus, indicating that different control mechanisms operate in these tissues. IGF-I mRNA is predominantly found in the liver. Its level in this organ rises well before levels of IGF-II fall. This suggests that distinct factors govern the expression of IGF-I and -II genes.     

Effects of temperature and photoperiod upon adult eclosion of the sweetpotato whitefly, Bemisia tabaci

A series of experiments were conducted to characterize patterns of eclosion by Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) to their adult stage and to determine how these patterns are influenced by certain environmental parameters. Under a constant temperature of 29.5±0.6°C and a photoperiod of 14:10LD, 90% of the adults emerged from their pupal cases between 0600 and 0930 h (with lights on occurring at 0600 h). Few emerged during hours of darkness. The peak time of adult emergence was delayed when temperatures were fluctuated. Under a series of constant temperatures, a significant inverse correlation was found between the time of median emergence (i.e., eclosion of 50% of the total number of adults) and temperature (P<0.001). No emergence was observed at temperatures below 17±0.3°C. Emergence patterns persisted under conditions of continuous light and continuous darkness, suggesting the presence of a circadian system.

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Zusammenfassung Um das Verständnis über den Lebenslauf von Bemisia tabaci zu ergänzen, wurde eine Serie von Experimenten durchgeführt, deren Zweck die Charakterisierung des Ausschlüpfvorgangs in das Endstadium war und die Feststellung, wie dieser Vorgang von gewissen Umweltparametern beeinflusst wird. Bei einer konstanten Temperatur von 29.5±0.6°C und einem Beleuchtungszyklus von 14: 10 LD (Licht/Dunkelheit) schlüpften 90% der Ausgewachsenen zwischen 0600 Uhr and 0930 Uhr (ab 0600 Uhr mit Licht) aus ihren Puppenhüllen aus. Wenig Ausschlüpfen geschah während der unbeleuchteten Stunden. Der Höhepunkt des Ausschlüpfens wurde bei wechselnden Temperaturen verschoben. Bei einer Serie von gleichbleibenden Temperaturen wurde eine bedeutende inverse Korrelation zwischen der medianen Ausschlüpfzeit (d.h. 50% der gesamten Ausgewachsenen schlüpften aus) und der Temperatur festgestellt (P<0.001). Kein Ausschlüpfen wurde beobachtet bei Temperaturen unter 17°C. Das Ausschlüpfschema war gleichbleibend bei dauerndem Licht oder dauernder Dunkelheit, was auf das Vorhandensein eines circadianen Systems hinweist.

     

Experimentally induced defects of mitochondrial metabolism in rat skeletal muscle. Biological effects of the NADH: coenzyme Q reductase inhibitor diphenyleneiodonium.

An animal model for the human condition of mitochondrial myopathy has been established and characterized physiologically and biochemically. The NADH: coenzyme Q reductase inhibitor diphenyleneiodonium [Bloxham (1979) Biochem. Soc. Trans. 7, 103-106] was either infused acutely in vivo into rat hind limb or injected chronically into rats. Both modes of delivery resulted in a reduced muscle oxidative capacity and increased fatigue. Analysis of muscle metabolites by h.p.l.c. and 31P-n.m.r. indicated that ATP concentrations were similar to control values during periods of stimulation and these were maintained by the phosphocreatine pool. During the recovery period after muscle stimulation in the experimental animals the muscle pH remained depressed and the rate of phosphocreatine synthesis was markedly delayed as compared with controls. Factors thought to be involved in the fatigue response are discussed in relation to this model.