Baroreflex dysfunction in rats submitted to protein restriction

Earlier studies from the authors' laboratory showed that malnourishment induces alterations in the cardiovascular homeostasis increasing the basal mean arterial pressure and heart rate. In this study, the authors evaluated whether the sympathetic and parasympathetic efferent activities contribute to changes in the cardiovascular homeostasis through altered modulation of the arterial baroreflex of malnourished rats. After weaning, male Fischer rats were given 15% (Normal Protein--NP) or 6% (Low Protein--LP) protein diet for 35 d. The baroreflex gain and latency were evaluated before and after selective autonomic blockades in control and malnourished rats. It was observed that malnourishment affected the baroreflex gain in response to activation and deactivation of the arterial baroreflex. Moreover, malnourished rats showed increased baroreflex latency as compared to that of control rats. Regarding the autonomic efferent activity directed to the heart, the data showed increased sympathetic and decreased parasympathetic efferent activities in malnourished rats, and such alterations could be related to the observed changes in the arterial baroreflex gain as well as in the basal mean arterial pressure and heart rate.     

Mutagenic bypass of the butadiene-derived 2'-deoxyuridine adducts by polymerases eta and zeta

Butadiene is a ubiquitous environmental chemical carcinogen that when activated to its monoepoxide intermediate can react with the N3 position of cytosine, resulting in two stereoisomeric adducted bases that rapidly deaminate to N3 2′-deoxyuridine lesions. We have previously shown that replication of DNAs containing these adducts through mammalian cells resulted in 97% mutagenicity, predominantly C to T transitions. Since replicative DNA polymerases were blocked by these lesions in vitro, translesional polymerases were assessed for their ability to bypass these adducts. While polymerases ι, κ and ζ were significantly blocked one nucleotide prior to the lesion, pol η incorporated nucleotides opposite the adducts with a preference for insertion of a G or A. Following polymerase dissociation and reassociation, pol η was also able to extend primers with mispaired termini opposite the lesions, with extensions from the A and T mismatched primer termini being the most efficient. Pol ζ was also able to extend primers containing all mismatched nucleotides opposite the lesions, with the most efficient extension occurring off of the A mismatched primer.     

An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune etiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-alpha, IFN-alpha, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applied for dengue fever.     

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.     

Variability in tolerance to UV-B radiation among Beauveria spp. isolates

Solar radiation, particularly the UV-B component, negatively affects survival of entomopathogenic fungi in the field. In an effort to identify Beauveria spp. isolates with promise for use in biological control settings with high insolation, we examined 53 Beauveria bassiana isolates, 7 isolates of 4 other Beauveria spp. and Engyodontium albus (=Beauveria alba). The origins of these fungi varied widely as to host/substrate and country, but approximately 30% of these isolates were B. bassiana from ticks in Brazil. A preliminary trial with three B. bassiana isolates (Bb 19, CG 310 and CG 481) at several UV-B dosages indicated that 2h of weighted UV-B irradiance at 978mWm(-2) (providing a total dose of 7.04kJm(-2)) allowed separation of isolates into low, medium or high UV-B tolerance. This dose, therefore, was selected as a single dose to compare UV-B tolerances of all 60 Beauveria spp. isolates. There was high variability in tolerance to UV-B radiation among the B. bassiana isolates, ranging from virtually zero tolerance (e.g., Bb 03) to almost 80% tolerance (e.g., CG 228). In addition, surviving B. bassiana conidia demonstrated delayed germination; and this is likely to reduce virulence. Conidia of the other species were markedly more sensitive to UV-B, with E. albus (UFPE 3138) being the least UV-B tolerant. Among B. bassiana isolates originating from 0 degrees to 22 degrees latitudes, those from lower latitudes demonstrated statistically significant greater UV-B tolerances than those isolates from higher latitudes. Isolates from above 22 degrees , however, were unaffected by latitude of origin. A similar analysis based on host type did not indicate a correlation between original host and UV-B tolerance. The identification in this study of several B. bassiana isolates with relatively high UV-B tolerance will guide the selection of isolates for future arthropod microbial control experiments.     

Skp2-mediated p27(Kip1) degradation during S/G2 phase progression of adipocyte hyperplasia

