Ross River virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production

Pseudotyped retroviruses have important applications as vectors for gene transfer and gene therapy and as tools for the study of viral glycoprotein function. Recombinant Moloney murine leukemia virus (Mo-MuLV)-based retrovirus particles efficiently incorporate the glycoproteins of the alphavirus Ross River virus (RRV) and utilize them for entry into cells. Stable cell lines that produce the RRV glycoprotein-pseudotyped retroviruses for prolonged periods of time have been constructed. The pseudotyped viruses have a broadened host range, can be concentrated to high titer, and mediate stable transduction of genes into cells. The RRV glycoprotein-pseudotyped retroviruses and the cells that produce them have been employed to demonstrate that RRV glycoprotein-mediated viral entry occurs through endocytosis and that membrane fusion requires acidic pH. Alphavirus glycoprotein-pseudotyped retroviruses have significant advantages as reagents for the study of the biochemistry and prevention of alphavirus entry and as preferred vectors for stable gene transfer and gene therapy protocols.     

Mice overexpressing human caspase 3 appear phenotypically normal but exhibit increased apoptosis and larger lesion volumes in response to transient focal cerebral ischaemia

Caspase 3 activation has been implicated in cell death following a number of neurodegenerative insults. To determine whether caspase genes can affect the susceptibility of cells to neurodegeneration, a transgenic mouse line was created, expressing human caspase 3 under control of its own promoter. The human gene was regulated by the murine homeostatic machinery and human procaspase 3 was expressed in the same tissues as mouse caspase 3. These novel transgenic mice appeared phenotypically and developmentally normal and survived in excess of 2 years. Behavioural assessment using the 5-choice serial reaction time task found no differences from wild-type littermates. Caspase activity was found to be tightly regulated under physiological conditions, however, significantly larger lesions were obtained when transgenic mice were subjected to focal cerebral ischaemia/reperfusion injury compared to wild-type littermates. These data demonstrate that mice overexpressing human caspase 3 are essentially normal, however, they have increased susceptibility to degenerative insults.     

Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations

Nonsteroidal antiinflammatory drugs (NSAIDs) block prostanoid biosynthesis by inhibiting prostaglandin H(2) synthase (EC 1.14.99.1). NSAIDs are either rapidly reversible competitive inhibitors or slow tight-binding inhibitors of this enzyme. These different modes of inhibition correlate with clinically important differences in isoform selectivity. Hypotheses have been advanced to explain the different inhibition kinetics, but no structural data have been available to test them. We present here crystal structures of prostaglandin H(2) synthase-1 in complex with the inhibitors ibuprofen, methyl flurbiprofen, flurbiprofen, and alclofenac at resolutions ranging from 2.6 to 2.75 A. These structures allow direct comparison of enzyme complexes with reversible competitive inhibitors (ibuprofen and methyl flurbiprofen) and slow tight-binding inhibitors (alclofenac and flurbiprofen). The four inhibitors bind to the same site and adopt similar conformations. In all four complexes, the enzyme structure is essentially unchanged, exhibiting only minimal differences in the inhibitor binding site. These results argue strongly against hypotheses that explain the difference between slow tight-binding and fast reversible competitive inhibition by invoking global conformational differences or different inhibitor binding sites. Instead, they suggest that the different apparent modes of NSAID binding may result from differences in the speed and efficiency with which inhibitors can perturb the hydrogen bonding network around Arg-120 and Tyr-355.     

Increased heat sensitivity of photosynthesis in tobacco plants with reduced Rubisco activase

High temperature inhibits photosynthesis by several mechanisms including deactivation of Rubisco. The inhibition of photosynthesis by high temperature and its relationship to Rubisco deactivation was studied using tobacco (Nicotiana tabaccum L. cv W38) transformed with a Rubisco activase gene inserted in the antisense orientation and untransformed controls. High temperature (42 °C) reduced photosynthesis in both lines of plants. However, photosynthesis recovered nearly completely in wild-type plants and very little in plants lacking Rubisco activase. The F₀ level of chlorophyll fluorescence decreased and q_N increased in the control plants during heating. In the antisense plants, q_N was always high and F₀ increased slightly during heat stress. NADP-malate dehydrogenase activation was unaffected by heat stress in control plants but was increased in the transgenic plants, consistent with a high redox status in the chloroplast. In wild-type plants, the inhibition of photosynthesis could be explained by a reversible decarbamylation of Rubisco and an acceptor-side limitation imposed on photosynthetic electron transport. However, in the anti-activase plants, carbamylation was low and constant and could not explain how photosynthesis was reduced at high temperature. Because ribulose bisphosphate was saturating at high temperature, the reduction in photosynthesis must have been caused by some impairment of Rubisco function not reflected in measurements of activation state or carbamylation status. This in vivo Rubisco impairment was not relieved upon return to lower temperature. We speculate that the reversible decarbamylation of Rubisco at moderately high temperature may be a protective mechanism by which the plant avoids more serious effects on Rubisco and the rest of the photosynthetic apparatus.This revised version was published online in October 2005 with corrections to the Cover Date.     

