大鼠胰腺γ-氨基丁酸免疫组织化学定位

本文用ABC—GDN免疫组织化学方法,研究了γ-氨基丁酸(Gamma—Aminobutyric Acid,GABA)在大鼠胰腺的定位和分布,并用相邻切片法,观察它与胰岛素的共存关系。结果发现GABA免疫反应阳性细胞主要分布于胰腺内分泌部(胰岛)。在外分泌部亦有少许分布。大部分胰岛细胞呈GABA免疫反应阳性,集中位于胰岛的中央部。相邻连续切片免疫染色证实GABA与胰岛素共存于胰岛B细胞中。外分泌部胰腺GABA免疫反应阳性细胞,呈零散分布于腺泡和导管上皮间。本文为进一步探讨GABA在胰腺的生理作用提供了形态学依据。     

EFFECTS OF VASOACTIVE INTESTINAL PEPTIDE ON ACTIVITIES OF SUCCINIC DEHYDROGENASE AND ALKALINE PHOSPHATASE IN RAT HEPATOMA CELLS

Using cytochemical method,microspectrophotometry and image analysis,effects of va-soactive intestinal peptide(VIP)on activities of succinic dehydrogenase(SDH)and alkalinephosphatase(ALP)in rat hepatoma cells were studied in vitro.The results showed that thehepatoma cell expressed potent positive reactions of SDH and ALP,the positive positionswere located at the cell membranes and/or cytoplasm.Having been treated with VIP,ALPdecreased obviously in activity(P<0. 01,compared with hepatoma cells untreated by VIP).The sites of ALP activty were chiefly located at the cell membranes,particularly at the cell-cell contacts.Cultured rat hepatoma cells had intensive SDH activity in their cytoplasm.Compared with untreated eclls,there was no marked difference in the intensity of SDH activ-ity in VIP-treated hepatoma cells(P>0.05).     

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from aKlebsiella pneumoniae mutant strain

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA⁺ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.     

尼群地平对实验性糖尿病大鼠心肌收缩性的影响

腹腔注射链脲佐霉素（６５ｍｇ／ｋｇ）诱发Ｗｉｓｔａｒ大鼠糖尿病。糖尿病发病４周后,向饲料中加尼群地平（３０ｍｇ·ｋｇ－１·ｄ－１）。结果表明,糖尿病４周时大鼠心室舒张功能首先受损,８周后心室舒张和收缩功能均明显受累。尼群地平处理对糖尿病大鼠的心肌收缩性有一定的改善作用。提示尼群地平对大鼠糖尿病性心肌病有一定有益作用。     

Histochemical detection of monoamine oxidase and alcohol dehydrogenase activities in the Syrian hamster Harderian glands: existence of a sexual dimorphism

Summary Monoamine oxidase (MAO) and alcohol dehydrogenase (AD) activities were studied histochemically in the Syrian hamster Harderian gland using tryptamine as substrate and Nitroblue Tetrazolium as the final electron acceptor. No dark: light-related changes were observed. Male type I secretory cells showed an intense MAO reaction. Female type I cells exhibited a moderate MAO activity. Both male and female glands showed a moderate/intense AD-positive reaction. Male type II cells were lacking MAO and AD activities. MAO activity found in the hamster Harderian glands corresponded mainly to MAO type A since treatment with chlorgyline (0.01, 0.1 and 0.5mm) totally inhibited it. The possible role of these two enzymes in Harderian gland indolalkylamine metabolism is discussed.     

Early responses to Nod factors and mycorrhizal colonization in a non-nodulating Phaseolus vulgaris mutant

Legumes can acquire nitrogen through a symbiotic interaction with rhizobial bacteria. The initiation of this process is determined by a molecular dialogue between the two partners. Legume roots exude flavonoids that induce the expression of the bacterial nodulation genes, which encode proteins involved in the synthesis and secretion of signals called Nod factors (NFs). NFs signal back to the plant root and trigger several responses, leading to bacterial invasion and nodule formation. Here, we describe the molecular and cellular characterization of a Phaseolus vulgaris non-nodulating mutant (NN-mutant). Root hair cells of the NN-mutant plant respond with swelling and branching when inoculated with Rhizobium etli, albeit without curling induction. Furthermore, neither initiation of cell division in the outer cortex, nor entrapment of bacteria nor infection thread formation was observed. Both the bean wild-type and the NN-mutant responded with elevated intracellular calcium changes in the root hairs. Although the NN-mutant is deficient in early nodulin gene expression when inoculated with R. etli, it can be effectively colonized by arbuscular mycorrhizal fungi (Glomus intraradices). Our data indicate that the P. vulgaris NN-mutant is not blocked at the NFs early perception stage, but at later downstream stages between Ca²⁺ signaling and early nodulin induction. This supports the idea that both microsymbionts are perceived and trigger different downstream pathways in the host plant.     

Keratella distribution in North Patagonian lakes (Argentina)

The distribution of Keratella species from 15 different lakes in North Patagonia (Argentina) was analysed. The genus was not present at altitudes above 1000 m. K. tropica was restricted to Patagonian Plateau lakes with a comparatively high conductivity. A morphometric analysis of the widely distributed K. cochlearis was performed. Results showed three groups of K. cochlearis corresponding to Andean lakes, Patagonian Plateau lakes and a Patagonian Reservoir.     

Expression of Discoidin Domain Receptor 2 (DDR2) Extracellular Domain in Pichia Pastoris and Functional Analysis in Synovial Fibroblasts and NIT3T3 Cells

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained, After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II. Wei Zhang and Tianbing Ding equally contributed to this work.     

A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.