Crystals of an antibody FV fragment against an integral membrane protein diffracting to 1.28 Å resolution

The F_v fragment of a monoclonal antibody, 7E2 (IgG₁, κ, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli. Crystals suitable for highresolution X-ray analysis were obtained by microdialysis under low salt conditions. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 51.51 Å, b = 56.15 Å, c = 99.86 Å (1 Å = 0.1 nm) and contain one F _v fragment per asymmetric unit. Using synchrotron radiation diffraction data were collected up to 1.28 Å resolution. This high resolution is very unusual for a heterodimeric protein. The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics. © 1995 Wiley-Liss, Inc.     

Evidence for a size-sensing mechanism in animal cells

Continuously proliferating cells exactly double their mass during each cell cycle. Here we have addressed the controversial question of if and how cell size is sensed and regulated. We used erythroblasts that proliferate under the control of a constitutively active oncogene (v-ErbB) or under the control of physiological cytokines (stem cell factor, erythropoietin and v-ErbB inhibitor). The oncogene-driven cells proliferated 1.7 times faster and showed a 1.5-fold increase in cell volume. The two phenotypes could be converted into each other 24 h after altering growth factor signalling. The large cells had a higher rate of protein synthesis, together with a shortened G1 phase. Additional experiments with chicken erythroblasts and mouse fibroblasts, synchronized by centrifugal elutriation, provided further evidence that vertebrate cells can respond to cell size alterations (induced either through different growth factor signalling or DNA synthesis inhibitors) by compensatory shortening of the subsequent G1 phase. Taken together, these data suggest that an active size threshold mechanism exists in G1, which induces adjustment of cell-cycle length in the next cycle, thus ensuring maintenance of a proper balance between growth and proliferation rates in vertebrates.     

Nematicidal activity of fervenulin isolated from a nematicidal actinomycete, <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CMU-MH021, on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

An isolate of the actinomycete, Streptomyces sp. CMU-MH021 produced secondary metabolites that inhibited egg hatch and increased juvenile mortality of the root-knot nematode Meloidogyne incognita in vitro. 16S rDNA gene sequencing showed that the isolate sequence was 99% identical to Streptomyces roseoverticillatus. The culture filtrates form different culture media were tested for nematocidal activity. The maximal activity against M. incognita was obtained by using modified basal (MB) medium. The nematicidal assay-directed fractionation of the culture broth delivered fervenulin (1) and isocoumarin (2). Fervenulin, a low molecular weight compound, shows a broad range of biological activities. However, nematicidal activity of fervenulin was not previously reported. The nematicidal activity of fervenulin (1) was assessed using the broth microdilution technique. The lowest minimum inhibitory concentrations (MICs) of the compound against egg hatch of M. incognita was 30 μg/ml and juvenile mortality of M. incognita increasing was observed at 120 μg/ml. Moreover, at the concentration of 250 μg/ml fervenulin (1) showed killing effect on second-stage nematode juveniles of M. incognita up to 100% after incubation for 96 h. Isocoumarin (2), another bioactive compound produced by Streptomyces sp. CMU-MH021, showed weak nematicidal activity with M. incognita.     

Autosomal-Dominant Striatal Degeneration Is Caused by a Mutation in the Phosphodiesterase 8B Gene

Autosomal-dominant striatal degeneration (ADSD) is an autosomal-dominant movement disorder affecting the striatal part of the basal ganglia. ADSD is characterized by bradykinesia, dysarthria, and muscle rigidity. These symptoms resemble idiopathic Parkinson disease, but tremor is not present. Using genetic linkage analysis, we have mapped the causative genetic defect to a 3.25 megabase candidate region on chromosome 5q13.3-q14.1. A maximum LOD score of 4.1 (Θ = 0) was obtained at marker D5S1962. Here we show that ADSD is caused by a complex frameshift mutation (c.94G>C+c.95delT) in the phosphodiesterase 8B (PDE8B) gene, which results in a loss of enzymatic phosphodiesterase activity. We found that PDE8B is highly expressed in the brain, especially in the putamen, which is affected by ADSD. PDE8B degrades cyclic AMP, a second messenger implied in dopamine signaling. Dopamine is one of the main neurotransmitters involved in movement control and is deficient in Parkinson disease. We believe that the functional analysis of PDE8B will help to further elucidate the pathomechanism of ADSD as well as contribute to a better understanding of movement disorders.     

