A spectrophotometric assay for nitrate in an excess of nitrite.

Miranda et al. have developed a method for simultaneous evaluation of nitrate and nitrite concentrations using reduction of nitrate by vanadium(III) combined with detection by the acidic Griess reaction [K.M. Miranda, M.G. Espey, D.A. Wink, A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite, Nitric Oxide 5 (2001) 62-71]. The sensitivity of the nitrate assay decline if the mixture analyzed contains a large excess of nitrite relative to nitrate, for instance, in the case of oxidation products of nitric oxide (NO) in aerated solutions, or in sweat. By this reason nitrite should be removed before the nitrate assay, if [NO2-]>[NO3-]. Here we lay out an improved method allowing the above limitation to be erased, using sulfamic acid for nitrite removal. We also describe some modifications that enhance the reproducibility of the assay.     

Autonomic dysreflexia during sperm retrieval in spinal cord injury: influence of lesion level and sildenafil citrate.

Autonomic dysreflexia (AD) can occur during penile vibratory stimulation in men with spinal cord injury, but this is variable, and the association with lesion level is unclear. The purpose of this study was to characterize the cardiovascular responses to penile vibratory stimulation in men with spinal cord injury. We hypothesized that those with cervical injuries would demonstrate a greater degree of AD compared with men with thoracic injuries. We also questioned whether the rise in blood pressure could be attenuated by sildenafil citrate. Participants were classified as having cervical (n = 8) or thoracic (n = 5) injuries. While in a supine position, subjects were instrumented with an ECG, and arterial blood pressure was determined beat by beat. Subjects reported to the laboratory twice and received an oral dose of sildenafil citrate (25-100 mg) or no medication. Penile vibratory stimulation was performed using a handheld vibrator to the point of ejaculation. At ejaculation during the nonmedicated trials, the cervical group had a significant decrease in heart rate (-5-10 beats/min) and increase in mean arterial blood pressure (+70-90 mmHg) relative to resting conditions, whereas the thoracic group had significant increases in both heart rate (+8-15 beats/min) and mean arterial pressure (+25-30 mmHg). Sildenafil citrate had no effect on the change in heart rate or mean arterial pressure in either group. In summary, men with cervical injuries had more pronounced AD during penile vibratory stimulation than men with thoracic injuries. Administration of sildenafil citrate had no effect on heart rate or blood pressure during penile vibratory stimulation in men with spinal cord injury.     

Molecular diversity and phylogeny of indigenous <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> strains associated with <Emphasis Type="Italic">Trifolium repens</Emphasis> plants in Romania

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.     

Mechanism of Inhibition of Translation Termination by Blasticidin S

Understanding the mechanisms of inhibitors of translation termination may inform development of new antibacterials and therapeutics for premature termination diseases. We report the crystal structure of the potent termination inhibitor blasticidin S bound to the ribosomal 70S?release factor 1 (RF1) termination complex. Blasticidin S shifts the catalytic domain 3 of RF1 and restructures the peptidyl transferase center. Universally conserved uridine 2585 in the peptidyl transferase center occludes the catalytic backbone of the GGQ motif of RF1, explaining the structural mechanism of inhibition. Rearrangement of domain 3 relative to the codon-recognition domain 2 provides insight into the dynamics of RF1 implicated in termination accuracy.     

Direct effects of interleukin-7 on the function of human T cells <Emphasis Type="Italic">in vitro</Emphasis>

CD3+ T lymphocytes were isolated by positive magnetic separation from the peripheral blood of healthy donors. In the absence of any additional activating stimuli, interleukin-7 (IL-7) was shown to augment the levels of T cells expressing CD25 activation marker both in CD4-positive and in CD4-negative effector memory (CD45RA^-CD197^-) T cell subsets, as well as in terminally differentiated (CD45RA⁺CD197^-) T cells, without significantly affecting the activation status of naive (CD45RA⁺CD197⁺) and central memory (CD45RA^-CD197⁺) T cells. In addition, IL-7 noticeably enhanced the production of IL-2, interferon-γ (IFN-γ), and IL-10, but not IL-4, in T cells. The direct effects of IL-7 on T cell activation induced in vitro by MACSiBead? particles coated with CD2, CD3, and CD28 antibodies (Abs) were also investigated. Upon cell activation, IL-7 significantly augmented the levels of CD25+ T cells in naive (CD45RA+CD197+), central memory (CD45RA^-CD197⁺), and effector memory (CD45RA^-CD197^-) T-cell compartments. In addition, IL-7 facilitated activation of CD4^- (but not CD4⁺) terminally differentiated effector (CD45RA⁺CD197^-) T cells. Finally, IL-7 was found to upregulate the production of IL-2, IFN-γ, IL-4, and IL-10 by activated T cells. In conclusion, we speculate that IL-7 is capable of enhancing functional T cell activity without causing significant functional inbalance between various T cell subsets.     

MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory

Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion.     

Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.     

Verapamil contributes to the clastogenic effects of acrylamide, cyclophosphamide, and dioxidine on somatic cells of BALB/C and C57BL/6 mice

The chromosome aberration assay of metaphase bone marrow cells was used to study the clastogenic effects of acrylamide, cyclophosphamide, dioxidine, and their combinations with Verapamil (a calcium antagonist) in male BALB/C and C57BL/6 mice. Verapamil gavage at single (5 mg/kg) and repeated doses (2.5 and 5 mg/kg five times at 24-h intervals) significantly enhanced the clastogenic activity of acrylamide (50 and 100 mg/kg intraperitoneally) in BALB/C mice; in C57BL/6 mice, this effect was only observed when they received Verapamil at doses of 2.5 mg/kg for 5 days. Verapamil administered repeatedly (2.5–10 mg, gavage) significantly increased the clastogenic activity of cyclophosphamide (10 mg/kg intraperitoneally) in C57BL/6 mice. In BALB/C mice, this effect of Verapamil was only observed at a dose of 10 mg/kg (gavage). When injected intraperitoneally at a single dose of 0.1–0.4 mg/kg, Verapamil significantly enhanced the clastogenic activity of cyclophosphamide in mice of both strains. This calcium antagonist produced identical effects when administered to BALB/C mice intraperitoneally (2.5 and 5 mg/kg) and by gavage (5 mg/kg) and to C57BL/6 mice intraperitoneally (5 and 10 mg/kg) and by gavage (2.5 mg/kg). Repeated administration of Verapamil (at all doses tested) promoted the clastogenic effect of dioxidine (100 mg/kg intraperitoneally) on C57BL/6 mice, having no such influence on BALB/C mice. These results demonstrate the co-clastogenic activity of Verapamil in mice and suggest that its specific manifestations depend on the dose, method, and route of drug administration and the genotype of test animals.     

Synthesis and biological evaluation of 1,2-disubstituted carbonucleosides of 6-substituted purine and 8-azapurine.

A series of new one two substituted carbonucleoside analogues (OTC), with the purine and 8-azapurine base linked through a methylene group at the cyclopentane ring, were synthesized and evaluated for their activity against a number of viruses and tumor cells in vitro.