Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin. Clamping was maintained for 30 s (normothermia) or 2 min (25 degrees C) after adenovirus administration. Mice were killed 7 or 21 days later, and hearts were examined for EGFP expression. Compared with clamping the aorta alone and with no cooling, gene transfer was increased as follows: 1) 1.3-fold with hypothermia to extend dwell time; 2) 4.5-fold by clamping the aorta and the pulmonary artery; 3) 11.4-fold with nitroprusside administration; 4) 11.8-fold with serotonin addition, and 5) 14.3-fold with acetylcholine delivery. Gene expression remained substantial at 21 days, and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC(VI). Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC(VI) showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and increased left ventricular function after AC(VI) gene transfer.     

A complete library of amino acid alterations at N304 in Streptomyces clavuligerus deacetoxycephalosporin C synthase elucidates the basis for enhanced penicillin analogue conversion

N304 of Streptomyces clavuligerus deacetoxycephalosporin C synthase was mutagenized to alter its catalytic ability. Given that N304A, N304K, N304L, and N304R mutant enzymes exhibited significant improvements in penicillin analogue conversions, we advocate that replacement of N304 with residues with aliphatic or basic side chains is preferable for engineering of a hypercatalytic enzyme.     

Genomic analysis of mutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

When the absorption of light energy exceeds the capacity for its utilization in photosynthesis, regulation of light harvesting is critical in order for photosynthetic organisms to minimize photo-oxidative damage. Thermal dissipation of excess absorbed light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is induced rapidly in response to excess light conditions, and it is known that xanthophylls such as zeaxanthin and lutein, the transthylakoid pH gradient, and the PsbS protein are involved in this mechanism. Although mutants affecting NPQ and the biosynthesis of zeaxanthin and lutein were originally isolated and characterized at the physiological level in the unicellular green alga Chlamydomonas reinhardtii, the molecular basis of several of these mutants, such as npq1 and lor1, has not been determined previously. The recent sequencing of the C. reinhardtii nuclear genome has facilitated the search for C. reinhardtii homologs of plant genes involved in xanthophyll biosynthesis and regulation of light harvesting. Here we report the identification of C. reinhardtii genes encoding PsbS and lycopene ɛ-cyclase, and we show that the lor1 mutation, which affects lutein synthesis, is located within the lycopene ɛ-cyclase gene. In contrast, no homolog of the plant violaxanthin de-epoxidase (VDE) gene was found. Molecular markers were used to map the npq1 mutation, which affects VDE activity, as a first step toward the map-based cloning of the NPQ1 gene.     

Effect of Cryptobia salmositica-induced anorexia on feeding behavior and immune response in juvenile rainbow trout Oncorhynchus mykiss

At 10 degrees C, rainbow trout Oncorhynchus mykiss (n = 13 per group) infected with Cryptobia salmositica Katz, 1951 became anorexic at 3 wk post-infection (w.p.i.), with feed-intake decreasing significantly from 1.33 to 0.94% body weight (b.w.). Anorexia was most severe at 4 w.p.i. (0.80% b.w.), coinciding with peak parasitemia (9.2 x 10(6) parasites ml blood(-1)) and anemia. At 8 w.p.i., fish had recovered their appetite although they still had contained detectable parasites (6.8 x 10(5) parasites ml(-1)) and were anemic (pack cell volume, PCV, of 24.4%). However at 5 degrees C, anorexia occurred at 5 w.p.i. (0.81% b.w.), and was most severe at 7 w.p.i. (0.40% b.w.). At 8 w.p.i. (0.43% b.w.), fish displayed high parasitemia (4.6 x 10(6) parasites ml(-1)) and low PCV (10.8%). Fish at 5 degrees C had lower gastric evacuation (GE) rates (GE48h) than 10 degrees C fish, however there were no differences between infected and naive fish at both temperatures. Before anorexia, there was no significant correlation between mean share of meal (MSM, a measure of how food was partitioned within a group) and coefficient of variation in feeding but this became significant during anorexia (p = 0.02 and p = 0.0002 at 10 and 5 degrees C respectively). Significant correlations were detected between b.w. and MSM before onset of anorexia at 10 degrees C (p = 0.005) and 5 degrees C (p = 0.02); this was maintained at 10 degrees C (p = 0.001) but not at 5 degrees C (p = 0.98). Fish on an anorexic diet (0.93% b.w.) responded well at 10 degrees C to a live C. salmositica vaccine; this could partly be due to constant antigenic stimulation by the live vaccine.     

