Protection by herpes simplex virus glycoprotein D against Fas-mediated apoptosis: role of nuclear factor kappaB

Signals involved in protection against apoptosis by herpes simplex virus 1 (HSV-1) were investigated. Using U937 monocytoid cells as an experimental model, we have demonstrated that HSV-1 rendered these cells resistant to Fas-induced apoptosis promptly after infection. UV-inactivated virus as well as the envelope glycoprotein D (gD) of HSV-1, by itself, exerted a protective effect on Fas-induced apoptosis. NF-kappaB was activated by gD, and protection against Fas-mediated apoptosis by gD was abolished in cells stably transfected with a dominant negative mutant I-kappaBalpha, indicating that NF-kappaB activation plays a role in the antiapoptotic activity of gD in our experimental model. Moreover, NF-kappaB-dependent protection against Fas-mediated apoptosis was associated with decreased levels of caspase-8 activity and with the up-regulation of intracellular antiapoptotic proteins.     

UV-induced apoptosis is mediated independent of caspase-9 in MCF-7 cells: a model for cytochrome c resistance

The importance of the mitochondria in UV-induced apoptosis has become increasingly apparent. Following DNA damage cytochrome c and other pro-apoptotic factors are released from the mitochondria, allowing for formation of the apoptosome and subsequent cleavage and activation of caspase-9. Active caspase-9 then activates downstream caspases-3 and/or -7, which in turn cleave poly(ADP)-ribose polymerase (PARP) and other down-stream targets, resulting in apoptosis. In an effort to understand the mechanisms of Akt-mediated cell survival in breast cancer, we studied the effects of insulin-like growth factor (IGF)-I treatment on UV-treated MCF-7 human breast cancer cells. Apoptosis was induced in MCF-7 cells after UV treatment, as measured by caspase-7 and PARP cleavage, and IGF-I co-treatment protected against this response. Surprisingly caspase-9 cleavage was unchanged with UV and/or IGF-I treatment. Using MCF-7 cells overexpressing caspase-3 we have shown that resistance of caspase-9 to cleavage was not altered by the expression of caspase-3. Furthermore, overexpression of caspase-9 did not enhance PARP or caspase-7 cleavage after UV treatment. Because caspase-8 was activated with UV treatment alone, we believe that UV-induced apoptosis in MCF-7 cells occurs independently of cytochrome c and caspase-9, supporting the existence of a cytoplasmic inhibitor of cytochrome c in MCF-7 cells. We anticipate that such inhibitors may be overexpressed in cancer cells, allowing for treatment resistance.     

Reaction and substrate specificity of recombinant pig kidney Dopa decarboxylase under aerobic and anaerobic conditions

Dopa decarboxylase (DDC) catalyzes as main reaction the stereospecific CO(2) abstraction from L-Dopa and L-5-hydroxytryptophan (5-HTP), generating the corresponding aromatic amines, dopamine and serotonin, respectively. Side reactions with turnover time of minutes are also catalyzed by the enzyme. In particular, DDC exhibits half-transaminase activity toward D-aromatic amino acids and oxidative deaminase activity toward aromatic amines. The latter reaction could represent a new activity for this class of enzymes. Studies on the effect exerted by O(2) on reaction specificity of DDC revealed that under anaerobic conditions decarboxylation of L-aromatic amino acids takes place with a k(cat) approximately half of that measured in the presence of O(2), and is accompanied by a decarboxylation-dependent transamination, whereas oxidative deamination of aromatic amines is replaced by half-transamination. Half-transamination of D-aromatic amino acids is unaffected by the presence or absence of O(2). Some structural elements relevant for the control of reaction and substrate specificity of DDC have been identified by means of limited tryptic digestion and site-directed mutagenesis studies. All together, the data indicate that the chemical nature of the substrate, the presence of O(2), the integrity of a mobile loop, the absence of perturbation in the coenzyme-binding cleft and pH are important requirements for the achievement of a closed conformational state where the highest level of reaction specificity is reached.     

Kinetic properties of the glucose 6-phosphatase of the liver from arthritic rats

According to previous reports, adjuvant-induced arthritic rats present reduced activities of the hepatic glucose 6-phosphatase. A kinetic study was done in order to characterize this phenomenon. Microsomes were isolated from livers of arthritic and control rats (Holtzman strain) and the glucose 6-phosphatase was measured at various temperatures (13-37 degrees C) and glucose 6-phosphate concentrations. Irrespective of the temperature, the enzyme from arthritic rats presented a reduction of both V(max) and K(M). Detergent treatment of liver microsomes from control rats increased the activity, but no increase was found when microsomes from arthritic rats were treated in the same way. The mannose 6-phosphatase activity of detergent-treated microsomes from arthritic rats was only 25% of the activity found with detergent-treated microsomes from control rats. Without detergent treatment, the mannose 6-phosphatase activities of both control and arthritic rats were minimal. The activation energy, derived from V(max), was not changed by arthritis. In vivo arthritic rats presented higher hepatic glucose 6-phosphate concentrations, a phenomenon that is consistent with a reduced activity of glucose 6-phosphatase. It was concluded that in arthritic rats, the hydrolase is probably reduced, without a similar change in the translocase activity.     

Association of specific language impairment (SLI) to the region of 7q31

FOXP2 (forkhead box P2) was the first gene characterized in which a mutation affects human speech and language abilities. A common developmental language disorder, specific language impairment (SLI), affects 6%-7% of children with normal nonverbal intelligence and has evidence of a genetic basis in familial and twin studies. FOXP2 is located on chromosome 7q31, and studies of other disorders with speech and language impairment, including autism, have found linkage to this region. In the present study, samples from children with SLI and their family members were used to study linkage and association of SLI to markers within and around FOXP2, and samples from 96 probands with SLI were directly sequenced for the mutation in exon 14 of FOXP2. No mutations were found in exon 14 of FOXP2, but strong association was found to a marker within the CFTR gene and another marker on 7q31, D7S3052, both adjacent to FOXP2, suggesting that genetic factors for regulation of common language impairment reside in the vicinity of FOXP2.     

Toward consistent and productive complex media for industrial fermentations: studies on yeast extract for a recombinant yeast fermentation process

Yeast extract (YE) is commonly used as a key component in the complex media for industrial fermentations. However, the lot-to-lot variation of this raw material frequently requires extensive "use testing" of many lots to identify only the few that support desired fermentation performance. Through extensive fermentation studies and chemical analyses, we have identified adenine and two metabolizable carbon sources, trehalose and lactate, as the principle components in YE that affect the production of a recombinant protein antigen by a yeast strain. Adenine is required for culture growth and the relationship between biomass and measured adenine can be expressed by a Michaelis-Menten model, while the slowly metabolized trehalose serves to maintain the energy supply to the continued antigen synthesis. The rapidly utilized lactate exerts an indirect positive effect by sparing some of the accumulated ethanol from being consumed for growth to being utilized in the product formation. The effects of these YE components are mutually dependent. Based on the database generated from 40 lots at laboratory scale, a relatively high level of carbon sources in YE (trehalose plus lactate, >9.5% w/w) and an intermediate level of adenine (0.14-0.24% w/w) appear to be the minimal requirement of a good lot for this recombinant yeast fermentation. Many poor lots were improved in lab fermenters by rational supplementation of trehalose, lactate, or adenine to compensate for their insufficiencies. At the large production scale, predictions based on adenine and trehalose/lactate contents in various YE lots used correlated reasonably well with culture growth and antigen yield, illustrating the feasibility of such a simple chemical/biochemical analysis as a rapid and reliable initial screening tool. Without incurring any compositional change to an established manufacturing medium, this study demonstrates an effective approach to achieve consistency in fermentations employing complex nutrients and to improve fermentation productivities supported by suboptimal lots of raw material.     

Evolution of additive and nonadditive genetic variance in development time along a cline in Drosophila serrata

Abstract. Latitudinal clines provide natural systems that may allow the effect of natural selection on the genetic variance to be determined. Ten clinal populations of Drosophila serrata collected from the eastern coast of Australia were used to examine clinal patterns in the trait mean and genetic variance of the life-history trait egg-to-adult development time. Development time significantly lengthened from tropical areas to temperate areas. The additive genetic variance for development time in each population was not associated with latitude but was associated with the population mean development time. Additive genetic variance tended to be larger in populations with more extreme development times and appeared to be consistent with allele frequency change. In contrast, the nonadditive genetic variance was not associated with the population mean but was associated with latitude. Levels of nonadditive genetic variance were greatest in the region of the cline where the gradient in the change in mean was greatest, consistent with Barton's (1999) conjecture that the generation of linkage disequilibrium may become an important component of the genetic variance in systems with a spatially varying optimum.     

Palmitate acutely raises glycogen synthesis in rat soleus muscle by a mechanism that requires its metabolization (Randle cycle)

The acute effect of palmitate on glucose metabolism in rat skeletal muscle was examined. Soleus muscles from Wistar male rats were incubated in Krebs-Ringer bicarbonate buffer, for 1 h, in the absence or presence of 10 mU/ml insulin and 0, 50 or 100 microM palmitate. Palmitate increased the insulin-stimulated [(14)C]glycogen synthesis, decreased lactate production, and did not alter D-[U-(14)C]glucose decarboxylation and 2-deoxy-D-[2,6-(3)H]glucose uptake. This fatty acid decreased the conversion of pyruvate to lactate and [1-(14)C]pyruvate decarboxylation and increased (14)CO(2) produced from [2-(14)C]pyruvate. Palmitate reduced insulin-stimulated phosphorylation of insulin receptor substrate-1/2, Akt, and p44/42 mitogen-activated protein kinases. Bromopalmitate, a non-metabolizable analogue of palmitate, reduced [(14)C]glycogen synthesis. A strong correlation was found between [U-(14)C]palmitate decarboxylation and [(14)C]glycogen synthesis (r=0.99). Also, palmitate increased intracellular content of glucose 6-phosphate in the presence of insulin. These results led us to postulate that palmitate acutely potentiates insulin-stimulated glycogen synthesis by a mechanism that requires its metabolization (Randle cycle). The inhibitory effect of palmitate on insulin-stimulated protein phosphorylation might play an important role for the development of insulin resistance in conditions of chronic exposure to high levels of fatty acids.     

Treponema denticola cystalysin catalyzes beta-desulfination of L-cysteine sulfinic acid and beta-decarboxylation of L-aspartate and oxalacetate

Pyridoxal 5'-phosphate-dependent cystalysin from Treponema denticola catalyzes the beta-displacement of the beta-substituent from both L-aspartate and L-cysteine sulfinic acid. The steady-state kinetic parameters for beta-desulfination of L-cysteine sulfinic acid, k(cat) and K(m), are 89+/-7 s(-1) and 49+/-9 mM, respectively, whereas those for beta-decarboxylation of L-aspartate are 0.8+/-0.1 s(-1) and 280+/-70 mM. Moreover, cystalysin in the pyridoxamine 5'-phosphate form has also been found to catalyze beta-decarboxylation of oxalacetate as shown by consumption of oxalacetate and a concomitant production of pyruvate. The k(cat) and K(m) of this reaction are 0.15+/-0.01 s(-1) and 13+/-2 mM, respectively. Possible mechanistic and physiological implications are discussed.     

Steady-state assemblages of phytoplankton in four temperate lakes (NE U.S.A.)

For four temperate lakes (Northeast U.S.A.) we identify periods of persistent phytoplankton assemblages and investigate the ecological conditions that correlate to these persistent assemblages. Periods of persistent assemblages, here considered as steady-state phases, were defined according to equilibrium criteria (two or three coexisting species, contributing to 80% of the standing biomass, for at least 2 weeks) defined by Sommer et al. (1993, Hydrobiologia 249: 1–7). For all four lakes, samples were taken weekly during the ice-free season and phytoplankton attributes (biomass, assemblages, diversity, species richness, change rates) and abiotic variables (temperature, I^* – as light mean in the mixing zone – z_mix, and nutrients) were analysed. Chodikee (CH), an eutrophic and rapidly flushed lake, did not show any persistent phase. The remaining three lakes showed single steady-state phases that occurred at varying times during the ice-free season. Steady-state phases occurred during early stratification in late spring in the stably stratified oligotrophic Mohonk Lake (MO), in the late summer stratification in the meso-eutrophic Stissing Lake (ST), and during spring mixing in Wononscopomuc Lake (WO). MO showed a 3-week period with dominance of F assemblage (Botryococcus braunii, Willea wilhelmii and Eutetramorus planctonicus), characteristic for clear epilimnia, tolerant to low nutrient and sensitive to high turbidity. For three weeks, ST had a stable assemblage with dominance of Lo(Woronichinia sp.), common assemblage in summer epilimnion of mesotrophic lakes and sensitive to prolonged or deep mixing; and P, assemblage able to live in eutrophic epilimnia with mild light and sensitive to stratification and silica depletion. In contrast, the mesotrophic Wononscopomuc Lake (WO) showed persistent assemblages during a 4-week period of spring circulation, when a dinoflagellate (Lo) was co-dominant with Nitzschia acicularis (C). The latter species is characteristic for mesotrophic lakes, tolerant to low light and sensitive to stratification and silica depletion. Both Lo and P assemblages, among seven others, had before been quoted, in literature, as dominant in maturing stages. We could not find consistent statistical differences between the periods classified as steady-state and non-steady-state. However, the data demonstrated that prolonged period of both mixing and stratification can maintain dominant assemblages. Although, historically sensed as opposite mechanisms, both mixing and stratification, if persistent, were observed maintaining dominant assemblages because both scenarios are characterized by environmental constancy.