Molecular determinants of voltage-dependent slow inactivation of the Ca2+ channel.

Ba(2+) current through the L-type Ca(2+) channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the alpha(1C) subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba(2+) current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed approximately 45% of the sustained Ba(2+) or Ca(2+) current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (pi(f) approximately 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca(2+)-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca(2+) sensors of inactivation.     

Studies on the regeneration-restore and karyotype of protoplast in Metarhizium anisopliae var. majus

Abstract:  The characteristics and regeneration-restore of protoplasts and its karyotype of an insect pathological fungus, Metarhizium anisopliae var. majus were studied. Among the protoplasts, 25.3% were without a nucleus, and 74.7% contained a nucleus. Among the nucleus protoplasts, 53.6% contained a single nucleus. The regeneration-restore of protoplasts was of three distinct shapes. Considering the frequency of regeneration and the growing speed of the colony, 0.7 mol/l glucose was the optimum as osmotic stabilizer of culture medium in the regeneration-restore of the protoplasts. The chromosomal DNA molecules of M. anisopliae var. majus have been separated into seven bands by pulsed-field gel electrophoreses. Using the Schizosaccharomyces pombe chromosomes as size standard, the size of chromosomal DNA was estimated to be 1.1–6.5 Mb and its karyotype exhibited polytypism among strains.     

Ⅱ型限制性核酸内切酶NcrⅠ的研究

 在肉色诺卡氏菌C-212株Nocardia carnea C-212中筛选到一种Ⅱ型限制性核酸内切酶NcrⅠ,经与BglⅡ的λDNA降解物的酶谱比较,以及酶识别特异性和切割位点的检测,证明了NcrⅠ是已知的限制酶BglⅡ的同切限制酶,而且其切割位点也与BglⅡ相同,其为:     

EFFECTS OF HELPER PROTEINS ENCODED BY p19 AND orf1-orf2 GENES ON Cyt1Aa PROTEIN EXPRESSION IN ACRYSTALLIFEROUS STRAIN OF BACILLUS THURINGIENSIS

A series of plasmids were constructed to examine the effects of p19 and orf1‐orf2 genes from Bacillus thuringiensis on Cyt1Aa synthesis and inclusion formation. The plasmids expressed the cyt1Aa gene along with either p19 or orf1‐orf2, or each of them coordinatively with p20 in the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. No effect on the expression of Cyt1Aa protein was found when P19 or Orf1‐Orf2 co‐expressed with Cyt1Aa. However, when including p20 gene, the constructs with p19 or orf1‐orf2 gene produced lower yield of Cyt1Aa proteins than without p19 or orf1‐orf2 gene. Electron microscopy observation and bioassay showed that P19 and Orf1‐Orf2 have no influence on the crystal size and toxicity of Cyt1Aa protein. It is presumed that P19 and Orf1‐Orf2 might have negative effects on Cyt1Aa synthesis in B. thuringiensis.     

A Wuchiapingian (Late Permian) brachiopod fauna from an exotic block in the Indus–Tsangpo suture zone, southern Tibet, and its palaeobiogeographical and tectonic implications

A brachiopod fauna including 19 species of 17 genera from an exotic block in the Indus–Tsangpo suture zone in southern Tibet is described and illustrated. The brachiopod fauna is dominated by Martinia elegans and two new taxa: Jinomarginifera lhazeensis gen. et sp. nov. and Zhejiangospirifer giganteus sp. nov. The fauna is closely comparable with those from the middle and upper parts of the Wargal Formation and the Chhidru Formation in the Salt Range of Pakistan, the Chitichun Limestone in southern Tibet, and the Basleo area of West Timor, and these correlations suggest a Wuchiapingian age. The fauna exhibits substantial links with both peri–Gondwanan and Cathaysian faunas, which may imply that it is a seamount biota originally located in the southern margin of the Neotethys during the Late Permian, and was later (in the early Cenozoic) displaced and became sandwiched into younger marine deposits in the collision process between India and Eurasia.     

Intraspecific molecular variation inHieracium sect.Alpina (Asteraceae), an apomictic group

Intraspecific variation of four agamospecies ofHieracium sect.Alpina was studied using RAPD and isozyme techniques. No variation in either multiprimer RAPD or multi-enzyme phenotypes was observed withinH. holosericeum, suggesting that this widespread species consists of only a single genotype. A low level of within-population isozyme variation was seen inH. tenuifrons andH. calenduliflorum, the origin of which appears to be consistent with somatic mutation. Most isozyme and all RAPD variation in these two species was partitioned between populations. A strong correlation with geography suggests that its cause may be due to polytopic (-polyphyletic?) origin or perhaps to mutation and dispersal. The most variable species wasH. alpinum, in which isozyme variation occurred mostly within populations rather than between them, suggesting occasional sexual events or that the parents ofH. alpinum were heterozygous. RAPD variation in this species, in contrast, was partitioned between Scottish and Swiss populations, suggesting the existence of geographical races.     

Antitumor agents-CLXXV. Anti-tubulin action of (+)-thiocolchicine prepared by partial synthesis

(+)-Thiocolchicine (2b) was prepared from (±)-colchicine (1) in a five-step reaction sequence that included chromatographic separation of appropriate camphanylated diastereomers. Acid hydrolysis of the (+)-diastereomer, followed by acetylation, yielded the desired product 2b. (+)-Thiocolchicine has 15-fold lower inhibitory activity against tubulin polymerization than (−)-thiocolchicine, and is 29-fold less potent for inhibiting growth of human Burkitt lymphoma cells. The enantiomer 2a, prepared from the (−)-camphanylated diastereomer, had potent activity in all assays comparable to that of (−)-thiocolchicine prepared by other methods. These results support the hypothesis that the proper configuration of colchicine-related compounds is an important requirement for their anti-tubulin action.     

Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule.

A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.     

In vivo mechanical properties of leukocytes during adhesion to venular endothelium

Transient deformations of leukocytes (WBCs) were studied during their saltation along post-capillary venous endothelium (EC) in mesentery of the rat. During intermittent adhesion of WBCs to EC, prevailing fluid shear stresses, tau wall, resulted in a stepwise loading of the WBC upon attachment with a transient increase in length, L(t), and reduction in height, H(t). Measurements of L(t) and H(t) from frame-by-frame analysis of video recordings were modelled as the simple shear of a standard linear viscoelastic solid to facilitate calculation of the elastic (k1, k2) and viscous (mu) elements with k1 in parallel with serial elements k2 and mu. The magnitude of tau wall was determined from measurements of red cell velocity within the venule. During the spontaneous adhesion of WBCs, a value of cell viscosity (mu) of 45 Poise was determined. Stimulating adhesion by topical application of the chemoattractant FMLP resulted in a 15-fold increase of mu to 668 Poise. Transient deformations during topical application of cytochalesin B to disrupt actin fibers within the WBC, yielded a 40% reduction in k1, compared to an 80% reduction with colchicine which disrupts the microtubule structure. Thus, colchicine treated cells appear to be twice as deformable as cells treated with cytochalesin. During adhesion stimulated by the cytokine Interleukin-1, mu increased 50% without changes in k1 and k2, possibly due to slight activation of the WBC.