Pericardial and pericardioperitoneal canal relationships to cardiac function in the white sturgeon (Acipenser transmontanus)

Sturgeons are primitive bony fishes and their hearts have structural features found in other primitive fishes. Sturgeons have a pericardioperitoneal canal (PPC), a one-way conduit into the peritoneum. A PPC also occurs in elasmobranchs (sharks and rays) and studies with that group demonstrate that pericardial pressure and pericardial fluid loss via the PPC affect stroke volume. A study of white sturgeon (Acipenser transmontanus) heart function was conducted to test for a comparable PPC and pericardial effects. White sturgeon-elasmobranch heart-function similarities include biphasic ventricular filling, a comparable operational pericardial pressure (-0.03 kPa), and a strongly negative pressure (-0.2 to -0.6 kPa) with complete pericardial fluid withdrawal. Differences include the white sturgeon's relatively smaller atrium and ventricle but a larger conus arteriosus. Although white sturgeon heart size is also smaller, its pericardial volume is disproportionately less (2.4 to 2.7 vs. 3.5 to 5.4 ml kg(-1) in elasmobranchs), meaning it has less scope for increasing stroke volume upon PPC fluid release. These differences may reflect the phylogenetic progression from the less complex operation of the elasmobranch heart, which lacks sympathetic innervation and has a mechanically mediated (PPC) stroke volume, to the condition in the more derived bony fishes which have sympathetic and parasympathetic regulation of both stroke volume and heart rate.     

Investigations of oocyte in vitro maturation within a mouse model

This study attempted to develop a 'less meiotically competent' murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze-thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.     

Increasing terrestrial vegetation activity in China, 1982–1999

Variations in vegetation activity during the past 18 years in China were investigated using the normalized difference vegetation index (NDVI) derived from the 3rd generation time series dataset of NOAA-AVHRR from 1982 to 1999. In order to eliminate the effects of non-vegetation factors, we characterized areas with NDVI < 0.1 as “sparsely vegetated areas” and areas with NDVI ≥0.1 as “vegetated areas”. The results showed that increasing NDVI trends were evident, to varying extents, in almost all regions in China in the 18 years, indicating that vegetation activity has been rising in recent years in these regions. Compared to the early 1980s, the vegetated area increased by 3.5% by the late 1990s, while the sparsely vegetated area declined by 18.1% in the same period. The national total mean annual NDVI increased by 7.4% during the study period. Extended growing seasons and increased plant growth rates accounted for the bulk of these increases, while increases in temperature and summer rainfall, and strengthening agricultural activity were also likely important factors. NDVI changes in China exhibited relatively large spatial heterogeneity; the eastern coastal regions experienced declining or indiscernibly rising trends, while agricultural regions and western China experienced marked increases. Such a pattern was due primarily to urbanization, agricultural activity, regional climate characteristics, and different vegetation responses to regional climate changes.     

Unique actinomycin D binding to self-complementary d(CXYGGCCY'X'G) sequences: duplex disruption and binding to a nominally base-paired hairpin

Actinomycin D (ACTD) has been shown to bind weakly to the sequence -GGCC-, despite the presence of a GpC site. It was subsequently found, however, that d(CATGGCCATG) binds relatively well to ACTD but exhibits unusually slow association kinetics, contrary to the strong-binding -XGCY- sites. In an effort to elucidate the nature of such binding and to delineate the origin of its interesting kinetic behavior, studies have now been extended to include oligomers with the general sequence motifs of d(CXYGGCCY′X′G)₂. It was found that analogous binding characteristics are observed for these self-duplex decamers and comparative studies with progressively base-truncated oligomers from the 5′-end led to the finding that d(GGCCY′X′G) oligomers bind ACTD considerably stronger than their parent decamers and exhibit 1:1 drug/strand binding stoichiometry. Melting profiles monitored at the drug spectral region indicated additional drug binding prior to the onset of eventual complex disruptions with near identical melting temperatures for all the oligomers studied. These results are consistent with the notion that the related oligomers share a common strong binding mode of a hairpin-type, with the 3′-terminus G folding back to base-pair with the C base of GGC. A binding scheme is proposed in which the oligomers d(CXYGGCCY′X′G) exist predominantly in the duplex form and bind ACTD initially at the central GGCC weak site but subsequently disrupt to accommodate the stronger hairpin binding and thus the slow association kinetics. Such a mechanism is supported by the observation of distinct biphasic fluorescence kinetic traces in the binding of 7-amino-ACTD to these duplexes.