Effects of Bucephalus sp. (Trematoda: Bucephalidae) on Perna perna mussels from a culture station in Ratones Grande Island,Brazil

This study reports the prevalence of Bucephalus sp. in Perna perna populations from a culture station of southern Brazil and its effect on the mussel reproductive tissue and immune system. The prevalence of Bucephalus sp. in P. perna (n = 1871) was considered low (3.1%) and did not seasonally vary. Histological sections of the mantle of infected mussels revealed a marked (80%) reduction of the reproductive tissue that was severe even in mussels exhibiting a moderate infection degree. The total (THC) and differential (DHC) hemocyte counts were lower in infected mussels (3.9 x 10(6) hem/ml; granular hemocytes = 33%) as compared with non-infected animals (5.5 x 10(6) hem/ml; granular hemocytes = 40%). The plasma protein concentration did not vary upon infection. Hemocyte infiltration was significantly higher only in mussels with a very heavy infection degree. The parasite sporocysts were never seen encapsulated by the host hemocytes. Our results indicate that Bucephalus sp. promotes a severe castration in its host and apparently evades the mussel immune system.     

Glucose transport and utilization are altered in the brain of rats deficient in n-3 polyunsaturated fatty acids

Long-chain polyunsaturated (n-3) fatty acids have been reported to influence the efficiency of membrane receptors, transporters and enzymes. Because the brain is particularly rich in docosahexaenoic acid (DHA, 22:6 n-3), the present study addresses the question of whether the 22:6 n-3 fatty acid deficiency induces disorder in regulation of energy metabolism in the CNS. Three brain regions that share a high rate of energy metabolism were studied: fronto-parietal cortex, hippocampus and suprachiasmatic nucleus. The effect of the diet deficient in n-3 fatty acids resulted in a 30-50% decrease in DHA in membrane phospholipids. Moreover, a 30% decrease in glucose uptake and a 20-40% decrease in cytochrome oxidase activity were observed in the three brain regions. The n-3 deficient diet also altered the immunoreactivity of glucose transporters, namely GLUT1 in endothelial cells and GLUT3 in neurones. In n-3 fatty acid deficient rats, GLUT1-immunoreactivity readily detectable in microvessels became sparse, whereas the number of GLUT3 immunoreactive neurones was increased. However, western blot analysis showed no significant difference in GLUT1 and GLUT3 protein levels between rats deficient in n-3 fatty acids and control rats. The present results suggest that changes in energy metabolism induced by n-3 deficiency could result from functional alteration in glucose transporters.     

Involvement of monoaminergic system in the antidepressant-like effect of the hydroalcoholic extract of Siphocampylus verticillatus

The antidepressant-like effect of the hydroalcoholic extract obtained from aerial parts of Siphocampylus verticillatus, a Brazilian medicinal plant, was investigated in two models of depression in mice and against synaptosomal uptake of serotonin, noradrenaline and dopamine. The immobility times in the forced swimming test (FST) and in the tail suspension test (TST) were significantly reduced by the extract (dose range 100-1000 mg/kg, i.p.), without accompanying changes in ambulation when assessed in an open-field. In addition when given orally the extract was also effective in reducing the immobility time in the TST. The efficacy of extract in the TST was comparable to that of the tricyclic antidepressant imipramine (15 mg/kg, i.p.) and with fluoxetine (32 mg/kg, i.p.). The anti-immobility effect of the extract (600 mg/kg, i.p.) assessed in the TST was not affected by pre-treatment with naloxone (1 mg/kg, i.p., a non-selective opioid receptor antagonist) or L-arginine (750 mg/kg, i.p., a nitric oxide precursor). In contrast, the extract (600 mg/kg, i.p.) antidepressant-like effect was significantly reduced by pre-treatment of animals with p-chlorophenylalanine (PCPA, 100 mg/kg, i.p., an inhibitor of serotonin synthesis), sulpiride (50 mg/kg, i.p., a selective D2 receptor antagonist), prazosin (62.5 microg/kg, i.p., an alpha1 adrenoreceptor antagonist) or by guanosine 5'-monophosphate (GMP, 250 mg/kg, i.p., a nucleotide known to block some actions elicited by NMDA). The biochemical data show that the extract of S. verticillatus inhibited in a graded manner the uptake of monoamines. However, at the IC50 level, the extract was approximately 3.2 to 3.4-fold more potent and also more efficacious in inhibiting the synaptosomal uptake of noradrenaline and serotonin than dopamine. Taken together these data demonstrate that the extract of S. verticillatus elicited a significant antidepressant-like effect, when assessed in the TST and FST in mice. Its action seems to involve an interaction with adrenergic, dopaminergic, glutamatergic and serotonergic systems.     

Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence

We describe a cis element that dramatically increases the expression levels of exogenous genes in baculovirus-infected insect cells. This 21 bp sequence element is derived from a 5' untranslated leader sequence of a lobster tropomyosin cDNA (L21). By using a transfer vector carrying L21, the expression levels of tropomyosin and luciferase were 20- and seven-fold higher with L21 than without L21, respectively. L21 has both the Kozak sequence and the A-rich sequence found in the polyhedrin leader sequence. We assume that both sequence elements are essential for the enhancement of protein expression in the baculovirus-based expression system.     

Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.     

Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Sub-lethal concentrations of activated complement increase rat lymphocyte glutamine utilization and oxidation while lethal concentrations cause death by a mechanism involving ATP depletion

Nucleated cells are more resistant to complement-mediated cell death than anucleated cells such as erythrocytes. There are few reports concerning the metabolic response of nucleated cells subjected to sub-lethal complement attack. It is possible that the rate of utilization of specific metabolic fuels by the cell is increased to enhance cell defence. We have measured the maximum activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase and glutaminase in rat mesenteric lymphocytes exposed to sub-lethal concentrations of activated complement (present in zymosan-activated serum, ZAS). These enzymes were carefully selected as they indicate changes of flux in glycolysis, TCA cycle, pentose phosphate pathway and glutaminolysis, respectively. The only enzyme activity to change on exposure of lymphocytes to ZAS was glutaminase, which was enhanced approximately by two-fold. Although rates of both glutamine and glucose utilization were enhanced by exposure to ZAS, only the rate of oxidation of glutamine was increased. Complement kills anucleated cells by simple osmotic lysis. However, it is likely that some nucleated cells will display characteristics of an ordered death mechanism and we have demonstrated that the concentration of lymphocyte ATP is dramatically decreased by activated complement. Nevertheless, the extent of cell death could be significantly reduced by the addition of inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). We conclude that glutamine metabolism is not only important for lymphocyte proliferative responses but is also important for cell defence from sub-lethal concentrations of activated complement. The rapid rate of complement-induced lymphocyte death reported here is suggested to be a consequence of over-activation of the nuclear enzyme PARP and ATP depletion.     

Bacteriocin production by spray-dried lactic acid bacteria

AIMS: Cell survival and antagonistic activity against Listeria innocua, Listeria monocytogenes and Staphylococcus aureus were investigated after spray-drying three bacteriocin-producing strains of lactic acid bacteria: Carnobacterium divergens, Lactobacillus salivarius and Lactobacillus sakei. METHODS AND RESULTS: Bacterial cell concentrates were spray-dried and stored at 4 degrees C and 18 degrees C and 0.3% ERH (equilibrium relative humidity). Enumeration and antagonistic activity were evaluated before and after spray-drying and at regular intervals during storage. CONCLUSIONS: A higher survival rate was obtained when survival was performed at 4 degrees C. With the exception of Carnobacterium divergens which lost the inhibitory activity against Staph. aureus after drying, antagonistic production was not affected by the process nor by the storage. Of the three species studied, Lact. salivarius showed the highest resistance to the spray-drying and storage processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray-drying is a potentially useful process for large scale production of dried powders containing viable organisms with antagonistic activity against pathogens.     

Ethnicity and type 2 diabetes in Rio de Janeiro,Brazil, with a review of the prevalence of the disease in Amerindians

To what extent can ethnic factors contribute to the prevalence of type 2 diabetes and impaired glucose tolerance (IGT) in an urban Brazilian population? Conversely, how can environmental factors such as diet change these prevalences in a given ethnic group, in this case Brazilian Indians? To answer these questions estimates of ethnic admixture in Afro- and Euro-Brazilians from Rio de Janeiro, Brazil, were established using eight genetic systems and compared with the prevalences of these conditions obtained previously. This information was integrated with results obtained inside and outside of Brazil. The similarity of prevalences for type 2 diabetes and IGT in Afro- and Euro-Brazilians may be related to the extensive gene flow that occurred between them and to similar socioeconomic levels in the samples investigated. On the other hand, changes in the traditional diet are probably conditioning the appearance of diabetes among Brazilian and other South American Indians.     

Isolation of viable Toxoplasma gondii from naturally infected aborted bovine fetuses

Neospora caninum and Toxoplasma gondii are related parasites. The former is a common cause of abortion in dairy cattle. The latter has not been conclusively demonstrated in bovine fetuses. During the course of attempts to isolate N. caninum from aborted fetuses, T. gondii was isolated from 2 aborted fetuses, 1 from Portugal and 1 from the United States. Both isolates were made by bioassay of fetal brains in mice. The fetus from Portugal was about 5 mo in gestational age, and the fetus from the United States was a full-term stillborn.