p27(Kip1), an important regulator of Cdk2 activity and G1/S transition, is tightly regulated in a cell-type and condition-specific manner to integrate mitogenic and differentiation signals governing cell cycle progression. We show that p27 protein levels progressively declined from mid-G1 through late-G2 phase as density-arrested 3T3-L1 preadipocytes synchronously reentered the cell cycle during early stages of adipocyte differentiation. This dramatic fall in p27 protein accumulation was due, at least in part, to a decrease in protein stability. Specific inhibitors of the 26S proteasome were shown to completely block the decrease in p27 protein levels throughout G1, increase the abundance of ubiquitylated p27 protein, and inhibit G1/S transition resulting in G1 arrest. It is further demonstrated that p27 was phosphorylated on threonine 187 during S phase progression by Cdk2 and that phosphorylated p27 was polyubiquitylated and degraded. Furthermore, we demonstrate that Skp2 and Cks1 dramatically increased during S/G2 phase progression concomitantly with the maximal fall in p27 protein. Complete knockdown of Skp2 with RNA interference partially prevented p27 degradation equivalent to that observed with Cdk2 blockade suggesting that the SCF(Skp2) E3 ligase and other proteasome-dependent mechanisms contribute to p27 degradation during preadipocyte replication. Interestingly, Skp2-mediated p27 degradation was not essential for G1/S or S/G2 transition as preadipocytes shifted from quiescence to proliferation during adipocyte hyperplasia. Finally, evidence is presented suggesting that elevated p27 protein in the absence of Skp2 was neutralized by sequestration of p27 protein into Cyclin D1/Cdk4 complexes.     

C1 inhibitor-mediated protection from sepsis

C1 inhibitor (C1INH) protects mice from lethal Gram-negative bacterial LPS-induced endotoxin shock and blocks the binding of LPS to the murine macrophage cell line, RAW 264.7, via an interaction with lipid A. Using the cecal ligation and puncture (CLP) model for sepsis in mice, treatment with C1INH improved survival in comparison with untreated controls. The effect was not solely the result of inhibition of complement and contact system activation because reactive center-cleaved, inactive C1INH (iC1INH) also was effective. In vivo, C1INH and iC1INH both reduced the number of viable bacteria in the blood and peritoneal fluid and accelerated killing of bacteria by blood neutrophils and peritoneal macrophages. In vitro, C1INH bound to bacteria cultured from blood or peritoneal fluid of mice with CLP-induced sepsis, but had no direct effect on bacterial growth. However, both C1INH and iC1INH enhanced the bactericidal activity of blood neutrophils and peritoneal exudate leukocytes. C1INH-deficient mice (C1INH-/- mice) subjected to CLP had a higher mortality than did wild-type littermate mice. Survival of C1INH-/- mice was significantly increased with two doses of C1INH, one given immediately following CLP, and the second at 6 h post-CLP. C1INH may be important in protection from sepsis through enhancement of bacterial uptake by, and/or bactericidal capacity of, phagocytes. Treatment with C1INH may provide a useful additional therapeutic approach in some patients with peritonitis and/or sepsis.     

Malnutrition enhances cardiovascular responses to chemoreflex activation in awake rats

Several studies in the literature suggest that low-protein intake is associated with increases in sympathetic efferent activity and cardiovascular disease. Among the possible mechanisms, changes in the neurotransmission of cardiovascular reflexes have been implicated. Therefore, the present study comprised the evaluation of chemoreflex responsiveness in rats subjected to a low-protein diet during the 35 days after weaning. As a result, we observed that malnourished rats presented higher levels of baseline mean arterial pressure and heart rate and exhibited a mild increase in the pressor response to chemoreflex activation. They also exhibited a massive bradycardic response to chemoreflex activation. Interestingly, bilateral ligature of the carotid body arteries further increased baseline mean arterial pressure and heart rate in malnourished animals. The data suggest severe autonomic imbalance and/or change in the central interplay between neural and cardiovascular mechanisms.     

Red-green color vision impairment in Duchenne muscular dystrophy

The present study evaluated the color vision of 44 patients with Duchenne muscular dystrophy (DMD) (mean age 14.8 years; SD 4.9) who were submitted to a battery of four different color tests: Cambridge Colour Test (CCT), Neitz Anomaloscope, Ishihara, and American Optical Hardy-Rand-Rittler (AO H-R-R). Patients were divided into two groups according to the region of deletion in the dystrophin gene: upstream of exon 30 (n=12) and downstream of exon 30 (n=32). The control group was composed of 70 age-matched healthy male subjects with no ophthalmological complaints. Of the patients with DMD, 47% (21/44) had a red-green color vision defect in the CCT, confirmed by the Neitz Anomaloscope with statistical agreement (P<.001). The Ishihara and the AO H-R-R had a lower capacity to detect color defects--5% and 7%, respectively, with no statistical similarity between the results of these two tests nor between CCT and Anomaloscope results (P>.05). Of the patients with deletion downstream of exon 30, 66% had a red-green color defect. No color defect was found in the patients with deletion upstream of exon 30. A negative correlation between the color thresholds and age was found for the controls and patients with DMD, suggesting a nonprogressive color defect. The percentage (66%) of patients with a red-green defect was significantly higher than the expected <10% for the normal male population (P<.001). In contrast, patients with DMD with deletion upstream of exon 30 had normal color vision. This color defect might be partially explained by a retina impairment related to dystrophin isoform Dp260.     

Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: late expression of beta1-adrenergic receptor shifts norepinephrine specificity in the adult tissue

Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.