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.     

Effect of nitric oxide in the nucleus isthmi on the hypoxic and hypercarbic drive to breathing of toads

Nucleus isthmi (NI) is a mesencephalic structure of the amphibian brain that has been reported to participate in CO(2) chemoreception and in the ventilatory response to hypoxia. However, no information exists about the modulators and/or mediators involved. In the present study, we assessed the participation of nitric oxide (NO) in the hypoxic and hypercarbic drive to breathing, specifically in the NI. We compared the ventilatory and cardiovascular responses with hypoxia and hypercarbia after microinjecting 100 nmol/0.5 microliter of N(G)-nitro-L-arginine methyl ester (L-NAME; an NO synthase blocker) into the NI of toads (Bufo paracnemis). L-NAME had no effect under resting conditions. Hypoxia elicited an increase in ventilation in control and vehicle toads by elevating tidal volume (V(T)). Hypercarbia caused hyperventilation in all groups due to an increase in both V(T) and frequency. The microinjection of L-NAME into the NI elicited an increase in ventilatory response to hypoxia and hypercarbia due to a higher V(T.) We conclude that NO in the NI has an inhibitory effect when the respiratory drive is high, acting on V(T).     

Effect of Chronic Manganese Treatment on Adenosine Tissue Levels and Adenosine A2a Receptor Binding in Diverse Regions of Mouse Brain

In the present study the effects of chronic manganese (Mn) treatment on adenosine A_2a receptor binding in mouse brain have been assessed. Male albino mice were divided in two groups: In the Mn-treated group, the animals were injected intraperitoneally (i.p.) with MnCl₂ (5 mg/kg/day) five days per week during 9 weeks; in the control group, they were injected likewise with a saline solution. A significant decrease of the Kd without alteration of Bmax in the cerebellum and, an increase of the Kd and Bmax in hippocampus of mice treated with Mn were found. Also, an increase of Kd in frontal cortex was observed. The binding parameters in caudate nucleus, olfactory bulb and hypothalamus were not altered by Mn. A significant decrease in the adenosine concentration in caudate nucleus, olfactory bulb and hypothalamus, without significant changes in hippocampus, frontal cortex and cerebellum was also detected. These findings suggest that chronic administration of Mn could affect adenosine receptor function and turnover, depending on the brain region analyzed.     

Antipyretic effect of arginine vasotocin in toads

Arginine vasotocin (AVT) is a nonmammalian analog of the mammalian hormone arginine vasopressin (AVP). These peptides are known for their antidiuretic and pressor effects. More recently, AVP has been recognized as an important antipyretic molecule in mammals. However, no information exists about the role of AVT in febrile ectotherms. We tested the hypothesis that AVT is an antipyretic molecule in the toad Bufo paracnemis. Toads equipped with a temperature probe were placed in a thermal gradient, and preferred body temperature was recorded continuously. A behavioral fever was observed after lipopolysaccharide (LPS) was injected systemically (200 microg/kg). Systemically injected AVT (300 pmol/kg) alone caused no significant change in body temperature, but abolished LPS-induced fever. Moreover, a smaller dose of AVT (10 pmol/kg), which did not affect LPS-induced fever when injected peripherally, abolished fever when injected intracerebroventricularly. We therefore conclude that AVT plays an antipyretic role in the central nervous system, by means of behavior, in an ectotherm, a fact consistent with the notion that AVT/AVP elicits antipyresis by reducing the thermoregulatory set point.