Nitric oxide synthase-mediated phytoalexin accumulation in soybean cotyledons in response to the Diaporthe phaseolorum f. sp. meridionalis elicitor

Phytoalexin biosynthesis is part of the defense mechanism of soybean (Glycine max) plants against attack by the fungus Diaporthe phaseolorum f. sp. meridionalis (Dpm), the causal agent of stem canker disease. The treatment of soybean cotyledons with Dpm elicitor or with sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in a high accumulation of phytoalexins. This response did not occur when SNP was replaced by ferricyanide, a structural analog of SNP devoid of the NO moiety. Phytoalexin accumulation induced by the fungal elicitor, but not by SNP, was prevented when cotyledons were pretreated with NO synthase (NOS) inhibitors. The Dpm elicitor also induced NOS activity in soybean tissues proximal to the site of inoculation. The induced NOS activity was Ca(2+)- and NADPH-dependent and was sensitive to the NOS inhibitors N(G)-nitro-L-arginine methyl ester, aminoguanidine, and L-N(6)-(iminoethyl) lysine. NOS activity was not observed in SNP-elicited tissues. An antibody to brain NOS labeled a 166-kD protein in elicited and nonelicited cotyledons. Isoflavones (daidzein and genistein), pterocarpans (glyceollins), and flavones (apigenin and luteolin) were identified after exposure to the elicitor or SNP, although the accumulation of glyceollins and apigenin was limited in SNP-elicited compared with fungal-elicited cotyledons. NOS activity preceded the accumulation of these flavonoids in tissues treated with the Dpm elicitor. The accumulation of these metabolites was faster in SNP-elicited than in fungal-elicited cotyledons. We conclude that the response of soybean cotyledons to Dpm elicitor involves NO formation via a constitutive NOS-like enzyme that triggers the biosynthesis of antimicrobial flavonoids.     

Selective inhibition of protein kinases blocks the formation of a new axis,the beginning of budding,in Hydra

In Hydra, head regeneration and bud formation appear to be very similar processes. The fact that there are genes whose expression is specific for one of the two processes suggests that they do not have identical molecular bases. We analyzed the signal transduction pathways regulating bud development using inhibitors of protein kinase C, Src, PI3K and ERK. The four inhibitors reversibly blocked bud formation in Hydra when applied before stage 1. Once the bud reached stage 3, three of them had no effect and the bud developed normally. The inhibitors blocked the expression of Budhead, an early head marker, and of CnOtx which are specific for bud formation. The results are in agreement with the central role of a signaling pathway mediated by Src on bud development.     

Interleukin-dependent modulation of HLA-DR expression on CD4and CD8 activated T cells

Summary Interleukins (IL) regulate different T-cell surface Ag known as activation markers that have distinct functional roles. In this paper, while studying the influence of some cytokines(IL-12, IL-2 and IL-4) on the expression of several markers [CD69,CD25, CD26, CD3, human leukocyte antigen (HLA-DR), CD45R0] in in vitro activated human T lymphocytes, we observed two groups of donors responding to phytohaemagglutinin (PHA) activation with high or low HLA-DRAg expression. We also found that CD4 and CD8 populations had different HLA-DR densities under PHA activation (particularly the high HLA-DR-expressing group). Interleukins, in a dose-dependent manner (IL-2 partially),upregulated these HLA-DR levels. In 5 day cultures, IL-12 and IL-2 enhanced the CD8/CD4 ratio of activated T cells,which was responsible, in part, for the IL-dependent HLA-DR upregulation.IL-12 and IL-2 also upregulated the HLA-DR expression at the molecular level on CD8, and IL-12 downregulated it on CD4 cells. It seems that IL-4 upregulated HLA-DR by shortening the mitogen-dependent regulation kinetics. We hypothesize that the different effect of each IL on HLA-DR expression might be related to the regulation of the dose of antigenic peptide presentation and, thus, also influence TH1/TH2 dominance.     

