An isothermal titration calorimetric method to determine the kinetic parameters of enzyme catalytic reaction by employing the product inhibition as probe.

An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2',3'-cyclic monophosphate. Employing the inhibition of product as a probe, the K(m), K(i), k(c), and DeltaH(m) can be determined by two simple calorimetric measurements. First, the substrate was titrated into the cell containing high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations of product can be calculated from the peak height and the molar reaction heat. From Michaelis-Menten function in the presence of inhibitors, the relationship between K(m) and K(i) can be obtained. Then, the dissociation constant, which is equal to K(i), was measured by a regular ITC experiment. Thus, K(m) and k(c) can be calculated. The method developed here can be applied in other enzyme catalytic systems with inhibitive products.     

A comparison of the central gastrointestinal antitransit effects of morphine and bombesin in the mouse

The gastrointestinal motor effects of centrally-given morphine and bombesin were compared in mice. Both compounds produced a dose-related decrease in the propulsion of a marker along the gut when given by the intracerebroventricular (i.c.v.) or intrathecal (i.th.) routes. Co-administration of the same compound by both routes was found to produce a marked increase in potency for morphine, but only a slight increase in potency for bombesin. Isobolographic analysis of the gut effects of these compounds revealed a multiplicative brain-spinal cord interaction for morphine, but an additive interaction for bombesin. These results are consistent with the interpretation that morphine can act at either the level of the brain or the spinal cord, activating independent pathways which ultimately converge to alter gut propulsion. In contrast, spinal bombesin requires communication to supraspinal sites in order for its gut effects to occur, suggesting activation of a common outflow pathway from the central nervous system.     

Expression of dengue virus structural proteins and nonstructural protein NS1 by a recombinant vaccinia virus.

A recombinant vaccinia virus containing cloned DNA sequences coding for the three structural proteins and nonstructural proteins NS1 and NS2a of dengue type 4 virus was constructed. Infection of CV-1 cells with this recombinant virus produced dengue virus structural proteins as well as the nonstructural protein NS1. These proteins were precipitated by specific antisera and exhibited the same molecular size and glycosylation patterns as authentic dengue virus proteins. Infection of cotton rats with the recombinant virus induced NS1 antibodies in 1 of 11 animals. However, an immune response to the PreM and E glycoproteins was not detected. A reduced level of gene expression was probably the reason for the limited serologic response to these dengue virus antigens.     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.     

盾叶薯蓣地上部分的三个新甾体皂甙

从盾叶薯蓣Dioscorea zingiberensis Wright地上部分分离鉴定了四个甾体皂甙,经鉴定甙A为约莫皂甙元-3-O-[α-L-鼠李吡喃糖基(1→2)]-β-D-葡萄吡喃糖甙;甙B为24α-羟基约莫皂甙元-3-O-[α-L-鼠李吡喃糖基(1→2)]β-D-葡萄吡喃糖甙;甙C为约莫皂甙元-3-O-[α-L-鼠李吡喃糖基(1→2)][β-D-葡萄吡喃糖基(1→4)]-β-D-葡萄吡喃糖基;甙D为约莫皂甙元-3-O-[α-L-鼠李吡喃糖基(1→2)][β-D-葡萄吡喃糖基(1→3)]-β-D-葡萄吡喃糖甙。前三者为新化合物,分别命名为盾叶皂甙A_1、A_2、A_3(zingiberoside A_1、A_2、A_3),其中盾叶皂甙A_2的甙元为一新甾体皂甙元,命名为盾叶皂甙元(zingiberogenin)。     

Epidermal growth factor and insulin stimulate nuclear pore-mediated macromolecular transport in isolated rat liver nuclei

Fluorescence photobleaching was used to measure the effect of epidermal growth factor (EGF), insulin, and glucagon on the nuclear transport of fluorescent-labeled dextrans across the nuclear pore complex. EGF and insulin were found to stimulate transport approximately 200%, while boiling these polypeptide growth factors greatly diminished this enhancement activity. Glucagon demonstrated no enhancement effect. The nuclear transport enhancement effects were observed at EGF and insulin concentrations that elicit the various physiological responses, e.g., nanomolar range.     

Low-Level Microwave Irradiations Affect Central Cholinergic Activity in the Rat

Sodium-dependent high-affinity choline uptake was measured in various regions of the brains of rats irradiated for 45 min with either pulsed or continuous-wave low-level microwaves (2,450 MHz; power density, 1 mW/cm2; average whole-body specific absorption rate, 0.6 W/kg). Pulsed microwave irradiation (2-microseconds pulses, 500 pulses/s) decreased choline uptake in the hippocampus and frontal cortex but had no significant effect on the hypothalamus, striatum, and inferior colliculus. Pretreatment with a narcotic antagonist (naloxone or naltrexone; 1 mg/kg i.p.) blocked the effect of pulsed microwaves on hippocampal choline uptake but did not significantly alter the effect on the frontal cortex. Irradiation with continuous-wave microwaves did not significantly affect choline uptake in the hippocampus, striatum, and hypothalamus but decreased the uptake in the frontal cortex. The effect on the frontal cortex was not altered by pretreatment with narcotic antagonist. These data suggest that exposure to low-level pulsed or continuous-wave microwaves leads to changes in cholinergic functions in the brain.     

我国某些常见化石硅藻的环境分析

本文详细研究了中国海表层沉积物中的常见化石硅藻,结果表明,这些种的分布范围及数量变化随环境的不同而变化。因此,根据柱状样品中这些种类数量的变化,能够推断古气候变化,恢复古地理环境,再现海平面波动历程,进而划分、对比第四系地层。     

Evidence for a junctional feet-ryanodine receptor complex from sarcoplasmic reticulum

Heavy sarcoplasmic reticulum vesicles, labelled with the Ca2+ release channel probe [3H]ryanodine, were solubilized in detergent, then centrifuged through sucrose gradients. A single peak of ryanodine binding activity was observed with an apparent sedimentation coefficient of 30S. Electron microscopy of the peak fraction showed disk structures of 25-28 nm diameter and 10 nm thickness. Proteins specifically enriched in the peak fraction were the Mr 160,000 and 260,000 and junctional feet proteins (Mr 320,000 and 300,000). This suggests that the feet proteins and ryanodine receptor may be specifically associated into a large oligomeric complex comprising subunits of Mr 160,000-320,000.