Serotonergic mechanisms of the lateral parabrachial nucleus and cholinergic-induced sodium appetite

Central cholinergic mechanisms are suggested to participate in osmoreceptor-induced water intake. Therefore, central injections of the cholinergic agonist carbachol usually produce water intake (i.e., thirst) and are ineffective in inducing the intake of hypertonic saline solutions (i.e., the operational definition of sodium appetite). Recent studies have indicated that bilateral injections of the serotonin receptor antagonist methysergide into the lateral parabrachial nucleus (LPBN) markedly increases salt intake in models involving the activation of the renin-angiotensin system or mineralocorticoid hormones. The present studies investigated whether sodium appetite could be induced by central cholinergic activation with carbachol (an experimental condition where only water is typically ingested) after the blockade of LPBN serotonergic mechanisms with methysergide treatment in rats. When administered intracerebroventricularly in combination with injections of vehicle into both LPBN, carbachol (4 nmol) caused water drinking but insignificant intake of hypertonic saline. In contrast, after bilateral LPBN injections of methysergide (4 microg), intracerebroventricular carbachol induced the intake of 0.3 M NaCl. Water intake stimulated by intracerebroventricular carbachol was not changed by LPBN methysergide injections. The results indicate that central cholinergic activation can induce marked intake of hypertonic NaCl if the inhibitory serotonergic mechanisms of the LPBN are attenuated.     

Participation of host cell actin filaments during interaction of trypomastigote forms of Trypanosoma cruzi with host cells

The involvement of actin filaments from the host cell on the process of invasion of trypomastigote forms of Trypanosma cruzi was analyzed in seven different cell lines. Prior incubation of all cell lines with cytochalasin D, under conditions which interfere with actin filaments, markedly inhibited parasite internalization and increased parasite attachment. Attached parasites were readily ingested following washing of the drug-treated cells. Cytochalasin treatment interfered with the distribution of actin filaments of the host cell as evaluated by visualization of the filaments using confocal laser scanning microscopy of cells incubated in the presence of FITC-phalloidin. Concentration of actin filaments could be observed in most, but not all, parasites in the process of internalization. We also treated LLCMK 2 and macrophage cells with Jasplakinolide, a drug that stabilizes actin filaments, before interaction with the trypomastigote forms. This drug partially inhibits parasite invasion into the cells. Prior incubation of the host cells in the presence of colchicine, which interfere with microtubules, also inhibited parasite internalization into the cells.     

Structure-function analysis of Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase domains

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.     

A new species,Litomosoides odilae n. sp (Nematoda: Onchocercidae) from Oligoryzomys nigripes (Rodentia: Muridae) in the rainforest of Misiones,Argentina

A new species of Litomosoides was collected from the abdominal cavity of Oligoryzomys nigripes (Rodentia: Muridae) in a semideciduous secondary rainforest of Misiones, Argentina. Litomosoides odilae n. sp. belongs to the carinii group and is characterized by the amphids displaced dorsally; buccal capsule with an anterior segment transparent and an annular asymmetrical thickening; esophagus divided, with the posterior glandular portion slightly wider than the muscular; male cloacal aperture strongly protruded; and microfilaria sheathed with an attenuated tail. The morphology of the new species, which is similar to that of L petteri, a parasite of marsupials in Brazil, suggests that host-switching events may have occurred in the diversification of this genus.     

The lower trapezius musculocutaneous flap revisited: versatile coverage for complicated wounds to the posterior cervical and occipital regions based on the deep branch of the transverse cervical artery

The clinical role of the lower trapezius musculocutaneous flap varies within the literature. Many describe its use in the reconstruction of the lateral neck and facial regions, but very few refer to its use in the posterior cervical and occipital regions. Different vascular pedicles have also been described and effectively used. A retrospective analysis was conducted, reviewing the authors' experience with 13 patients who suffered complex open wounds to the posterior cervical and occipital regions that were treated with a lower trapezius muscle or musculocutaneous flap. All flaps were based on the deep branch of the transverse cervical artery. This pedicle was used to support a relatively large skin segment over the distal portion of the lower trapezius muscle, a margin that, in the authors' experience, extends at least 1 cm beyond the muscular margin. Postoperatively, patients were evaluated based on complications, residual shoulder function, and aesthetic outcome. In addition to the clinical study, cadaveric dissection of the trapezius muscle was conducted on 22 specimens, and the vascular anatomy was confirmed by direct visualization. The authors' experience indicates that the lower trapezius musculocutaneous flap, when based on the deep branch of the transverse cervical artery, provides a reliable alternative for the reconstruction of complicated wounds in the posterior cervical and occipital regions, with the added capability of providing richly vascularized tissue to compromised wounds as far cephalad as the vertex of the skull.     

Characterization of Paracoccidioides brasiliensis atypical isolates by random amplified polymorphic DNA analysis

Two atypical Paracoccidioides brasiliensis strains (yeast form at room temperature) have been isolated from chronically infected patients living in Brazil. Different random primers were used to characterize these isolates and compare them to typical strains. The RAPD patterns allowed the differentiation of all the selected isolates. Their genetic distance ranged from 5% to 80% of non-shared bands depending on the strains and the primer used. The RAPD data were used to build a Wagner phenogram, which showed two major branched with more than 56% of genetic distance separating them. No significant difference was observed between the atypical isolates and the others suggesting that specific genes are involved in the dimorphism phenomenon.     

Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector

To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.     

Short-term physiological changes in turbot and seabream juveniles exposed to exogenous ammonia

Seabream and turbot juveniles (40-520 g) were exposed to constant exogenous NH5-N concentrations (1.27-4.27 mmol/l; pH, 8.15). In 96 hr acclimated fish, plasma TA-N (total ammonia nitrogen) contents were positively correlated to ambient ammonia concentrations. The LD50 were 2.2-2.5 mmol/l TA-N in both species. There were no marked osmoregulatory disturbances and plasma urea-N, thyroid hormones levels and gill (Na-K)-ATPase activities were only affected at the highest concentrations. Liver GOT, GPT and GIDH activity dose-response were low and species dependent. In cannulated and non-cannulated turbot exposed to half 96 hr LC50 (lethal ambient concentration for 50% of the population), there was a rapid, pronounced and prolonged increase in plasma TA-N, followed by an immediate decline when exogenous ammonia supply was stopped. Maximum loading and unloading were observed within 1-3 hr. Plasma cortisol levels indicated a stressful situation in exposed fish (150 ng/ml) and a quick recovery capacity. In dose and time response experiments, the most relevant physiological indicator of ammonia stress was blood TA-N content. Other parameters tested led either to transient or low amplitude responses except when fish approached death.