Adenosine and the acid-base state of vascular smooth muscle

Hog carotid artery media was incubated under conditions of normocapnia (95% O2-5% CO2) and hypercapnia (nominally 75% O2-25%CO2). The intracellular pH (pHi) was determined from the distribution of 14C-labeled 5,5-dimethyloxazoladine-2,4-dione, alpha- and beta-receptor antagonists were used to block the effects of endogenous catecholamines. With 5% CO2, adenosine had no effect on the pHi. High K+ (25mM) and dipyridamole (DPM) induced a cellular metabolic acidosis that was reversed by adenosine and not affected by 0.5 mM ca2+ or ouabain. Hypercapnia decreased the resting pHi from 7.30 to 6.79. Adenosine significantly attenuated this decrease. With high K+ or DPM, a similar degree of hypercapnia only depressed the pHi to 6.91 and 6.90, respectively. The alkalinizing effect of high K+ and DPM was not altered by 0.5 mM Ca2+, was partically reversed by ouabain, and was completely reversed by adenosine. These results suggest that, under normocapnic conditions, although adenosine relaxes the contraction associated with K+-depolarization, it does not do so by elevating cellular proton levels. However, adenosine may decrease a tissue's ability to attenuate a local respiratory acidosis characteristic of increased O2 demand, resulting in relaxation under hypercapnic conditions. In any case, this demonstrates an interaction, with respect to the acid-base state of the vascular smooth muscle cells, among adenosine, K+, and H+, all suggested components of the metabolic theory of blood flow autoregulation.     

Thrombin's enzymatic activity increases permeability of endothelial cell monolayers

Human alpha-thrombin increases the permeability of bovine pulmonary artery endothelial cell (CCL-209) monolayers. To determine if this increase is via an enzymatic or receptor-mediated mechanism, enzymatically active forms of alpha-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included alpha-thrombin and two digestion products, zeta-thrombin (chymotryptic product with 89% clotting activity) and gamma-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) alpha-thrombin and diisopropylphosphorofluoridate-(DIP) alpha-thrombin. Cell binding activity of alpha- and PPACK-alpha-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-alpha-thrombin. gamma-Thrombin, on the other hand, did not compete for binding of 125I-labeled alpha-thrombin. All enzymatic forms of alpha-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with alpha- or gamma-thrombin prevented the increase in permeability, further indicating that alpha-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of alpha-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of alpha-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion-binding site of alpha-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Nucleic Acid Blot Analyzer II. ANALYZE, an image analysis software package for molecular biology

The Nucleic Acid Blot Analyzer, a new instrument providing high-speed imaging of 32P labeled nucleic acids, captures, stores and presents images in digital form, thus lending itself to rapid data handling and analysis as well as replacing conventional X-ray film autoradiography for many applications. A software package called ANALYZE has been specifically designed for the instrument in order to provide automatic or semi-automatic analysis for molecular biological techniques. The software includes image display manipulation, quantitative and positional analysis, as well as file maintenance utilities. The specific application of the software/hardware to various techniques is presented.     

Cotransformation of temperature sensitivity and nutritional markers in Neisseria gonorrhoeae.

Cotransformation remains the only tool for establishing linkage in Neisseria gonorrhoeae. Because of the difficulty of inducing auxotrophic markers via mutagenesis in this species, most previous studies have utilized antibiotic resistance and naturally occurring (auxotypic) auxotrophic markers. We have succeeded in isolating auxotrophic and temperature-sensitive mutants. The temperature-sensitive mutants have been characterized by their growth on complex and defined media at 31, 37, and 40 degrees C. Two of the mutants exhibited an unusual pattern of temperature sensitivity--growth on the defined medium but absence of growth on the complex medium at 37 degrees C. Both mutants, however, were temperature-sensitive on the two media at 40 degrees C. We have demonstrated linkages between markers isolated in our laboratory and the auxotypic markers of the clinical isolate RUG208. Ts-2 exhibited 85 to 95% linkage to Arg- and his-2 exhibited 40% linkage to Val-. In addition weak linkages were shown between his-2 and Arg- (2 to 6%) and between Arg- and Val- (3 to 5%). Linkages among his-2, Arg-, and Val- which could be demonstrated when deoxyribonucleic acid from strain F62 was used to transform RUG208 were absent when F62 was used as recipient for RUG208 DNA. Our data are consistent with a tentative map order of his-2, Val-, Arg-, Ts-2.     

Barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)-induced growth inhibition of algae: a review

Many field and laboratory studies have attempted to explain the inhibitory effect of rotting barley on algae. Early field studies lacked controls and replication and results depended on visual observations. Such studies offer information on barley bale field construction and application rates. In the laboratory, discrepancies in the barley variety used, algal species tested, barley liquor preparation and phenol extraction methodologies existed. Inconsistencies have led to different growth responses for the same species of algae tested, i.e. with some studies finding an inhibitory response and other studies reporting an accelerated growth response of algae. Two successful forms of investigation have been identified: (a) using commercially available compounds, i.e. with known shikimate-pathway-producing phenols and acids, which can then be combined with algal assays of different algal species and (b) using commercially available algal species from which batch cultures are grown, which are then added to barley liquor of different ages. Algal growth may then be investigated using in vivo fluorescence and the filtrate can be analysed via HPLC/MS. The identification of allelochemicals, which range from phenolics to quinones within the Poaceae family of which barley is a member, has received a lot of attention in recent years.