Protein kinase C modulation of ventilatory response to hypoxia in nucleus tractus solitarii of conscious rats

This study aimedto determine the role of protein kinase C (PKC) in signal transductionmechanisms underlying ventilatory regulation in the nucleus tractussolitarii (NTS). Microinjection of phorbol 12-myristate 13-acetate intothe commissural NTS of nine chronically instrumented, unrestrained ratselicited significant cardiorespiratory enhancements that lasted for atleast 4 h, whereas administration of vehicle(n = 15) or the inactive phorbol ester 4-phorbol 12,13-didecanoate (n = 7)did not elicit minute ventilation (E)changes. Peak hypoxic Eresponses (10% O₂-balanceN₂) were measured in 19 additional animals after NTS microinjection of bisindolylmaleimide(BIM) I, a selective PKC inhibitor (n = 12), BIM V (inactive analog; n = 7),or vehicle (Con; n = 19). In Con,E increased from 139 ± 9 to 285 ± 26 ml/min in room air and hypoxia, respectively, and similarresponses occurred after BIM V. BIM I did not affect room airE but markedly attenuated hypoxia-induced E increases (128 ± 12 to 167 ± 18 ml/min; P < 0.02 vs. Con and BIM V). When BIM I was microinjected into the cerebellum(n = 4), cortex(n = 4), or spinal cord(n = 4),E responses were similar to Con.Western blots of subcellular fractions of dorsocaudal brain stemlysates revealed translocation of PKC, , , , , and  isoenzymes during acute hypoxia, and enhanced overall PKC activity wasconfirmed in the particulate fraction of dorsocaudal brain stem lysatesharvested after acute hypoxia. These studies suggest that, in the adultrat, PKC activation in the NTS mediates essential components of theacute hypoxic ventilatory response.

     

NMDA receptors mediate peripheral chemoreceptor afferent input in the conscious rat

N-methyl-D-aspartate(NMDA) glutamate receptors mediate critical components ofcardiorespiratory control in anesthetized animals. The role of NMDAreceptors in the ventilatory responses to peripheral and centralchemoreceptor stimulation was investigated in conscious, freelybehaving rats. Minute ventilation(E)responses to 10% O₂, 5%CO₂, and increasing intravenousdoses of sodium cyanide were measured in intact rats before and afterintravenous administration of the NMDA receptor antagonist MK-801 (3 mg/kg). After MK-801, eupcapnic tidal volume(VT) decreased while frequencyincreased, resulting in a modest reduction inE.Inspiratory time (TI) decreased, whereas expiratory time remained unchanged. TheE responsesto hypercapnia were qualitatively similar in control and MK-801conditions, with slight reductions in respiratory drive (VT/TI)after MK-801. In contrast, responses to hypoxia were markedly attenuated after MK-801 and were primarily due to reduced frequency changes, whereas VT wasunaffected. Sodium cyanide doses associated with significantEincreases were 5 and 50 µg/kg before and after MK-801,respectively. Thus 1-log shift to the right of individual dose-responsecurves occurred with MK-801. Selective carotid body denervation reducedE duringhypoxia by 70%, and residual hypoxic ventilatory responses wereabolished after MK-801. These findings suggest that, in conscious rats,carotid and other peripheral chemoreceptor-mediated hypoxic ventilatoryresponses are critically dependent on NMDA receptor activation and thatNMDA receptor mechanisms are only modestly involved during hypercapnia.

     

The Glyoxylate Cycle in an Arbuscular Mycorrhizal Fungus. Carbon Flux and Gene Expression

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of (13)C labeling of germinating spores and extraradical mycelium with (13)C(2)-acetate and (13)C(2)-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle.     

Nitrogen nutrition and regulation of cephalosporin production in Streptomyces clavuligerus.

When used as sole nitrogen source, certain amino acids (e.g., proline, asparagine) supported both growth and sporulation by Streptomyces clavuligerus streaked onto solid defined medium. Ammonium supported growth but suppressed sporulation. Amino nitrogen was best for cephalosporin production in liquid defined medium, although urea was almost as useful. A comparison of amino acids showed asparagine and glutamine to be the best nitrogen sources and arginine to be almost as good. Ammonium salts supported a somewhat lower growth rate than asparagine, but antibiotic production was very poor on these inorganic nitrogen sources. Addition of ammonium to asparagine did not affect growth rate but increased mycelial mass; cephalosporin production was reduced by about 75%. Antibiotic production was more closely associated with growth in the absence of ammonium than in its presence, indicating a strong inhibitory and (or) repressive effect of NH4+ on antibiotic production. Ammonium exerted its negative effect when added at 24h or earlier, i.e. before antibiotic formation began.     

Genetic recombination in the cephamycin producer,Streptomyces clavuligerus

Summary In order to study the mechanism of cephamycin production by streptomycetes and to use genetic recombination in strain development, we undertook genetic studies inStreptomyces lipmanii andS. clavuligerus. S. lipmanii crosses gave 0.005–1.3% prototroph-like colonies, but all segregated back to parental genotypes. Crosses ofS. clavuligerus resulted in lower frequencies of prototroph-like colonies, i.e., 0.00002–0.9%. In ade x ura and ade x his crosses, the recombinant progeny did not segregate back. In arg x ade and arg x his crosses, segregation occurred in about 50% of the progeny. These data demonstrate that true haploid recombinants occur in crosses ofS. clavuligerus. S. lipmanii yielded only heterokaryons and, therefore, is less suitable thanS. clavuligerus for further genetic study.     

Application of in vitro methods to study carbon uptake and transport by AM fungi

Just as multi-compartmented root chambers have advantages over standard plastic pots for the study of nutrient uptake by arbuscular mycorrhizal [AM] fungi in soil, so the split-plate in vitro system has advantages over the standard dual culture system for the study of the physiology of AM fungi. We used the split-plate culture system of Ri T-DNA transformed Daucus carota L. roots and Glomus intraradices Schenck & Smith, in which only the fungus has access to the distal compartment, to study the ability of germ tubes and extraradical and intraradical hyphae to take up ¹³C-labeled substrates. Labeled substrates were added to one side of the plate divider and plates were incubated for 8 weeks while the fungus proliferated on the side from which the root was excluded. Tissues then were recovered from the plate and examined via NMR spectroscopy. Results showed that the morphological phases of the fungus differed in their ability to take up these substrates, most notably that intraradical hyphae take up hexose while extraradical hyphae cannot. In addition, NMR studies indicated that intraradical hyphae actively synthesized lipids while extraradical hyphae did not. These data show that eventual axenic culture of AM fungi is more than a matter of finding the proper substrate for growth. Genetic regulation must be overcome to make extraradical hyphae behave like intraradical hyphae in terms of C uptake and metabolism. This revised version was published online in June 2006 with corrections to the Cover Date.