The effects of high temperature on isoprene synthesis in oak leaves

Isoprene emission from plants is highly temperature sensitive and is common in forest canopy species that experience rapid leaf temperature fluctuations. Isoprene emission declines with temperature above 35 °C but the temperature at which the decline begins varies between 35 and 44 °C. This variability is caused by the rate at which leaf temperature is increased during measurement with lower temperatures associated with longer measurement cycles. To investigate this we exposed leaves of red oak (Quercus rubra L.) to temperature regimes of 35–45 °C for periods of 20–60 min. Isoprene emission increased during the first 10 min of high temperature exposure and then decreased over the next 10 min until it reached steady state. This phenomenon was common at temperatures above 35 °C but was not noticeable at temperatures below that. The response was reversible within 30 min by lowering leaf temperature to 30 °C. Because there is no storage of isoprene inside the leaf, this behaviour indicates regulation of isoprene synthesis in the leaf. We demonstrated that the variability in isoprene decline results from regulation and explains the variability in the temperature response. This is consistent with our theory that isoprene protects leaves from damage caused by rapid temperature fluctuations.     

The metabolic origins of non-photorespiratory CO2 release during photosynthesis: a metabolic flux analysis

Respiration in the light (R_L) releases CO₂ in photosynthesizing leaves and is a phenomenon that occurs independently from photorespiration. Since R_L lowers net carbon fixation, understanding R_L could help improve plant carbon-use efficiency and models of crop photosynthesis. Although R_L was identified more than 75 years ago, its biochemical mechanisms remain unclear. To identify reactions contributing to R_L, we mapped metabolic fluxes in photosynthesizing source leaves of the oilseed crop and model plant camelina (Camelina sativa). We performed a flux analysis using isotopic labeling patterns of central metabolites during ¹³CO₂ labeling time course, gas exchange, and carbohydrate production rate experiments. To quantify the contributions of multiple potential CO₂ sources with statistical and biological confidence, we increased the number of metabolites measured and reduced biological and technical heterogeneity by using single mature source leaves and quickly quenching metabolism by directly injecting liquid N₂; we then compared the goodness-of-fit between these data and data from models with alternative metabolic network structures and constraints. Our analysis predicted that R_L releases 5.2 μmol CO₂ g⁻¹ FW h⁻¹ of CO₂, which is relatively consistent with a value of 9.3 μmol CO₂ g⁻¹ FW h⁻¹ measured by CO₂ gas exchange. The results indicated that ≤10% of R_L results from TCA cycle reactions, which are widely considered to dominate R_L. Further analysis of the results indicated that oxidation of glucose-6-phosphate to pentose phosphate via 6-phosphogluconate (the G6P/OPP shunt) can account for >93% of CO₂ released by R_L.     

Deregulation of the serum- and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure

Infertility and recurrent pregnancy loss (RPL) are prevalent but distinct causes of reproductive failure that often remain unexplained despite extensive investigations. Analysis of midsecretory endometrial samples revealed that SGK1, a kinase involved in epithelial ion transport and cell survival, is upregulated in unexplained infertility, most prominently in the luminal epithelium, but downregulated in the endometrium of women suffering from RPL. To determine the functional importance of these observations, we first expressed a constitutively active SGK1 mutant in the luminal epithelium of the mouse uterus. This prevented expression of certain endometrial receptivity genes, perturbed uterine fluid handling and abolished embryo implantation. By contrast, implantation was unhindered in Sgk1-/- mice, but pregnancy was often complicated by bleeding at the decidual-placental interface and fetal growth retardation and subsequent demise. Compared to wild-type mice, Sgk1-/- mice had gross impairment of pregnancy-dependent induction of genes involved in oxidative stress defenses. Relative SGK1 deficiency was also a hallmark of decidualizing stromal cells from human subjects with RPL and sensitized these cells to oxidative cell death. Thus, depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility, or predisposes to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage.     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.