Mitogenic activity of Cratylia mollis lectin on human lymphocytes.

The mitogenic effect of Cratylia mollis seed lectin preparations containing two (Cramoll 1,4) or one molecular form (Cramoll 1) showed activity similar to the well known T-cell mitogen, concanavalin A (Con A). The effect on human lymphocytes was analyzed through a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Inhibition of lymphocyte proliferation with methyl-alpha-d-mannoside (both preparations) indicated that the mitogenic effect involved carbohydrate lectin binding sites.     

The fall of a symbol? A high predation rate by the introduced horseshoe whip snake <Emphasis Type="Italic">Hemorrhois hippocrepis</Emphasis> paints a bleak future for the endemic Ibiza wall lizard <Emphasis Type="Italic">Podarcis pityusensis</Emphasis>

Invasive species currently account for a major threat to global biodiversity, and island ecosystems are among the most vulnerable, because of the frequency and success of species introductions on islands. Within Mediterranean islands, reptiles not only are frequently introduced species but are also among the most threatened because of these introductions. The Balearic archipelago is a good example of this, since only two of its current 16 species of reptiles are native. Thirteen years ago, the snake Hemorrhois hippocrepis was introduced by cargo in Ibiza island, and it is in expansion. Individuals obtained from an early eradication campaign showed a fast expression of phenotypic plasticity and acquired larger sizes than those of the source population, probably due to a high prey availability and predator scarcity. The species is thriving at the expense of a small variety of native and non-native prey, but the predation pressure on the endemic Podarcis pityusensis, the only native reptile in the island, is very high, as this lizard represents 56% of the prey in frequency, which might threaten its survival on the long term. Our results on the feeding ecology of the snake are of sufficient concern to justify the maintenance of actions to eradicate this invader.     

Hydrogel Environment Supports Cell Culture Expansion of a Grade IV Astrocytoma

Malignant astrocytomas are aggressive cancers of glial origin that can develop into invasive brain tumors. The disease has poor prognosis and high recurrence rate. Astrocytoma cell lines of human origin are an important tool in the experimental pathway from bench to bedside because they afford a convenient intermediate system for in vitro analysis of brain cancer pathogenesis and treatment options. We undertook the current study to determine whether hydrogel culture methods could be adapted to support the growth of astrocytoma cell lines, thereby facilitating a system that may be biologically more similar to in vivo tumor tissue. Our experimental protocols enabled maintenance of Grade IV astrocytoma cell lines in conventional monolayer culture and in the extracellular matrix hydrogel, Geltrex^?. Light and fluorescence microscopy showed that hydrogel environments promoted cellular reorganization from dispersed cells into multilayered aggregates. Transmission electron microscopy revealed the prevalence of autophagy and nuclear membrane distortions in both culture systems. Analysis of microarray Gene Expression Omnibus (GEO) DataSets highlighted expression of genes implicated in pathways for cancer progression and autophagy. A pilot quantitative polymerase chain reaction (qPCR) analysis of the autophagic biomarkers, Beclin 1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), with two reference genes (beta actin, ACTB; glyceraldehyde 3-phosphate dehydrogenase, GAPDH), uncovered a relative increase of BECN1 and LC3B in hydrogel cultures of astrocytoma as compared to the monolayer. Taken together, results establish that ultrastructural and molecular characteristics of autophagy are features of this astrocytoma cell line, and that hydrogel culture systems can afford novel opportunities for in vitro studies of glioma.     

Species, dynamics and population composition of phthirapteran in free-range chickens (Gallus gallus L.) in São Luis Island, State of Maranhão

The objective of this work was to identify the phthirapteran species, to determine the prevalence according to the anatomical region of the body and to know the dynamics and composition of the population of these ectoparasites in free-range chicken in S?o Luis Island, state of Maranh?o. Inspection was performed in 40 chickens and feathers were collected from the head, neck, wing, thigh, dorsal and ventral regions and cloacae. The phthirapteran species identified were: Menopon gallinae L., Menacanthus stramineus Nitzsch, Menacanthus pallidulus Neumann, Menacanthus cornutus Schommer (Menoponidae), and Lipeurus caponis L., Goniodes dissimilis Denny and Goniocotes gallinae De Geer (Philopteridae). L. caponis was collected from all regions sampled, including the head, which was the least infested region. The dorsum was the most infested, especially in the dry period of the year and where the greater parasitic diversity was observed, the wing and the head were the least infested regions. Considering the dynamics and the composition of the population the phthirapteran presented a prevalence of 85% of the sampled chickens, the mean intensity of infestation was 45.3 varying from <1 to 453. The egg stage was superior to the others life stages followed by nymphs and female adults, independent of the phthirapteran species.     