Neuroprotective effects of Argon are mediated via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1 in retinal ganglion cells

Retinal ischemia and reperfusion injuries (R‐IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti‐apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK‐1/2 dependent regulation of heat‐shock proteins. Inhalation of Argon (75 Vol%) was performed after R‐IRI on the rats′ left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat‐shock proteins ?70, ?90 and heme‐oxygenase‐1, mitogen‐activated protein kinases (p38, JNK, ERK‐1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova . Argon significantly reduced the R‐IRI‐affected heat‐shock protein expression (p < 0.05). While Argon significantly induced ERK‐1/2 expression (p < 0.001), inhibition of ERK‐1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme‐oxygenase‐1 (p < 0.05). R‐IRI‐induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK‐1/2 activation in Müller cells. We conclude, that Argon treatment protects R‐IRI‐induced apoptotic loss of RGC via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1.

     

A new method for the automated age-at-death evaluation by tooth-cementum annulation (TCA)

A valid age at death estimation is required in historical and also forensic anthropology. Tooth cementum annulation (TCA) is a method for age at death estimation of adult individuals. The method is based on light microscope images taken from tooth-root cross sections. The age is then estimated by manually counting the cementum incremental lines and adding this to the chronological age at the assumed point of tooth eruption. Manual line counting, however, is time consuming, potentially subjective and the number of individual counts is insufficient for statistical evaluations. Software developed for the automated evaluation of TCA images, that uses Fourier analysis and algorithms for image analysis and pattern recognition is presented here. It involves "line-by-line" scanning and the counting of gray scale peaks within a selected region-of-interest (ROI). Each scanning process of a particular ROI yields up to 400 counts that are subsequently statistically evaluated. This simple and time saving program seeks to substitute manual counting and supply consistent and reproducible results as well as reduce the demand of human error by eliminating unavoidable factors such as subjectivity and fatigue.     

Comparative analysis of the human angiotensin II type 1a receptor heterologously produced in insect cells and mammalian cells

Angiotensin II type 1a receptor (AT1aR) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT1aR in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The recombinant receptor was expressed at a level of 29-32 pmol/mg and it binds to angiontensin II with high affinity (Kd=0.98-1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the recombinant AT1aR could couple to endogenous Galphaq protein. Confocal laser scanning microscopy revealed that the recombinant receptor was predominantly localized in the plasma membrane and agonist induced internalization of the recombinant AT1aR was also observed. The recombinant AT1aR was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.     

银杏内生菌Chaetomium globosum ZY-22次生代谢产物分离鉴定

采用柱层析方法从银杏叶内生真菌Chaetomium globosum ZY-22的培养菌丝体提取物中分离得到脑苷脂B(1)、脑苷脂C(2)、尿囊素(3)、9(11)-去氢麦角甾醇过氧化物(4)以及4,6,8,22-四烯-3-酮-麦角甾烷(5)和球毛壳甲素(6)共6个次生代谢物;经波谱分析确定了6个化合物的结构,其中脑苷脂B、脑苷脂C和尿囊素是首次从内生真菌中得到;海虾致死试验结果显示,化合物1～6在10 μg/mL浓度下对丰年虾的致死率分别为1.6%、4.2%、7.4%、16.9%、12.8%、83.6%、表明球毛壳甲素对海虾表现出很强的毒性作用.     

Crystal structure and functional characterization of selenocysteine-containing glutathione peroxidase 4 suggests an alternative mechanism of peroxide reduction

Glutathione peroxidases (GPX) are anti-oxidative enzymes that reduce organic and inorganic hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. The human genome involves eight GPX genes and five of them encode for selenocysteine-containing enzymes. Among the human GPX-isoforms, GPX4 is unique since it is capable of reducing complex hydroperoxy ester lipids such as hydroperoxy phospholipids and hydroperoxy cholesterolesters. Using a number of genetically modified mouse strains the biological role of GPX4 has comprehensively characterized but the molecular enzymology is less well explored. This lack of knowledge is partly related to the fact that mammalian selenoproteins are not high-level expressed in conventional overexpression systems. To explore the structural and functional properties of human GPX4 we expressed this selenoprotein in a cysteine-auxotrophic E. coli strain using a semi-chemical expression strategy. The recombinant enzyme was purified in mg amounts from the bacterial lysate to electrophoretic homogeneity and characterized with respect to its protein-chemical and enzymatic properties. Its crystal structure was solved at 1.3?Å resolution and the X-ray data indicated a monomeric protein, which contains the catalytic selenium at the redox level of the seleninic acid. These data suggest an alternative reaction mechanism involving three different redox states (selenol, selenenic acid, seleninic acid) of the catalytically active selenocysteine.     

H5N1 whole-virus vaccine induces neutralizing antibodies in humans which are protective in a mouse passive transfer model

Background

Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge.

Methods

We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus.

Results

Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species.

Conclusions

These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines.