Convenient colorimetric and fluorometric assays for S-nitrosothiols

S-nitrosothiols have been shown to affect a number of physiological functions. Several techniques have been used to detect these species in biological systems, primarily by methods utilizing chemiluminescence. Since the apparatus required for measurement of chemiluminescence are not readily available in most laboratories, methods employing more conventional techniques such as uv-vis and fluorescence spectroscopy may be of greater use. Herein, we report the development of colorimetric and fluorometric methods for the reliable quantitation of S-nitrosothiols. Solutions containing sulfanilamide/N-(1-naphthyl)- ethylenediamine dihydrochloride or 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid), when exposed to S-nitrosoglutathione (GSNO), S-nitrosocysteine, or S-nitrosoacteylpenicillamine, resulted in no absorbance changes in the range of 400-800 nm. Exposure to HgCl2 or Cu(acetate)2 resulted in release of nitric oxide (NO) from the S-nitrosothiols. The liberated NO reacted subsequently with oxygen and formed a chemical species which reacted with either analysis solution, resulting in an increase in absorption between 400 and 800 nm. A plot of RSNO versus absorbance was linear for both mercury(II) and copper(II) ions where the slope in the presence of mercury ion was significantly greater than that for copper ion. The sensitivity was as low as 5 microM RSNO using HgCl2. The fluorometric method using 2, 3-diaminonaphthalene as the scavenger of the NOsolidusO2 products gave a sensitivity of 50 nM for GSNO. In addition, S-nitrosylated proteins were quantitated using the fluorometric technique. These methods provide accurate determination of low concentrations of S-nitrosothiols, utilizing conventional spectroscopic techniques available in most laboratories.     

Critical analysis of driving force for pure-step migration by vacancy supersaturation and crystal anisotropy

The gist of the paper is to introduce the other compelling factors for the boundary displacement which are dominant in the lower temperature regime such as non equilibrium vacancy concentration and anisotropy and to prove that the driving force due to the above factors is sufficient to induce grain boundary migration.     

Intracranial metyrapone stimulates CRF-ACTH axis in the teleost, Clarias batrachus: possible role of neurosteroids

Intracranial administration of metyrapone, a blocker of the enzyme 11-beta-hydroxylase, which is essential for the biosynthesis of corticosteroids, resulted in profound stimulation of the nucleus preopticus and the CRF-ACTH axis in the teleost, Clarias batrachus. It is suggested that the putative blockade of the neurosteroid biosynthesis following metyrapone might be responsible for this action. The present study for the first time uncovers the possibility of inhibitory regulation of the CRF-ACTH axis by metyrapone sensitive neurosteroids.     

Isoform-specific induction of nuclear free calcium oscillations by platelet-derived growth factor

Confocal laser scanning microscopy was used to analyze alterations in nuclear free calcium (Ca2+n) levels induced by platelet-derived growth factor (PDGF) isoforms in BALB/c3T3 fibroblasts loaded with the calcium-sensitive fluorescent indicator Fluo-3. Both AA-PDGF and BB-PDGF caused a transient increase in Ca2+n. Analysis of PDGF-induced Ca2+n alterations as a function of time revealed that BB-PDGF stimulation resulted in the generation of Ca2+n oscillations that diminished over time. The frequency of BB-PDGF-stimulated oscillations was modulated by extracellular Ca2+ and could not be mimicked by increasing intracellular inositol 1,4,5-trisphosphate levels in the absence of growth factor stimulation. Caffeine alone had no effect on Ca2+n levels, but exposure of cells to caffeine after BB-PDGF stimulation augmented Ca2+n oscillations, either by increasing the frequency or reinitiating preexisting oscillations. The genesis of these oscillations in Ca2+n appears to be in the region just outside of the nucleus, as perinuclear cytoplasmic free calcium (Ca2+i) increased just prior to Ca2+n. In contrast, AA-PDGF stimulation resulted in the generation of one or two irregular, transient Ca2+n spikes. Caffeine pretreatment followed by AA-PDGF stimulation resulted in Ca2+n oscillations very similar to those produced by BB-PDGF alone. Additionally, the AA-PDGF and BB-PDGF isoforms appeared to modulate distinct pools of cellular Ca2+, as BB-PDGF was still capable of inducing Ca2+n oscillations subsequent to prior induction of oscillations by AA-PDGF/caffeine. These PDGF isoform-specific changes in nuclear free Ca2+ could serve as a mechanism by which isoform-specific cellular signaling pathways may be manifested by the growth factors.     