Biochemical and molecular study of mentally retarded patient with partial deficiency of hypoxanthine-guanine phosphoribosyltransferase

Nucleotide metabolism was studied in erythrocytes of a mentally retarded child and family members. Partial hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency was found in the propositus and an asymptomatic maternal uncle. Studies in crude lysates demonstrated decreased apparent V(max) and slightly decreased apparent K(m) for hypoxanthine in both HPRT-deficient subjects. Genomic DNA analysis revealed a single nucleotide change with leucine-147 to phenylalanine substitution in both subjects; mother and grandmother were heterozygous carriers of the same defect. This new variant has been termed HPRT(Potenza). Increased erythrocyte concentration of NAD and rate of synthesis by intact erythrocytes were found in the patient; increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NAD synthetase (NADs) were demonstrated in erythrocyte lysates, with normal apparent K(m) for their substrates and increased V(max). These alterations were not found in any member of the family, including the HPRT-deficient uncle. These findings show multiple derangement of nucleotide metabolism associated with partial HPRT deficiency. The enzyme alteration was presumably not the cause of neurological impairment since no neurological symptoms were found in the HPRT-deficient uncle, whereas they were present in the propositus' elder brother who had normal HPRT activity.     

Dok-related protein negatively regulates T cell development via its RasGTPase-activating protein and Nck docking sites

Downstream of kinase (Dok)-related protein (DokR, also known as p56(dok)/FRIP/Dok-R) is implicated in cytokine and immunoreceptor signaling in myeloid and T cells. Tyrosine phosphorylation induces DokR to bind the signal relay molecules, RasGTPase-activating protein (RasGAP) and Nck. Here, we have examined the function of DokR during hematopoietic development and the requirement for RasGAP and Nck binding sites in its biological function. Retroviral-mediated expression of DokR in bone marrow cells dramatically inhibited their capacity to form colonies in vitro in response to the cytokines macrophage colony-stimulating factor and stem cell factor, whereas responses to interleukin-3 and granulocyte macrophage colony-stimulating factor were only weakly affected. When introduced into lethally irradiated mice, hematopoietic cells expressing DokR showed a drastically reduced capacity to repopulate lymphoid tissues. Most notably, DokR dramatically reduced repopulation of the thymus, in part by reducing the number of T cell precursors seeding in the thymus, but equally, through inhibiting the transition of CD4(-)CD8(-) to CD4(+)CD8(+) T cells. Consequently, the number of mature peripheral T cells was markedly reduced. In contrast, a minimal effect on B cell and myeloid lineage development was observed. Importantly, functional RasGAP and Nck binding sites were found to be essential for the biological effects of DokR in vitro and in vivo.     

Neurotransmission of autonomic components of aortic baroreceptor afferents in the NTS of awake rats

The effect of sequential blockade of N-methyl-D-aspartic acid (NMDA) receptors with DL-2-amino-5-phosphonopentanoic acid (AP-5) and non-NMDA receptors with 6,7-dinitroquinoxaline-2,3 dione (DNQX) in the nucleus tractus solitarii (NTS) on the cardiovascular responses to electrical stimulation (ES) of the aortic depressor nerve (ADN) was evaluated in awake rats. Two protocols were used. In protocol 1, bilateral microinjection of AP-5 into the NTS (n = 7) reduced the hypotensive response to ES of the ADN; subsequent microinjection of DNQX produced additional reduction in this response. AP-5 reduced the bradycardic response, and DNQX almost abolished this response. In protocol 2, bilateral microinjection of DNQX into the NTS (n = 6) reduced the hypotensive response, and subsequent microinjection of AP-5 significantly reduced this response. DNQX produced a significant reduction in bradycardic response, and AP-5 abolished this response. The data indicate that processing of the parasympathetic component of the NTS aortic baroreceptor afferents is mediated by both NMDA and non-NMDA receptors, whereas processing of the sympathoinhibitory component seems to be only partially mediated by ionotropic receptors.     

Pfannenstiel incision as an alternative approach for harvesting the rectus abdominis muscle for free-tissue transfer

