The role of nitric oxide in the regulation of the systemic and pulmonary vasculature of the rattlesnake, <Emphasis Type="Italic">Crotalus durissus terrificus</Emphasis>

The functional role of nitric oxide (NO) was investigated in the systemic and pulmonary circulations of the South American rattlesnake, Crotalus durissus terrificus. Bolus, intra-arterial injections of the NO donor, sodium nitroprusside (SNP) caused a significant systemic vasodilatation resulting in a reduction in systemic resistance (Rsys). This response was accompanied by a significant decrease in systemic pressure and a rise in systemic blood flow. Pulmonary resistance (Rpul) remained constant while pulmonary pressure (Ppul) and pulmonary blood flow (Qpul) decreased. Injection of L-Arginine (L-Arg) produced a similar response to SNP in the systemic circulation, inducing an immediate systemic vasodilatation, while Rpul was unaffected. Blockade of NO synthesis via the nitric oxide synthase inhibitor, L-NAME, did not affect haemodynamic variables in the systemic circulation, indicating a small contribution of NO to the basal regulation of systemic vascular resistance. Similarly, Rpul and Qpul remained unchanged, although there was a significant rise in Ppul. Via injection of SNP, this study clearly demonstrates that NO causes a systemic vasodilatation in the rattlesnake, indicating that NO may contribute in the regulation of systemic vascular resistance. In contrast, the pulmonary vasculature seems far less responsive to NO.     

Novel features of the rabbit transverse tubular system revealed by quantitative analysis of three-dimensional reconstructions from confocal images

With scanning confocal microscopy we obtained three-dimensional (3D) reconstructions of the transverse tubular system (t-system) of rabbit ventricular cells. We accomplished this by labeling the t-system with dextran linked to fluorescein or, alternatively, wheat-germ agglutinin conjugated to an Alexa fluor dye. Image processing and visualization techniques allowed us to reconstruct the t-system in three dimensions. In a myocyte lying flat on a coverslip, t-tubules typically progressed from its upper and lower surfaces. 3D reconstructions of the t-tubules also suggested that some of them progressed from the sides of the cell. The analysis of single t-tubules revealed novel morphological features. The average diameter of single t-tubules from six cells was estimated to 448 ± 172 nm (mean ± SD, number of t-tubules 348, number of cross sections 5323). From reconstructions we were able to identify constrictions occurring every 1.87 ± 1.09 μm along the principal axis of the tubule. The cross-sectional area of these constrictions was reduced to an average of 57.7 ± 27.5% (number of constrictions 170) of the adjacent local maximal areas. Principal component analysis revealed flattening of t-tubular cross sections, confirming findings that we obtained from electron micrographs. Dextran- and wheat-germ agglutinin-associated signals were correlated in the t-system and are therefore equally good markers. The 3D structure of the t-system in rabbit ventricular myocytes seems to be less complex than that found in rat. Moreover, we found that t-tubules in rabbit have approximately twice the diameter of those in rat. We speculate that the constrictions (or regions between them) are sites of dyadic clefts and therefore can provide geometric markers for colocalizing dyadic proteins. In consideration of the resolution of the imaging system, we suggest that our methods permit us to obtain spatially resolved 3D reconstructions of the t-system in rabbit cells. We also propose that our methods allow us to characterize pathological defects of the t-system, e.g., its remodeling as a result of heart failure.     

The cytotoxic effects of single-stranded telomere mimics on OMA-BL1 cells.

Telomerase is a ribonucleoprotein that adds 5'-d(TTAGGG)-3' hexameric repeats onto the 3' ends of chromosomes. High telomerase activity has been associated with immortal cells, transformed cells, mitogenic stimulation, and proliferative diseases. It is not clear what phenotype would be observed by transient inhibition of telomerase. Studies were designed to inhibit telomerase activity using a series of S-ODN telomere sequence motifs. The studies evaluated the length, hydrogen bonding, and sequence requirements of telomerase inhibition using the TRAP assay and a bioassay measuring cell viability following exposure to the compounds. In addition, we have also studied the role of the 3' end and secondary structure of telomere mimics on telomerase inhibition. Observations reveal that sensitivity to the S-ODNs may not require hybridization to an antisense target but required guanine nucleotides on the 3' end for cells in culture and telomerase inhibition in vitro. The importance of H bonding and the requirement for a free 3' end for the activity of these compounds has also been demonstrated. However, transient inhibition of telomerase is not cytotoxic to all immortal cells and is not sufficient to explain the mechanism of cytotoxicity of these short oligonucleotides.     

Nuclear organization of splicing small nuclear ribonucleoproteins in adenovirus-infected cells.

