Novel Roles for Kv7 Channels in Shaping Histamine-Induced Contractions and Bradykinin-Dependent Relaxations in Pig Coronary Arteries

Voltage-gated K_v7 channels are inhibited by agonists of G_q-protein-coupled receptors, such as histamine. Recent works have provided evidence that inhibition of vascular K_v7 channels may trigger vessel contractions. In this study, we investigated how K_v7 activity modulates the histamine-induced contractions in “healthy” and metabolic syndrome (MetS) pig right coronary arteries (CAs). We performed isometric tension and immunohistochemical studies with domestic, lean Ossabaw, and MetS Ossabaw pig CAs. We found that neither the K_v7.2/K_v7.4/K_v7.5 activator ML213 nor the general K_v7 inhibitor XE991 altered the tension of CA rings under preload, indicating that vascular K_v7 channels are likely inactive in the preloaded rings. Conversely, ML213 potently dilated histamine-pre-contracted CAs, suggesting that K_v7 channels are activated during histamine applications and yet partially inhibited by histamine. Immunohistochemistry analysis revealed strong K_v7.4 immunostaining in the medial and intimal layers of the CA wall, whereas K_v7.5 immunostaining intensity was strong in the intimal but weak in the medial layers. The medial K_v7 immunostaining was significantly weaker in MetS Ossabaw CAs as compared to lean Ossabaw or domestic CAs. Consistently, histamine-pre-contracted MetS Ossabaw CAs exhibited attenuated ML213-dependent dilations. In domestic pig CAs, where medial K_v7 immunostaining intensity was stronger, histamine-induced contractions spontaneously decayed to ~31% of the peak amplitude within 4 minutes. Oppositely, in Ossabaw CAs, where K_v7 immunostaining intensity was weaker, the histamine-induced contractions were more sustained. XE991 pretreatment significantly slowed the decay rate of histamine-induced contractions in domestic CAs, supporting the hypothesis that increased K_v7 activity correlates with a faster rate of histamine-induced contraction decay. Alternatively, XE991 significantly decreased the amplitude of bradykinin-dependent dilations in pre-contracted CAs. We propose that in CAs, a decreased expression or a loss of function of K_v7 channels may lead to sustained histamine-induced contractions and reduced endothelium-dependent relaxation, both risk factors for coronary spasm.     

Kin Discrimination in a Macropod Marsupial

Differential treatment of kin is ubiquitous in social animals. Parents often behave preferentially towards their dependent offspring. Species in several taxa also bias behaviour towards non-descendent kin. This latter phenomenon has not been demonstrated in marsupials, which are reportedly less social than eutherian mammals. We report the first evidence of non-parental kin-biased behaviour in a macropodid marsupial. Experimental pairing of individuals based on kinship reliably altered the rate of aggression between individuals in pairs of female tammar wallabies ( Macropus eugenii ). This effect is probably attributable to relatedness rather than to familiarity. Marsupial sociality may be substantially more complex than is currently recognized.     

Physiological responses of Sargasso Sea picoplankton to nanomolar nitrate perturbations

A study was conducted in July 1989 at three stations in thenorthern Sargasso Sea, where picoplankton (<1 µm)provided approximately half of the standing crop of chlorophyll.Temporal changes in the position of the nitracline at a singlelocation indicated that the vertical supply of nitrate was notat ‘steady-state’ and phytoplankton distributionstracked the nitracline. Our main experimental objective wasto examine the short-term effects of ecologically significantnitrate perturbations (+20 and +100 nM) on the physiologyof <1 µm communities growing at low (nanomolar)ambient nitrate concentrations. A chemiluminescent nitrate methodwas used to measure the time course (up to 4 h) of nitratedisappearance at in situ irradiance, in parallel with measurementsof photosynthetic ¹⁴CO₂ assimilation. Picoplankton growing at<60 nM nitrate rapidly responded to nanomolar nitratesupplements with luxury consumption and enhanced photosynthesisin proportion to their ambient nitrate environment. Light-saturatedSynechococcus populations from the most nitrate-depleted waters(13 nM) had doubled their cellular rate of photosynthesisafter 4 h, in response to a 20 nM nitrate pulse.     

Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.     

Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation

We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.     

Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.