Bovine floor plate explants secrete SCO-spondin

SCO-spondin is a large-molecular mass glycoprotein, secreted by the subcommissural organ (SCO), which has been implicated in neuronal development during ontogeny of the central nervous system. The expression of SCO-spondin is not restricted to the SCO but it also occurs in the floor plate, a key structure participating in neuronal differentiation and patterning of the neural tube. It has been postulated that SCO-spondin detected in the floor plate is released into the lumen of the neural tube, but this new route of secretion of floor plate cells needs to be further substantiated. For this purpose, we standardized long-term organ culture of bovine floor plate and performed morphological, immunological, biochemical, and gene expression analyses. The study of floor plate explants and their conditioned media allowed us to demonstrate that: (1) organ-cultured floor plate cells are actively secretory for up to 25 days; (2) SCO-spondin gene is actively transcribed and translated by the cultured floor plate cells; (3) SCO-spondin is released into the culture medium via the apical cell pole; and (4) upon release, SCO-spondin does not aggregate in the conditioned medium but remains soluble. Furthermore, in the cultured floor plate cells, SCO-spondin may be secreted through a route bypassing the Golgi apparatus.     

Characterization of a novel testicular form of human hormone-sensitive lipase

Hormone-sensitive lipase (HSL) is an esterase and lipase, which are essential for spermatogenesis. Two HSL mRNAs are expressed in human testis. A long form is encoded by a testis-specific exon and nine exons common to testis and adipocyte HSL. Here we show that the short-form 3.3-kb mRNA possesses a unique 5' end that is transcribed from a novel testis-specific exon. The corresponding protein is similar to the 775-amino-acid-long adipocyte HSL. Immunohistochemistry experiments on human testis sections revealed that the long form is strictly expressed in haploid germ cells whereas the short form is expressed in interstitial and tubular somatic cells as well as premeiotic germ cells.     

Progesterone blocks the estradiol-stimulated luteinizing hormone surge by disrupting activation in response to a stimulatory estradiol signal in the ewe

The preovulatory surges of GnRH and LH are activated by increased concentrations of circulating estradiol, but ovulation is blocked when progesterone concentrations are elevated. Although it is has been shown that this action of progesterone is due to a central inhibition of the GnRH surge, the mechanisms that underlie the blockade of the GnRH surge are poorly understood. In this study we investigated whether progesterone can block the estradiol-dependent activation stage of the GnRH surge induction process, and thus prevent expression of the LH surge. The results demonstrated that exposure to progesterone for half or the full duration of the activation stage can prevent the stimulation of LH surges by estradiol (experiment 1), whereas exposure to progesterone midway though a period of estradiol exposure, which in itself is sufficient to activate the surge, did not block the LH surge (experiment 2). These results suggest that progesterone 1) disrupts activation of the surge induction system in response to a stimulatory estradiol signal and 2) does not compromise the ability of animals to respond to a stimulatory estradiol signal applied immediately after progesterone exposure. Because the disruptive effects of activated progesterone in response to estradiol are rapid but transient, it may be that progesterone directly interferes with the activation of estradiol-responsive neural systems to block the GnRH/LH surge.     

Fructose inhibits apoptosis induced by reoxygenation in rat hepatocytes by decreasing reactive oxygen species via stabilization of the glutathione pool

Oxidative stress induces apoptosis in liver parenchymal cells. The present study demonstrates that the substitution of fructose for glucose as sole carbon source in the incubation medium reduced apoptosis due to reoxygenation up to 50% in cultured rat hepatocytes. This anti-apoptotic action of fructose cannot be explained by the effects of this sugar on the intracellular ATP concentration and the ATP/ADP ratio. Rather, the suppression of apoptosis by fructose seems to be a consequence of remarkably higher intracellular levels of glutathione observed during reoxygenation in fructose-fed hepatocytes in contrast to glucose-fed ones. With fructose as substrate, the generation of excess reactive oxygen species (ROS) during the initial phase of reoxygenation was strongly reduced. With respect to ROS reduction and stabilization of the cellular glutathione pool fructose was found as efficient as a pretreatment of glucose fed cells with N-acetyl-L-cysteine. The enhanced metabolization of ROS by the glutathione/glutathione peroxidase system in fructose-cultured hepatocytes under reoxygenation was expected to improve their mitochondrial status so that late events in the apoptotic pathway are suppressed. This could be confirmed by the reduced release of cytochrome c from mitochondria into the cytosol as well as by the observed decrease of caspase-3 activity during reoxygenation.     

