Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator

The blood anion nitrite contributes to hypoxic vasodilation through a heme-based, nitric oxide (NO)-generating reaction with deoxyhemoglobin and potentially other heme proteins. We hypothesized that this biochemical reaction could be harnessed for the treatment of neonatal pulmonary hypertension, an NO-deficient state characterized by pulmonary vasoconstriction, right-to-left shunt pathophysiology and systemic hypoxemia. To test this, we delivered inhaled sodium nitrite by aerosol to newborn lambs with hypoxic and normoxic pulmonary hypertension. Inhaled nitrite elicited a rapid and sustained reduction ( approximately 65%) in hypoxia-induced pulmonary hypertension, with a magnitude approaching that of the effects of 20 p.p.m. NO gas inhalation. This reduction was associated with the immediate appearance of NO in expiratory gas. Pulmonary vasodilation elicited by aerosolized nitrite was deoxyhemoglobin- and pH-dependent and was associated with increased blood levels of iron-nitrosyl-hemoglobin. Notably, from a therapeutic standpoint, short-term delivery of nitrite dissolved in saline through nebulization produced selective, sustained pulmonary vasodilation with no clinically significant increase in blood methemoglobin levels. These data support the concept that nitrite is a vasodilator acting through conversion to NO, a process coupled to hemoglobin deoxygenation and protonation, and evince a new, simple and inexpensive potential therapy for neonatal pulmonary hypertension.     

PlasmoDB: the Plasmodium genome resource. A database integrating experimental and computational data

PlasmoDB (http://PlasmoDB.org) is the official database of the Plasmodium falciparum genome sequencing consortium. This resource incorporates the recently completed P. falciparum genome sequence and annotation, as well as draft sequence and annotation emerging from other Plasmodium sequencing projects. PlasmoDB currently houses information from five parasite species and provides tools for intra- and inter-species comparisons. Sequence information is integrated with other genomic-scale data emerging from the Plasmodium research community, including gene expression analysis from EST, SAGE and microarray projects and proteomics studies. The relational schema used to build PlasmoDB, GUS (Genomics Unified Schema) employs a highly structured format to accommodate the diverse data types generated by sequence and expression projects. A variety of tools allow researchers to formulate complex, biologically-based, queries of the database. A stand-alone version of the database is also available on CD-ROM (P. falciparum GenePlot), facilitating access to the data in situations where internet access is difficult (e.g. by malaria researchers working in the field). The goal of PlasmoDB is to facilitate utilization of the vast quantities of genomic-scale data produced by the global malaria research community. The software used to develop PlasmoDB has been used to create a second Apicomplexan parasite genome database, ToxoDB (http://ToxoDB.org).     

Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase

The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) support rapid growth of a variety of Pseudomonas strains when provided as the sole source of carbon and nitrogen. All key enzymes of glutamate metabolism were detected in P. fluorescence, with glutaminase and asparaginase showing the highest specific activities. A periplasmic glutaminase/asparaginase activity (PGA) was found in all pseudomonads examined, including a number of root-colonizing biocontrol strains. The enzyme was purified and shown to be identical with the ansB gene product described previously. In addition to PGA, P. fluorescens contains a cytoplasmic asparaginase with marked specificity for Asn. PGA is strongly and specifically induced by its substrates (Asn, Gln) but also by the reaction products (Asp, Glu). In addition, PGA is subject to efficient carbon catabolite repression by glucose and by citrate cycle metabolites. A mutant of P. putida KT2440 with a disrupted ansB gene was unable to utilize Gln, whereas growth of the mutant on other amino acids was normal.     

Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3′-hydroxyl and 5′-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.     

Single-chain Tet transregulators

We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse ‘tet-on’ phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.     

Assessing functional divergence in EF-1alpha and its paralogs in eukaryotes and archaebacteria

A number of methods have recently been published that use phylogenetic information extracted from large multiple sequence alignments to detect sites that have changed properties in related protein families. In this study we use such methods to assess functional divergence between eukaryotic EF-1α (eEF-1α), archaebacterial EF-1α (aEF-1α) and two eukaryote-specific EF-1α paralogs—eukaryotic release factor 3 (eRF3) and Hsp70 subfamily B suppressor 1 (HBS1). Overall, the evolutionary modes of aEF-1α, HBS1 and eRF3 appear to significantly differ from that of eEF-1α. However, functionally divergent (FD) sites detected between aEF-1α and eEF-1α only weakly overlap with sites implicated as putative EF-1β or aminoacyl-tRNA (aa-tRNA) binding residues in EF-1α, as expected based on the shared ancestral primary translational functions of these two orthologs. In contrast, FD sites detected between eEF-1α and its paralogs significantly overlap with the putative EF-1β and/or aa-tRNA binding sites in EF-1α. In eRF3 and HBS1, these sites appear to be released from functional constraints, indicating that they bind neither eEF-1β nor aa-tRNA. These results are consistent with experimental observations that eRF3 does not bind to aa-tRNA, but do not support the ‘EF-1α-like’ function recently proposed for HBS1. We re-assess the available genetic data for HBS1 in light of our analyses, and propose that this protein may function in stop codon-independent peptide release.     