We have studied the effect of adenovirus infection on the nuclear organization of splicing small nuclear ribonucleoproteins (snRNPs) in HeLa cells. In uninfected HeLa cells, snRNPs are widespread throughout the nucleoplasm but also are concentrated in specific nuclear structures, including coiled bodies, interchromatin granules, and perichromatin fibrils. We have used immunofluorescence microscopy to study the localization of splicing snRNPs relative to centers of viral DNA synthesis and accumulation identified with antiserum against the viral 72,000-molecular-weight single-stranded DNA-binding protein (72K protein). Splicing snRNPs were independently detected with both monoclonal and polyclonal antibodies specific for common snRNP antigens, snRNP-specific proteins, and the snRNA-specific 2,2,7-trimethylguanosine 5' cap structure. We have examined infected cells 2 to 24 h after infection, and, in the majority of these cells, we observed no colocalization of the snRNP and 72K-protein staining patterns. In the late phase, snRNPs were found to markedly concentrate in discrete clusters that were distinct from the centers of viral DNA synthesis and accumulation identified with anti-72K protein. We have treated cells with hydroxyurea at various times after infection to inhibit aspects of the virus infectious program. We have found that the accumulation of snRNP clusters is correlated with late gene expression rather than with DNA synthesis or early gene expression. Finally, we show that the late-phase snRNP clusters colocalize with a monoclonal antibody that primarily stains interchromatin granules. These results suggest that the centers of snRNP concentration in late-phase infected cells are likely to correspond to interchromatin granule clusters.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

Electrochemical investigation into the redox activity of Fe(II)/Fe(III) in the presence of nicotine and possible relations to neurodegenerative diseases

The biological relevance of Fe(II)/Fe(III) is becoming evermore apparent, especially in relation to its potential role in the progression of neurodegenerative diseases such as Parkinson's and Alzheimer's disease. The reported relationship between smoking and a reduced incidence of neurodegenerative disorders prompted this work. In order to investigate whether nicotine can interact with iron, we have studied the electrochemical behaviour of a Fe(II)/Fe(III) redox couple in the presence of nicotine. Solubility issues and lack of available nonreacting salts of nicotine necessitated studies being conducted at low pH values. Cyclic voltammetry experiments revealed a definite alteration in the electrochemical behaviour of the Fe(II)/Fe(III) redox couple suggesting the capability of nicotine to complex with free iron and, hence, reduce its reactivity. This is evident from a slower rate of heterogeneous electron transfer, ks, and a shift from reversible to quasi-reversible behaviour, as characterised from the diffusion coefficient (D), the full width half maximum (FWHM), DeltaEp and Ef. Additional complexation titrations, pH ranging from 1 to 7, confirm a weak complexation reaction occurring between Fe(III) and nicotine.     

Molecular analysis of geographic patterns of eukaryotic diversity in Antarctic soils

We describe the application of molecular biological techniques to estimate eukaryotic diversity (primarily fungi, algae, and protists) in Antarctic soils across a latitudinal and environmental gradient between approximately 60 and 87 degrees S. The data were used to (i) test the hypothesis that diversity would decrease with increasing southerly latitude and environmental severity, as is generally claimed for "higher" faunal and plant groups, and (ii) investigate the level of endemicity displayed in different taxonomic groups. Only limited support was obtained for a systematic decrease in diversity with latitude, and then only at the level of a gross comparison between maritime (Antarctic Peninsula/Scotia Arc) and continental Antarctic sites. While the most southerly continental Antarctic site was three to four times less diverse than all maritime sites, there was no evidence for a trend of decreasing diversity across the entire range of the maritime Antarctic (60 to 72 degrees S). Rather, we found the reverse pattern, with highest diversity at sites on Alexander Island (ca. 72 degrees S), at the southern limit of the maritime Antarctic. The very limited overlap found between the eukaryotic biota of the different study sites, combined with their generally low relatedness to existing sequence databases, indicates a high level of Antarctic site isolation and possibly endemicity, a pattern not consistent with similar studies on other continents.     

Reduction of ferric iron by acidophilic heterotrophic bacteria: evidence for constitutive and inducible enzyme systems in Acidiphilium spp

AIMS: To compare the abilities of two obligately acidophilic heterotrophic bacteria, Acidiphilium acidophilum and Acidiphilium SJH, to reduce ferric iron to ferrous when grown under different culture conditions. METHODS AND RESULTS: Bacteria were grown in batch culture, under different aeration status, and in the presence of either ferrous or ferric iron. The specific rates of ferric iron reduction by fermenter-grown Acidiphilium SJH were unaffected by dissolved oxygen (DO) concentrations, while iron reduction by A. acidophilum was highly dependent on DO concentrations in the growth media. The ionic form of iron present (ferrous or ferric) had a minimal effect on the abilities of harvested cells to reduce ferric iron. Whole cell protein profiles of Acidiphilium SJH were very similar, regardless of the DO status of the growth medium, while additional proteins were present in A. acidophilum grown microaerobically compared with aerobically-grown cells. CONCLUSIONS: The dissimilatory reduction of ferric iron is constitutive in Acidiphilium SJH while it is inducible in A. acidophilum. SIGNIFICANCE AND IMPACT OF THE STUDY: Ferric iron reduction by Acidiphilium spp. may occur in oxygen-containing as well as anoxic acidic environments. This will detract from the effectiveness of bioremediation systems where removal of iron from polluted waters is mediated via oxidation and precipitation of the metal.