TNP-470 inhibits oxidative stress, nitric oxide production and nuclear factor kappa B activation in a rat model of hepatocellular carcinoma

The objective of this study is to determine if treatment with the angiogenesis inhibitor TNP-470 results in impairment of oxidative stress, inhibition of nuclear factor kappa B (NF-κB) activation and decrease of nitric oxide production in an experimental model of rat hepatocarcinogenesis. Tumour was induced by diethylnitrosamine and promoted by two-thirds hepatectomy plus acetaminofluorene administration. Experiments were carried out at 28 weeks after initiating the treatment. TNP-470 was administered at 30 mg/kg, three times per week from 20 to 28 weeks. Carcinomatous tissue growing outside dysplastic nodules and a marked expression of placental glutathione S-transferase were detected in rats with induced carcinogenesis. Liver concentrations of thiobarbituric acid reactive substances, reduced glutathione (GSH) and glutathione disulfide (GSSG) were significantly higher than those of controls and there was a significant increase in the GSSG/GSH ratio. Tumour growth was accompanied by augmented expression of inducible nitric oxide synthase, activation of (NF-κB) and proteolysis of IkappaB. All these effects were absent in animals receiving TNP-470. Our results indicate that TNP-470 inhibits oxidative stress, nitric oxide production and NF-κB activation induced by experimental hepatocarcinogenesis. These changes would contribute to the beneficial effects of TNP-470 in cancer treatment.     

Vitiligo: complex segregation and linkage disequilibrium analyses with respect to microsatellite loci spanning the HLA

Familial clustering and linkage disequilibrium studies suggest that genetic factors predispose to vitiligo, although a clear transmission pattern and cosegregation of vitiligo with specific mutations have not been demonstrated. We collected pedigree data on vitiligo from a set of 56 multigeneration families belonging to the Paisa community from Antioquia, Colombia, with the goal of applying the unified model of complex segregation and linkage disequilibrium analyses to test the hypotheses of the existence of a major gene predisposing to vitiligo and that allelic or haplotype polymorphisms of microsatellite loci at 6p21.3-21.4 spanning HLA (D6S276, D6S265, D6S273, and D6S291) are associated with this predisposition. Minimum sibship sample size to discriminate dominant and recessive inheritance models was largely accomplished. Between the 15 models of complex segregation used, the one that best fitted the data was that of a major dominant gene and the existence of strong environmental effects acting on the recessive genotype. The penetrance and risk estimations discriminated two sets of vitiligo patients: those with early onset of vitiligo cosegregating with a dominant mode of inheritance without environmental effects, and those with late onset of vitiligo cosegregating with the recessive genotype and being influenced by environmental effects. After establishing the normal distribution of allelic frequencies and performing multiple comparisons correction, the linkage disequilibrium analysis suggested that a major genetic factor could be located at 6p21.3-21.4, because we detected significant case-control differences for allele 122 at D6S265 ( Pc=0.0264) and significant linkage disequilibrium between loci D6S276 and D6S273 in the cases but not in the controls. We cannot explain these results as a consequence of evolutionary forces or as genetic stratification acting differentially on cases and controls, because there was neither deviation from the Hardy-Weinberg expectations nor genetic subdivision between cases and controls, as theta; (non-biased F(ST)) was not significantly different from 0.     

