Incomplete protection, but suppression of virus burden, elicited by subunit simian immunodeficiency virus vaccines.

We compared the efficacy of immunization with either simian immunodeficiency virus (SIV) Env glycoprotein (Env), Env plus Gag proteins (Gag-Env), or whole inactivated virus (WIV), with or without recombinant live vaccinia vector (VV) priming, in protecting 23 rhesus macaques (six vaccine and two control groups) from challenge with SIVmac251 clone BK28. Vaccination elicited high titers of syncytium-inhibiting and anti-Env (gp120/gp160) antibodies in all vaccinated macaques and anti-Gag (p27) antibodies in groups immunized with WIV or Gag-Env. Only WIV-immunized macaques developed anticell (HuT78) antibodies. After homologous low-dose intravenous virus challenge, we used frequency of virus isolation, provirus burden, and change in antibody titers to define four levels of resistance to SIV infection as follows. (i) No infection ("sterilizing" immunity) was induced only in WIV-immunized animals. (ii) Abortive infection (strong immunity) was defined when virus or provirus were detected early in the postchallenge period but not thereafter and no evidence of virus or provirus was detected in terminal tissues. This response was observed in two animals (one VV-Env and one Gag-Env). (iii) Suppression of infection (incomplete or partial immunity) described a gradient of virus suppression manifested by termination of viremia, declining postchallenge antibody titers, and low levels (composite mean = 9.1 copies per 10(6) cells) of provirus detectable in peripheral blood mononuclear cells or lymphoid tissues at termination (40 weeks postchallenge). This response occurred in the majority (8 of 12) of subunit-vaccinated animals. (iv) Active infection (no immunity) was characterized by persistent virus isolation from blood mononuclear cells, increasing viral antibody titers postchallenge, and high levels (composite mean = 198 copies per 10(6) cells) of provirus in terminal tissues and blood. Active infection developed in all controls and two of three VV-Gag-Env-immunized animals. The results of this study restate the protective effect of inactivated whole virus vaccines produced in heterologous cells but more importantly demonstrate that a gradient of suppression of challenge virus growth, reflecting partial resistance to SIV infection, is induced by subunit vaccination. The latter finding may be pertinent to studies with human immunodeficiency virus vaccines, in which it is plausible that vaccination may elicit significant suppression of virus infection and pathogenicity rather than sterilizing immunity.     

Aluminium polyphosphate complexes in the mycorrhizal basidiomyceteLaccaria bicolor: A27Al-nuclear magnetic resonance study

The techniques of ²⁷Al- and ³¹P-nuclear magnetic resonance (NMR) spectroscopy were used to investigate the interactions of aluminium with intracellular ligands within the mycelium of the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton (S238). The vegetative mycelium was grown on medium containing 0.5 mM AlCl₃ for 0.5 to 3 d. The ²⁷Al-NMR spectra showed that aluminium was rapidly taken up and accumulated into polyphosphate complexes in the vacuole. Comparison with Al-polyphosphate complexes obtained in vitro on model systems indicated that Al forms at least three mixed-solvation complexes with Pi and polyphosphates, that there is more than one complex present under any set of conditions, and that the equilibrium between these complexes shifts dramatically with Al concentration in the medium. The high phosphate concentrations in the growth medium favoured the accumulation of the Al-polyphosphate complexes. When mycelium containing Al-polyphosphate complexes was transferred to Al-free nutrient solution for 9 d, the Alpolyphosphate complexes were not remobilized. The sequestration of Al in the polyphosphate complexes could therefore make a significant contribution to the protection of mycorrhizal plants against aluminium toxicity.Abbreviations NMR nuclear magnetic resonance - PolyP polyphosphate(s) - PP₁ terminal phosphate of PolyP - PP₃ middle phosphate of PolyP We thank Prof. Daniel Canet (Laboratoire de Méthodologie RMN, University of Nancy I, Vandceuvre-lès-Nancy, France) for his constant encouragement and Christine Delaruelle for skilled technical assistance in growing the fungal cultures. This work was supported by a research grant from the Commission of the European Communities (STEP-CT90-0059, Role of Ectomycorrhiza in Stress Tolerance of Forest Trees) to F.M. and a travel grant from the Institut National de la Recherche Agronomique to I.K.; R.C. is a recipient of a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada.     

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

Heterogeneity of twoBeauveria bassiana strains revealed by biochemical tests,protein profiles and bio-assays ofLeptinotarsa decemlineata (Col.: Chrysomelidae) andColeomegilla macultata lengi (Col.: Coccinellidae) larvae

Biochemical profiles on API Rapid CH^* strips and protein profiles on polyacrylamide gels in the presence of sodium dodecyl sulfate were used to distinguish two strains of the entomopathogenic fungusBeauveria bassiana (Balsamo) Vuillemin, ARSEF 2991 and ATCC 44860. Next, the toxicity of these two strains was determined at concentrations of 10², 10⁴, 10⁶ and 10⁸ blastospores/ml on larvae of the Colorado potato beetleLeptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) and of its predator, the spotted ladybird beetle,Coleomegilla maculata lengi Timberlake (Coleoptera: Coccinellidae). Both strains were highly toxic toL. decemlineata larvae. However, the two strains exhibited different levels of toxicity forC. maculata larvae: ARSEF 2991 was toxic, whereas ATCC 44860 caused little coccinellid larval mortality.

