Liquid chromatographic assay for riluzole in mouse plasma and central nervous system tissues

An isocratic, reversed-phase high-performance liquid chromatographic procedure (HPLC) was developed for determination of the neuroprotective agent riluzole in mice plasma, brain and spinal cord. The procedure is based on isolation of the compound and the internal standard from plasma and central nervous system tissues using a Bakerbond spe C8 cartridge, with satisfactory recovery and specificity. Separation was on a C18 column, coupled with an UV detector at 263 nm. The assay was linear over a wide range, with a lower limit of quantification of 100 ng ml(-1) or g(-1) using 0.1 ml of plasma and about 100mg of brain tissue. The precision and accuracy were within the acceptable limits for an HPLC assay. The method is currently used to support pharmacological studies of the activity of riluzole when given in combination with other potential neuroprotective agents in an animal model of familiar amyotrophic lateral sclerosis (SOD1-G93A transgenic mice).     

The role of calcium in signal transduction processes in Sertoli cells

Sertoli cells play a pivotal role in regulation and maintenance of spermatogenesis. They are hormonally regulated predominantly by follicle-stimulating hormone (FSH) and testosterone (T). Although FSH and T have distinct mechanisms of action they act synergistically in promoting spermatogenesis. Stimulation of freshly isolated Sertoli cells with FSH evokes a prompt rise in cytosolic calcium which is quantitatively reproduced by cAMP. The cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP. Calcium homeostasis of Sertoli cells may also be regulated by cAMP-independent metabolism. Vasoactive testicular paracrine hormones such as angiotensin II (AII) and vasopressin acting via inositol triphosphate generation induce cytosolic calcium rise predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. Investigations involving androgens action on cytosolic calcium reveal a common mechanism of action between the peptide and steroid regulators of Sertoli cell function, indicating that cytosolic calcium ions may represent a unifying biochemical mechanism that could explain the synergism of FSH and T. Androgens rapidly and specifically increase cytosolic calcium, consistent with a plasma membrane site of action. This argues for the possible existence of a short term non-genomic signaling pathway in hormonal regulation of Sertoli cell function in addition to the classical longer term, slower genomic response.     

Early response to bleomycin is characterized by different cytokine and cytokine receptor profiles in lungs

The sensitivity to the fibrosis-inducing effect of bleomycin varies considerably from species to species, the reasons for which are unknown. The variability of the response in different strains of mice is well documented. Recent evidence indicates that the upregulated expression of cytokines and cytokine receptors may be involved. We evaluated the expression pattern of some cytokines and their receptors in C57Bl/6J bleomycin-sensitive and Balb/C bleomycin-resistant mice. Animals from both strains received, under ether anesthesia, either saline (50 microl) or bleomycin (0.1 U/50 microl) intratracheally. At various times after the treatment, the lungs were analyzed for cytokines and cytokine receptors by histochemistry and their mRNA by RNase protection assay. A significantly increased expression of TNF-alpha and IL-1beta was observed in both strains. However, an upregulated lung expression for TNF-alpha and IL-1 receptors was observed in C57Bl/6J-sensitive animals only. This profile is evident from 63 h onward. In addition to TNF-alpha, bleomycin administration also resulted in the upregulated expression of TGF-beta in the lungs of both strains at 8 h and in an enhanced expression of TGF-beta receptors I and II in C57Bl/6J mice only. The upregulation of TGF-beta receptor expression was preceded in this strain by an increased expression of IL-4, IL-13, and IL-13 receptor-alpha (at 8 h after bleomycin) and followed by an upregulation of gp130 and IL-6. The difference we observed in the cytokine receptor profile may offer an additional explanation for the different fibrogenic response of the two mouse strains to bleomycin.     

Upregulation of Neuronal Nitric Oxide Synthase in in Vitro Stellate Astrocytes and in Vivo Reactive Astrocytes After Electrically Induced Status Epilepticus

Neuronal nitric oxide synthase (nNOS) is a constitutively expressed and calcium-dependent enzyme. Despite predominantly expressed in neurons, nNOS has been also found in astrocytes, although at lower expression levels. We have studied the regulation of nNOS expression in cultured rat astrocytes from cortex and spinal cord by Western blotting and immunocytochemistry. nNOS was not detectable in cultured astrocytes grown in serum-containing medium (SCM), but was highly expressed after serum deprivation. Accordingly, calcium-dependent NOS activity and both intracellular nitrite levels and nitrotyrosine immunoreactivity after glutamate stimulation were higher in serum-deprived astrocytes than in cells grown in SCM. Serum deprivation induced a modification of astrocytes morphology, from flat to stellate. nNOS upregulation was also observed in reactive astrocytes of rat hippocampi after electrically induced status epilepticus, as demonstrated by double-labeling experiments. Thus, nNOS upregulation occurs in both in vitro stellate and in vivo reactive astrocytes, suggesting a possible involvement of glial nNOS in neurological diseases characterized by reactive gliosis.     

