Production and characterization of human renin antibodies with region-oriented synthetic peptides

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.     

Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli

A cosmid gene bank of Vibrio cholerae 395, classical Ogawa, was screened in Escherichia coli HB101 for expression of the vibrio neuraminidase (NANase) gene nanH (N-acylneuraminate glycohydrolase). Positive clones were identified by their ability to cleave the fluorogenic NANase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Seven NANase-positive clones were detected after screening 683 cosmid isolates with a rapid, qualitative plate assay method. The nanH gene was subcloned from one of the cosmids and was located within a 4.8-kilobase-pair BglII restriction endonuclease fragment. Evidence that nanH was the NANase structural gene was obtained by transposon mutagenesis and by purification and comparison of the cloned gene product with the secreted NANase purified from the parent V. cholerae strain. The sequence of the first 20 amino-terminal amino acids of the secreted NANase purified from V. cholerae was determined by automated Edman degradation and matched perfectly with the amino acid sequence predicted from nucleotide sequencing of nanH. The sequence data also revealed the existence of a potential signal peptide that was apparently processed from NANase in both V. cholerae and E. coli. In contrast to V. cholerae, E. coli nanH+ clones did not secrete NANase into the growth medium, retaining most of the enzyme in the periplasmic compartment. Kinetic studies in V. cholerae showed that nanH expression and NANase secretion were temporally correlated as cells in batch culture entered late-exponential-phase growth. Similar kinetics were observed in at least one of the E. coli nanH+ clones, suggesting that nanH expression in E. coli might be controlled by some of the same signals as in the parent V. cholerae strain.     

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[14C]choline and contained [3H]inulin or [125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 microM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 microM), the drug also reduced the internalization, while a high concentration (22 microM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.     

RATES OF FLORAL EVOLUTION: ADAPTATION TO BUMBLEBEE POLLINATION IN AN ALPINE WILDFLOWER,POLEMONIUM VISCOSUM

Animal pollinators are thought to shape floral evolution, yet the tempo of this process has seldom been measured. I used the prediction equation of quantitative genetics, R = h²S , to predict the rate at which a change in pollinator abundance may have caused divergence in floral morphology of the alpine skypilot, Polemonium viscosum. A selection experiment determined the rate at which such divergence can actually proceed. Corolla flare in this species increases by 12% from populations pollinated by a wide assemblage of insect visitors to those pollinated only by bumblebees. To simulate the evolutionary process giving rise to this change, I used a pollinator selection experiment. Plants with broad flowers set significantly more seeds than plants with narrow flowers under bumblebee pollination but had equivalent fecundity when visited by other insects or hand-pollinated. Bumblebee-mediated selection for broad corolla flare intensified from 0.07 at seed set to 0.17 at progeny establishment. Maternal parent-offspring regression yielded a confidence interval of 0.22–1.00 for trait heritability. Given these parameter estimates, the prediction equation shows that broadly flared flowers of bumblebee-pollinated P. viscosum could have evolved from narrower ones in a single generation. This prediction is matched by an observed 9% increase in offspring corolla flare after a single bout of bumblebee-mediated selection, relative to offspring of unselected controls. Findings show that plant populations can adapt rapidly to abrupt changes in pollinator assemblages.     

Lipid-mediated impairment of normal energy metabolism in the isolated perfused diabetic rat heart studied by phosphorus-31 NMR and chemical extraction

The relationship between extracellular palmitate and the accumulation of long-chain fatty-acyl coenzyme A with that of high-energy phosphate metabolism was investigated in the isolated perfused diabetic rat heart. Hearts were perfused with a glucose/albumin buffer supplemented with 0, 0.5, 1.2 or 2.0 mM palmitate. ³¹P-NMR was used to analyze phosphocreatine and ATP metabolism during 1 h of constant-flow recirculation perfusion. At the end of perfusion, frozen samples were taken for chemical analysis of high-energy phosphates and the free and acylated fractions of coenzyme A and carnitine. Perfusion of diabetic hearts with palmitate, unlike control hearts, caused a time-dependent and concentration-dependent reduction in ATP, despite normal and constant phosphocreatine. Concentrations of acid-soluble coenzyme A, long-chain-acyl coenzyme A and total tissue coenzyme A were elevated in palmitate-perfused diabetic hearts, while the total tissue carnitine pool was decreased. Increases in long-chain-acyl coenzyme A correlated with the reduction in myocardial ATP. This reduction in ATP could not be adequately explained by alterations in heart rate, perfusion pressure or vascular resistance.     