Chlorobium Tepidum: Insights into the Structure,Physiology, and Metabolism of a Green Sulfur Bacterium Derived from the Complete Genome Sequence

Green sulfur bacteria are obligate, anaerobic photolithoautotrophs that synthesize unique bacteriochlorophylls (BChls) and a unique light-harvesting antenna structure, the chlorosome. One organism, Chlorobium tepidum, has emerged as a model for this group of bacteria primarily due to its relative ease of cultivation and natural transformability. This review focuses on insights into the physiology and biochemistry of the green sulfur bacteria that have been derived from the recently completed analysis of the 2.15-Mb genome of Chl. tepidum. About 40 mutants of Chl. tepidum have been generated within the last 3 years, most of which have been made based on analyses of the genome. This has allowed a nearly complete elucidation of the biosynthetic pathways for the carotenoids and BChls in Chl. tepidum, which include several novel enzymes specific for BChl c biosynthesis. Facilitating these analyses, both BChl c and carotenoid biosynthesis can be completely eliminated in Chl. tepidum. Based particularly on analyses of mutants lacking chlorosome proteins and BChl c, progress has also been made in understanding the structure and biogenesis of chlorosomes. In silico analyses of the presence and absence of genes encoding components involved in electron transfer reactions and carbon assimilation have additionally revealed some of the potential physiological capabilities, limitations, and peculiarities of Chl. tepidum. Surprisingly, some structural components and biosynthetic pathways associated with photosynthesis and energy metabolism in Chl. tepidum are more similar to those in cyanobacteria and plants than to those in other groups of photosynthetic bacteria.     

Distribution of turbidity detection times produced by single cell-generated bacterial populations

The distributions of the times to turbidity for wells inoculated with single cells of Listeria innocua were determined in different environmental conditions (pH 4.5 to 7 and with 0.5% to 8% of NaCl at 30 degrees C). It was established by statistical analysis that the main source of the variability of the detection times, T, is the variability of individual lag times. A linear relation dev(T) approximately T was observed between the detection times and their standard deviation. At slow growth, other sources of variability became increasingly significant.     

GAD2 on chromosome 10p12 is a candidate gene for human obesity

The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11–12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of γ-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake. A case-control study (575 morbidly obese and 646 control subjects) analyzing GAD2 variants identified both a protective haplotype, including the most frequent alleles of single nucleotide polymorphisms (SNPs) +61450 C>A and +83897 T>A (OR = 0.81, 95% CI [0.681–0.972], p = 0.0049) and an at-risk SNP (−243 A>G) for morbid obesity (OR = 1.3, 95% CI [1.053–1.585], p = 0.014). Furthermore, familial-based analyses confirmed the association with the obesity of SNP +61450 C>A and +83897 T>A haplotype (χ² = 7.637, p = 0.02). In the murine insulinoma cell line βTC3, the G at-risk allele of SNP −243 A>G increased six times GAD2 promoter activity (p < 0.0001) and induced a 6-fold higher affinity for nuclear extracts. The −243 A>G SNP was associated with higher hunger scores (p = 0.007) and disinhibition scores (p = 0.028), as assessed by the Stunkard Three-Factor Eating Questionnaire. As GAD2 is highly expressed in pancreatic β cells, we analyzed GAD65 antibody level as a marker of β-cell activity and of insulin secretion. In the control group, −243 A>G, +61450 C>A, and +83897 T>A SNPs were associated with lower GAD65 autoantibody levels (p values of 0.003, 0.047, and 0.006, respectively). SNP +83897 T>A was associated with lower fasting insulin and insulin secretion, as assessed by the HOMA-B% homeostasis model of β-cell function (p = 0.009 and 0.01, respectively). These data support the hypothesis of the orexigenic effect of GABA in humans and of a contribution of genes involved in GABA metabolism in the modulation of food intake and in the development of morbid obesity.     

Optimization of chitin extraction from shrimp shells

The aim of this paper is to define optimal conditions for the extraction of chitin from shrimp shells. The kinetics of both demineralization and deproteinization with, in the latter case, the role of temperature are studied. The characterization of the residual calcium and protein contents, the molecular weights, and degrees of acetylation (DA) allows us to propose the optimal conditions as follows. The demineralization is completely achieved within 15 min at ambient temperature in an excess of HCl 0.25 M (with a solid-to-liquid ratio of about 1/40 (w/v)). The deproteinization is conveniently obtained in NaOH 1 M within 24 h at a temperature close to 70 degrees C with no incidence on the molecular weight or the DA. In these conditions, the residual content of calcium in chitin is below 0.01%, and the DA is almost 95%.     

Effects of increased salinity on gravitaxis in Euglena gracilis

The unicellular freshwater flagellate Euglena gracilis regulates its position in the water column by means of phototactic and gravitactic behavior. Recent experiments have revealed that the cells switch between negative and positive gravitaxis depending upon environmental stimuli such as solar radiation. In this study, the effect of increased salinity on gravitaxis in Euglena gracilis was investigated. In some experiments it was found that salt concentrations up to 5 gL-1 (in some experiments 10 gL-1) increased the motility, velocity and precision of negative gravitactic orientation. Higher salt concentrations decreased all these parameters. At concentrations of about 15 gL-1, cells which did not become immobile, switched from negative to positive gravitaxis. Positive gravitaxis persisted for several hours or even days when the cells were transferred back to standard culture medium. Most of the cells in cultures exposed to salt concentrations above 20 gL-1 lost their motility (partial formation of palmella stages) but recovered when transferred back to standard medium or de-ionised water. Post recovery, the cells showed pronounced positive gravitaxis. Additional investigations on the pigmentation, revealed that the cells showed a complete loss of a carotenoid shoulder in the spectrum, which reappeared when the cells were brought back to standard medium.