Genetic determinants of Rous sarcoma virus particle size

The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant deltaNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.     

Pillaring of smectites using an aluminium oligomer: A study of pillar density and thermal stability

Two smectite samples having different layer charges were pillared using hydroxy aluminium oligomers at a OH/Al ratio of 2.5 and at pH 4.3 to 4.6. Pillaring was carried out at different conditions such as ageing, temperature and base addition time of the pillaring solution, and also in the presence of nonionic surfactant polyoxyethylene sorbitanmonooleate (Tween-80). The primary objective of preparing at different conditions was to introduce varied quantities of aluminium oligomer between the layers and to study its effect on the properties of the pillared products. A simple method has been followed to estimate the amount of interlayer aluminium. A quantity called pillar density number (PDN) based on the ratio of interlayer Al adsorbed to CEC of the parent clay has been effectively used to evaluate the nature of the resulting pillared product. PDN, for a given clay, was found to correlate well with the sharpness of the d(0001) peaks for the air dried samples. The calculated number of pillars, varied from 3.00 × 10¹⁸ to 5.32 × 10¹⁸ per meq charge. The present study shows that a higher value of PDN is indicative of better thermal stability. Pillar density number may be conveniently used as a measure of the thermal stability of pillared samples.     

Limb salvage using free tissue transfer following meningococcal septicemia

Over the past 10 years, I have been privileged to conduct educational forums for audiences containing many recovering alcoholics or otherwise chemically dependent persons. In these forums about the addictive diseases and their treatment and research possibilities, significant interaction with the audience members occurs. During these interactions, certain anecdotal phenomena seem to predominate. The repetitive nature of these reports suggests the need for systematic investigation. As with editorial comments in major medical journals, observed phenomena and unanswered questions from those afflicted can be valuable in the generation of testable hypotheses. Perhaps the ideas presented herein will be useful in the development of future research on alcohol abuse and alcohol dependence.     

Characteristics of Alumina Powders Prepared by Spray-Drying of Boehmite Sol

Boehmite sol prepared from aluminum nitrate has been spray-dried to obtain micrometer-size spherical particles consisting of submicrometer crystallites. The spray-dried powder was further washed with solvents of varying polarities such as acetone, 2-propano1, and 2-methyl-2-propanol. Particle-size distribution, morphology, density, compaction, and sintering characteristics of powders washed with different solvents are reported. The effect of posttreatments on the boehmite-sol-derived powders toward reducing agglomeration and obtaining high-density bodies is discussed.     

Hydrogen induced cracking of a tappet adjusting screw

The electroplated tappet adjusting screws used in diesel engines failed during initial bend testing. The analysis of the failure showed that the fracture was nucleated from the subsurface of the screw. The fracture surface was intergranular at the ID and OD region and microvoid coalesence in the center. The improper baking after electroplating of the screw led to H₂-induced blistering/cracking. The high strength of the threaded region of the adjusting screw increased the failure propensity.     

Processing and Properties of Sol–Gel-Derived Alumina/Silicon Carbide Nanocomposites

Dense alumina/5 vol% SiC nanocomposites were prepared by sol–gel processing using nanosized (180 nm) precoated SiC powders and a commercial boehmite sol. The SiC powder was precoated with boehmite by a controlled heterogeneous precipitation from an aluminum nitrate solution. The coated SiC powder was then dispersed in a boehmite sol, gelled, calcined, and densified by gas pressure sintering under argon atmosphere at 7–8 MPa pressure. The dependence of the calcination conditions on densification, the effect of seeding on the microstructural development, as well as the mechanical behavior of the sintered specimens, are presented and discussed.