Phenotype of CYP2C19 and CYP3A4 by determination of omeprazole and its two main metabolites in plasma using liquid chromatography with liquid-liquid extraction

We present a new simple and reliable HPLC method for measuring omeprazole and its two main metabolites in plasma. This can be used for studying CYP2C19 and CYP3A4 genetic polymorphisms using omeprazole as the probe drug. Omeprazole, hydroxyomeprazole and omeprazole sulfone were extracted from plasma samples with phosphate buffer and dichloromethane-ether (95:5). HPLC separation was achieved using an Ultrasphere ODS C(18) (Beckman) column. The mobile phase was acetonitrile-phosphate buffer (24:76, pH 8), containing nonylamine at 0.015%. Retention times were 9.5 min for omeprazole, 3.25 min for hydroxyomeprazole, 7.4 min for omeprazole sulfone and 6.27 min for internal standard (phenacetine). Detection (UV at 302 nm) of analytes was linear in the range from 96 to 864 ng/ml. This is useful for calculating metabolic index for CYP2C19 and CYP3A4 in adults and children. This method is stable, reproducible, improves resolution and has practical advantages such as low cost.     

Binding of caspase-3 prodomain to heat shock protein 27 regulates monocyte apoptosis by inhibiting caspase-3 proteolytic activation

Caspase-3 is an essential executioner of apoptosis responsible for regulating many important cellular processes, among them the number of circulating monocytes, central players in the innate immune response. The activation of caspase-3 requires its processing from an inactive precursor. Here we show that the small heat shock protein 27 (Hsp27) associates with caspase-3 and protein-protein interaction experiments in vivo and with purified proteins demonstrate a direct interaction between Hsp27 and the amino-terminal prodomain of caspase-3. Using an in vitro caspase-3 activation assay, our results further establish that the interaction of Hsp27 with the caspase-3 prodomain inhibits the second proteolytic cleavage necessary for caspase-3 activation, revealing a novel mechanism for the regulation of this effector caspase. Hsp27 expression in monocytes is constitutive. Consistent with a central role of Hsp27 in blocking caspase-3 activation, Hsp27 down-regulation by double-stranded RNA interference induces apoptosis of macrophages, whereas Hsp27 overexpression increases the life span of monocytes by inhibiting apoptosis. Highlighting the importance of cell partitioning in the regulation of apoptosis, immunofluorescence, and subcellular fractionation studies revealed that whereas both caspase-3 and Hsp27 are cytoplasmic in fresh monocytes (i.e. not undergoing apoptosis), Hsp27 moves to the nucleus during apoptosis, a relocalization that can be blocked by promoting the differentiation of monocytes to macrophages or by inhibiting cell death. These results reveal a novel mechanism of caspase-3 regulation and underscore a novel and fundamental role of Hsp27 in the regulation of monocyte life span.     

<Emphasis Type="Italic">Mucor rouxii</Emphasis> Rho1 protein; characterization and possible role in polarized growth

We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70–85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the β-1,3-glucan synthase complex in fungi in which β-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of β-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of β-1,3-glucans on the regulation of morphology of zygomycetes.     

Cell proliferation during the early compartmentalization of the Xenopus laevis inner ear

The auditory and vestibular endorgans of the inner ear which are essential for the senses of hearing and balance form early during development when the otocyst undergoes a period of rapid growth and compartmentalization. Here we show the spatial and temporal patterns of proliferating cells in the Xenopus laevis inner ear as this organ develops from an otic vesicle at stage 31 until stage 47, an age at which compartmentalization and the initial appearance of sensory structures are evident. Sites of new cell production were identified in specimens at stages 31, 37, 42, 45 and 47 using immunohistochemical methods to detect bromodeoxyuridine (BrdU) incorporation three hours after exposure to this thymidine analogue. Cells undergoing terminal mitosis at stages 37, 42 and 45 were detected by exposing specimens at these stages to BrdU and permitting development to proceed until stage 47. Our results show that while newly replicating cells are uniformly distributed throughout the stage 31 otic vesicle, they are spatially restricted in stages 37 through 45, with few dividing cells visible in the central patches of the emerging sensory epithelia. In contrast, no clear proliferative pattern was discerned at stage 47. BrdU-positive cells that had undergone terminal mitosis at stage 37, 42 and 45 were detected in the central regions of nascent sensory epithelia at stage 47. These findings are consistent with a developmental mechanism in which cells undergoing terminal mitosis during early X. laevis stages contribute to sensory epithelia and in which cell mixing and migration are features of inner ear compartmentalization.