Oral administration of trans-resveratrol to guinea pigs increases cardiac DT-diaphorase and catalase activities,and protects isolated atria from menadione toxicity

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine. It has antioxidant and antiproliferative activities, and has been shown to induce NAD(P)H:quinone oxidoreductase, also known as DT-diaphorase, in cultured mouse hepatoma cells. DT-diaphorase is a detoxifying enzyme for quinone-containing substances, due to its ability to prevent their one-electron reduction and the consequent generation of reactive oxygen species (ROS). The aim of the present study was to investigate whether oral administration of trans-resveratrol to guinea pigs (60 mg/l in tap water for 16 days, ad libitum) increases cardiac DT-diaphorase and, consequently, reduces the response of isolated atria to 2-methyl-1,4-naphthoquinone (menadione), the positive inotropic effect of which is related to the amount of ROS generated by its cardiac metabolism. In the cardiac tissue of resveratrol-treated animals, DT-diaphorase activity was significantly higher than that measured in control animals, the V(max) of the enzyme reaction being 75.47 +/- 3.87 and 50.73 +/- 0.63 nmoles/mg protein/min, respectively (p < 0.05). Resveratrol administration also significantly increased the activity of cardiac catalase (32.20 +/- 2.39 vs. 25.14 +/- 3.85 units/mg protein in treated and control animals, respectively; p < 0.001). As a consequence, menadione metabolism by the cardiac homogenate obtained from resveratrol-treated animals generated a smaller amount of ROS and, in electrically driven left atria, menadione produced a significantly lower increase in the force of contraction than in atria isolated from control animals. These results indicate that oral administration of resveratrol exerts cardioprotection against ROS-mediated menadione toxicity.     

Cytosine-block telomeric type DNA-binding activity of hnRNP proteins from human cell lines

Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence. All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP). Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif. Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence. In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.     

Water-in-oil macroemulsions sustain long-term viability of microbial cells in organic solvents

Extremely stable water-in-oil macroemulsions have been obtained by dispersing water in isooctane in the presence of lecithin. Either prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and Rhodotorula minuta) cells hosted in these water-in-oil macroemulsions are viable for weeks despite the consistent excess of organic solvent (ranging from 70 to 84%, v/v) in these ternary systems. Conjugation occurs upon mixing macroemulsions containing F(+) or F(-) Escherichia coli strains, indicating consistent mass transfer between the water droplets. Populations of yeasts hosted in water-in-oil macroemulsion feature a higher frequency of cells aggregation when compared with the corresponding populations suspended in homogeneous aqueous media.     

Loss of preferred mates forces female satin bowerbirds (Ptilonorhynchus violaceus) to increase mate searching

Variation in mate choice among females can have important consequences for the operation of sexual selection, and can result from differences in the way females search for mates. Our previous work indicates that female satin bowerbirds Ptilonorhynchus violaceus alter their mate-searching patterns according to long-term experience. Females which mate with very attractive males mate with the same males in the following year, thereby reducing their search. In contrast, females which fail to encounter very attractive males typically reject their previous mates and search for more attractive males in the following year, thereby increasing their search. Here we report results from a natural experiment consistent with these observations. Five males, including the most attractive male of 1997, failed to re-establish display sites in 1998, most probably dying over winter. We monitored the mate-searching behaviour of females which mated with these males in 1997 to determine how the loss of attractive mates affects subsequent mate-searching patterns. Females which lost their mates sampled more males compared with their own search patterns in 1997 and with faithful females in 1998. Results from this natural experiment indicate that the loss of attractive and preferred mates forces females to increase their search and provide evidence that long-term experience with males shapes mate-searching behaviour.     

Prevalence of human herpesvirus (HHV)-8 infection among carriers of cardiovascular disease

Infectious agents, such as herpesviruses, have been hypothesized to be involved in development of atheromatous plaque. The study aim was to evaluate the possibility that HHV-8 infection could be an additional risk factor for the establishment of cardiovascular disease. HHV-8 seroprevalence was determined by immunofluorescence in a population of cardiovascular disease patients (n=50) as compared to an age- and sex-matched group of control subjects (n=47); HHV-8 genome was detected in DNA extracted from circulating PBMC and from atheromatous lesions by PCR with primers specific for the minor virus capsid gene (ORF 26). The seroprevalence of HHV-8 was significantly increased in the patients as compared to the control population, while the presence of HHV-8 genome was observed in PBMC from 2 patients and 1 control. Virus-specific DNA was found in 2 out of 4 atheromatous plaques. The higher seroprevalence in patients suffering from vascular diseases as compared to age-and sex-matched controls suggests that HHV-8 infection could be an additional risk factor for the establishment of cardiovascular disease, although the data on the persistence of viral DNA in PBMC or in the arterial lesions are too exiguous to definitively support this hypothesis. More extensive studies are needed to define the exact role of HHV-8 infection in the establishment and progression of atheromatous lesions.     

Interpenetrating network formation in agarose--kappa-carrageenan gel composites

Thermal, mechanical, turbidity, and microscope evidence is provided which strongly suggests molecular interpenetrating network (IPN) formation by mixtures of the seaweed polysaccharides agarose and kappa-carrageenan. Over a range of ionic strength, and potassium content, there is no evidence for synergistic coupling of the networks, and simple phase separation (demixing) can definitely be ruled out. At low ionic strength, where the agarose gels first, differential scanning calorimetry evidence shows some influence of the carrageenan on the agarose ordering enthalpy, particularly at higher polymer concentrations. As the potassium level is increased, however, and the order of gelling is reversed, this effect disappears. Cure behavior for the systems at high ionic strength can be described as a simple summation of the pure component contributions. At low ionic strength, on the other hand, the modulus behavior is more complex, suggesting either a modification, in the mixture, of the kappa-carrageenan gelling parameters or a more complex modulus additivity rule.