Mal de Meleda (MDM) caused by mutations in the gene for SLURP-1 in patients from Germany,Turkey, Palestine,and the United Arab Emirates

Mal de Meleda (MDM) or keratosis palmoplantaris transgrediens of Siemens is an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis with an onset in early infancy. There is no associated involvement of other organs; however, a spectrum of clinical presentations with optional and variable features has been described. Mutations in the ARS (component B)-81/s gene ( LY6LS) on chromosome 8q24-qter, which encodes SLURP-1, have recently been identified in patients with MDM. Here, we have analyzed four MDM families for mutations in SLURP-1. In a large Palestinian pedigree with multiple consanguinity, patients are homozygous for a new mutation that substitutes an arginine for a conserved glycine residue at position 86. A different mutation in Turkish patients results in the same amino acid exchange. Some remarkable similarities are seen in the clinical picture of patients from both families. Patients of an Emirati Bedouin family have a homozygous alteration of the translation initiation codon. In a German family with no known consanguinity, we have shown pseudodominant inheritance. Three affected children and their affected mother are homozygous for the missense mutation W15R. Our findings indicate that the MDM type of transgressive PPK is caused by SLURP-1 mutations in patients from various origins and demonstrate allelic heterogeneity for mutations in SLURP-1.     

Tetrahydrobiopterin restores endothelial dysfunction induced by an oral glucose challenge in healthy subjects

An oral glucose challenge causes transient impairment of endothelial function, probably because of increased oxidative stress. During oxidative stress, endothelial nitric oxide (NO) synthase (eNOS) becomes uncoupled because of decreased bioavailability of tetrahydrobiopterin (BH4), an essential cofactor of eNOS. Therefore, we examined whether an acute supplement of BH4 could restore endothelial dysfunction induced by an oral glucose challenge. Healthy subjects were examined in 53 experiments. Forearm blood flow was measured by venous occlusion plethysmography. Dose-response studies were obtained during intra-arterial infusion of serotonin to elicit endothelium-dependent, NO-specific vasodilation and during sodium nitroprusside (SNP) infusion to elicit endothelium-independent vasodilation. Subjects were examined before (fasting) and 1 and 2 h after an oral glucose challenge (75 g) with serotonin (n = 10) and SNP (n = 8). On different days (6R)-5,6,7,8-tetrahydro-l-biopterin dihydrochloride (6R-BH4; n = 10), the active cofactor of eNOS or its stereoisomer (6S)-5,6,7,8-tetrahydro-l-biopterin sulfate (6S-BH4; n = 10), which is inactive as a cofactor, was added 10 min (500 microg/min) before and during the 1-h postchallenge serotonin dose-response study. In vitro studies showed that 6R-BH4 and 6S-BH4 were equipotent antioxidants. Serotonin response was reduced by 24 +/- 7% (at the highest dose) at 1 h postchallenge compared with fasting (P = 0.001) and was restored 2 h postchallenge. The reduction was reversed by the administration of 6R-BH4 but not by 6S-BH4. SNP responses were slightly increased 1 and 2 h postchallenge (increased by 15 +/- 13% at third dose 2 h postchallenge, P = 0.0001). An oral glucose challenge causes transient, NO-specific, endothelial dysfunction, which may be reversed by BH4. Transient postprandial endothelial dysfunction may be partly explained by reduced bioavailability of BH4 and NO.     

Effects of truncated angiotensins in humans after double blockade of the renin system

Angiotensins different from ANG II exhibit biological activities, possibly mediated via receptors other than ANG II receptors. We studied the effects of 3-h infusions of ANG III, ANG-(1-7), and ANG IV in doses equimolar to physiological amounts of ANG II (3 pmol. kg-1. min-1), in six men on low-sodium diet (30 mmol/day). The subjects were acutely pretreated with canrenoate and captopril to inhibit aldosterone actions and ANG II synthesis, respectively. ANG II infusion increased plasma angiotensin immunoreactivity to 53 +/- 6 pg/ml (+490%), plasma aldosterone to 342 +/- 38 pg/ml (+109%), and blood pressure by 27%. Glomerular filtration rate decreased by 16%. Concomitantly, clearance of endogenous lithium fell by 66%, and fractional proximal reabsorption of sodium increased from 77 to 92%; absolute proximal reabsorption rate of sodium remained constant. ANG II decreased sodium excretion by 70%, potassium excretion by 50%, and urine flow by 80%, whereas urine osmolality increased. ANG III also increased plasma aldosterone markedly (+45%), however, without measurable changes in angiotensin immunoreactivity, glomerular filtration rate, or renal excretion rates. During vehicle infusion, plasma renin activity decreased markedly ( approximately 700 to approximately 200 mIU/l); only ANG II enhanced this decrease. ANG-(1-7) and ANG IV did not change any of the measured variables persistently. It is concluded that 1) ANG III and ANG IV are cleared much faster from plasma than ANG II, 2) ANG II causes hypofiltration, urinary concentration, and sodium and potassium retention at constant plasma concentrations of vasopressin and atrial natriuretic peptide, and 3) a very small increase in the concentration of ANG III, undetectable by usual techniques, may increase aldosterone secretion substantially.