The rectus abdominis muscle has been one of the most commonly used donor tissues for free-flap reconstruction of defects in the extremities and in selected head and neck patients. The rectus abdominis has provided adequate soft-tissue mass with predictable anatomy and results for the majority of its applications in free-flap reconstruction. Harvesting of this muscle has typically been done through a paramedian or midline incision, which has left a lengthy notable scar on a patient's abdomen. To avoid the late aesthetic deformity associated with this typical approach for the rectus abdominis, we began harvesting the muscle through a Pfannenstiel incision. Patients were initially selected based on young age and limited soft-tissue requirements. With additional experience, this technique was extended to include all healthy patients regardless of age. Also, soft-tissue limitations no longer became an issue, as we learned the entire rectus abdominis muscle could be harvested from this approach. An extended Pfannenstiel incision was made from the ipsilateral anterior superior iliac spine to the lateral border of the contralateral rectus abdominis. A superiorly based flap was raised to expose the full length of the anterior rectus sheath from pubis to costal margin. In our earlier patients, a periumbilical incision was made for presumed easier access, but we discovered this was an unnecessary maneuver. With the anterior sheath fully exposed, the muscle was harvested and the sheath repaired in a routine manner. The elevated abdominal flap was returned to its anatomic position and closed over a suction drain. Since 1993, 10 patients have undergone a Pfannenstiel approach for harvesting of the rectus abdominis muscle. The mean age was 16. The areas requiring coverage included a traumatic elbow defect, seven traumatic lower extremity defects, one lower extremity sarcoma defect, and one lower extremity septic joint defect. Mean follow-up for these patients was 12 months. There were no flap failures. One patient developed an arterial thrombosis on postoperative day 5 and was treated with successful revision. There were no abdominal wall complications. Cosmesis was judged as good in all patients. We would recommend avoiding this approach in heavy or moderate smokers, diabetic patients, and patients with significant obesity. The Pfannenstiel approach to the rectus abdominis muscle has allowed for complete harvest of the muscle, improved aesthetic results compared with alternative techniques, and avoidance of donor-site morbidityin healthy patients.     

In vitro activity evaluation of Parkia pendula seed lectin against human cytomegalovirus and herpes virus 6.

Human cytomegalovirus (HCMV) in vitro infectivity was inhibited by Parkia pendula seed lectin (PpeL) in contrast to human herpes virus 6 (HHV-6) which was not affected. The antiviral activity was detected for HCMV in human embryo lung (HEL) cells using a microtechnique in culture plates. The assay showed a reduction of cellular infectivity from approximately 95%, at a concentration of 150microg/mL with minimal cytotoxicity (25%). Also, a reduction of 75% was observed in HEL cells at a concentration of 75microg/mL without toxic effect. The reduction on infectivity was observed even after virus pre-adsorption to cells suggesting that this action should occur after virus penetration, in the intracellular replication phase. MT4 lymphocytes and cord blood mononuclear cells (CBMC) were used to evaluate the lectin effect on HHV-6 following the same technique. Lectin concentrations with few or no toxic effects on lymphocytes did not show inhibitory action of HHV-6 cytopathic effect. The results obtained with PpeL demonstrate that it may have an impact in the design of pharmacological strategies to infection of cytomegalovirus.     

Siaα2-3Galβ1- Receptor Genetic Variants Are Associated with Influenza A(H1N1)pdm09 Severity

Different host genetic variants may be related to the virulence and transmissibility of pandemic Influenza A(H1N1)pdm09, influencing events such as binding of the virus to the entry receptor on the cell of infected individuals and the host immune response. In the present study, two genetic variants of the ST3GAL1 gene, which encodes the Siaα2-3Galβ1- receptor to which influenza A(H1N1)pdm09 virus binds for entry into the host cell, were investigated in an admixed Brazilian population. First, the six exons encoding the ST3GAL1 gene were sequenced in 68 patients infected with strain A(H1N1)pdm09. In a second phase of the study, the rs113350588 and rs1048479 polymorphisms identified in this sample were genotyped in a sample of 356 subjects from the northern and northeastern regions of Brazil with a diagnosis of pandemic influenza. Functional analysis of the polymorphisms was performed in silico and the influence of these variants on the severity of infection was evaluated. The results suggest that rs113350588 and rs1048479 may alter the function of ST3GAL1 either directly through splicing regulation alteration and/or indirectly through LD with SNP with regulatory function. In the study the rs113350588 and rs1048479 polymorphisms were in linkage disequilibrium in the population studied (D’ = 0.65). The GC haplotype was associated with an increased risk of death in subjects with influenza (OR = 4.632, 95% CI = 2.10;1.21). The AT haplotype was associated with an increased risk of severe disease and death (OR = 1.993, 95% CI = 1.09;3.61 and OR 4.476, 95% CI = 2.37;8.44, respectively). This study demonstrated for the first time the association of ST3GAL1 gene haplotypes on the risk of more severe disease and death in patients infected with Influenza A(H1N1)pdm09 virus.