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Résumé Les profils biochimiques sur galeries API Rapid CH^* et les profils protéiques sur gels de polyacrylamide ont été utilisés pour distinguer deux souches du champignon entomopathogèneBeauveria bassiana (Balsamo) Vuillemin. La toxicité de ces deux souches a été déterminée à des concentrations de 10², 10⁴, 10⁶ et 10⁸ blastospores/ml sur des larves du doryphore,Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) et de la coccinelle maculéeColeomegilla maculata lengi Timberlake (Coleoptera: Coccinellidae). Les deux souches deB. bassiana se sont avérées actives à l'égard des larves deL. decemlineata. Toutefois la souche ARSEF 2991 s'est avérée pathogène pour les larves deC. maculata, alors que la souche ATCC 44860 a provoqué une faible mortalité des larves.

     

日本落叶松引种区林地棕壤肥力的多变量分析

本研究从日本落叶松引种区林地棕壤9个主要理化性状及与之配套的17株优势木材料入手,采用多变量分析方法筛选出影响该地区日本落叶松生长的主要肥力因子为氮及磷,並据此将该林地棕壤划分为9个肥力类型。在进行聚类分析时,本研究的创新在于以任意二样本点在各主成份上的坐标值为变量,以各主成份的贡献率为权重,重新定义欧氏距离公式,对主成份坐标值聚类,使其更为合理、直观。同时采用回归分析拟合出理化性状间的多个回归方程。此研究为该林地科学施肥、速生丰产、永续利用提供了可靠的数量依据。     

HBIG,无环鸟苷,干扰素联合对慢性乙型肝炎抗病毒效应观察

本文报道血清ＨＢＶ复制标志阳性的慢乙肝５４例,随机分为治疗组及对照组各２７例进行ＨＢＩＧ、无环鸟苷、干扰素联合近、远期抗病毒效应观察。治疗组为无环鸟苷第一周按２５～２０ｍｇ／ｋｇ／ｄ计后改１７～１５ｍｇ／ｋｇ／ｄ×５３天,共６０天;人白细胞干扰素１×１０６Ｕ肌注每周３次×４周,后改１．０×１０６Ｕ肌注每周２次×６周,共１０周;ＨＢＩＧ４００Ｕ肌注隔日１次,共１０周,对照组仅给予一般“保肝”药物。其中治疗组１８例,对照组１９例进行治后半年到２年追踪观察,结果近、远期ＨＢｃＡｇ、ＤＮＡＰ、ＨＢＶ－ＤＮＡ阴转率治疗组均高于对照组,其中治疗组近、远期ＨＢｃＡｇ,ＨＢＶ－ＤＮＡ阴转率均达４０％以上,明显高于对照组（Ｐ＜０．０５～０．０１）,治疗组近、远期各有４例及２例ＨＢｓＡｇ阴转,而对照组则无一例阴转,从近、远期综合抗病毒效应观察,治疗组全阴率分别为３３．３％、４４．４％,而对照组分别为３．７９％及０％,Ｐ＜０．０１,治疗组无明显毒副反应。对比单用无环鸟苷,全阴率３１．８％;无环鸟苷加干扰素两药联合全阴率３７．５％,均有所提高,达到４４．４％,值得进一步研究。     

底物膜技术检测精子顶体酶活性的研究

应用底物膜技术检测１３０例正常精液,精子顶体酶活性百分率的正常值下限为５７％。４５９例不孕症病人精液分析,无精症２５例,其余４３４例中７５％精子顶体酶活性正常。实验表明精子密度对数值与顶体酶活性百分率之间有正相关,ｒ＝０．８４（Ｐ＜０．０１）,回归方程为顶体酶活性百分率ｙ＝４８．４３％＋（８．９％）（ｌｏｇ精子计数）。活动精子百分率与顶体酶活性之间有密切正相关,ｒ＝０．９６７,（Ｐ＜０．０１）,顶体酶活性ｙ＝３８．６％＋０、３６ｘ％。前向活跃直线运动精子百分率与顶体酶活性之间也有密切相关．ｒ＝０．９６,（Ｐ＜０．０１）,顶体酶活性ｙ＝３４．２１％＋０．６１ｘ％。     

大熊猫消化道消化吸收区段游离面的扫描电镜观察

对大熊猫消化道的消化、吸收区段作扫描电镜观察后表明：（１）胃粘膜上皮细胞排列疏松,细胞表面具微绒毛,（２）十二指肠绒毛呈指状,表面凸凹不乎;绒毛表面具丰富的微绒毛,微绒毛表面粗糙,末端膨大。（３）直肠段具丰富的绒毛结构,表面不平滑,具颗粒状物质,但无微绒毛存在：直肠腺丰富。