Microbial Metal Tolerance in Bermuda Carbonate Sediments

The recovery of aerobic heterotrophic bacteria from Bermuda carbonate sediments on metal-supplemented media varied as much as 44-fold over a 15-cm depth. Distributional relationships with sulfate-reducing bacteria and sediment character indicated that metal tolerance was a function of metal bioavailability.     

Molecular Modelling of the Antagonist Binding Site on the Adenosine A,Receptor

Abstract

With the aid of molecular modelling both adenosine and adenosine A, receptor antagonists belonging to various chemical classes were compared. A model for the antagonist binding site was developed. As a consequence 1H-imidazo[4, 5-c]-quinolin-4-amines were synthesized, constituting a novel class of potent non-xanthine adenosine receptor antagonists.     

Xanthine-7-Ribosides as Adenosine Receptor Antagonists: Further Evidence for Adenosine'S Anti Mode Of Binding

Abstract

With the aid of computer graphics methods, we recently developed a model for the antagonist binding site of the adenosine A₁ receptor (J. Med. Chem. 1990, 33, 1708-1713). According to this model, xanthines should bind to the receptor in a flipped orientation, i.e. the ring atoms N1, N3, N7 and N9 in adenosine coincide with C2, C6, N9 and N7 respectively in theophylline (FIG. la and 1 b). This implicates that the domain where the ribose moiety of adenosine binds must be adjacent to N7 in xanthines, and furthermore that xanthine-7-ribosides should have affinity for the receptor. To further explore the role of the orientation of the ribose moiety in binding to the receptor, we have synthesized and determined the A₁ affinity of the 7-ribosides of theophylline, 13-dipropylxanthine and 1,3-dibutylxanthine (FIG. 1c). The orientation of the ribose moiety was studied with H-NMR spectroscopy and theoretical chemical calculations.     

Chronic vitamin E deficiency promotes vitamin C deficiency in zebrafish leading to degenerative myopathy and impaired swimming behavior

We hypothesized that zebrafish (Danio rerio) undergoing long-term vitamin E deficiency with marginal vitamin C status would develop myopathy resulting in impaired swimming. Zebrafish were fed for 1 y a defined diet without (E ?) and with (E +) vitamin E (500 mg α-tocopherol/kg diet). For the last 150 days, dietary ascorbic acid concentrations were decreased from 3500 to 50 mg/kg diet and the fish sampled periodically to assess ascorbic acid concentrations. The ascorbic acid depletion curves were faster in the E ? compared with E + fish (P < 0.0001); the estimated half-life of depletion in the E ? fish was 34 days, while in it was 55 days in the E + fish. To assess swimming behavior, zebrafish were monitored individually following a “startle-response” stimulus, using computer and video technology. Muscle histopathology was assessed using hematoxylin and eosin staining on paramedian sections of fixed zebrafish. At study end, E ? fish contained 300-fold less α-tocopherol (p < 0.0001), half the ascorbic acid (p = 0.0001) and 3-fold more malondialdehyde (p = 0.0005) than did E + fish. During the first minute following a tap stimulus (p < 0.05), E + fish swam twice as far as did E ? fish. In the E ? fish, the sluggish behavior was associated with a multifocal, polyphasic, degenerative myopathy of the skeletal muscle. The myopathy severity ranged from scattered acute necrosis to widespread fibrosis and was accompanied by increased anti-hydroxynonenal staining. Thus, vitamin E deficiency in zebrafish causes increased oxidative stress and a secondary depletion of ascorbic acid, resulting in severe damage to muscle tissue and impaired muscle function.     

A high density physical map of chromosome 1BL supports evolutionary studies,map-based cloning and sequencing in wheat

Background

As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning.

Results

Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome.

Conclusions

Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing.