Dynamics of the ventilatory response to central hypoxia in cats

The dynamics of the effect of central hypoxia on ventilation were investigated by the technique of artificial perfusion of the brain stem in alpha-chloralose-urethan-anesthetized cats. A two-channel roller pump and a four-way valve allowed switching the gas exchanger into and out of the extracorporeal circuit which controlled the brain stem perfusion. When isocapnic hypoxia (arterial PO2 range 18-59 Torr) was limited to the brain stem, a decline in ventilation was consistently found. In 12 cats 47 steps into and 48 steps out of central hypoxia were made. The ventilatory response was fitted using least squares with a model that consisted of a latency followed by a single-exponential function. The latencies for the steps into and out of hypoxia were not significantly different (P = 0.14) and were 32.3 +/- 4.0 and 25.1 +/- 3.6 (SE) s, respectively. The time constant for the steps into hypoxia (149.7 +/- 8.5 s) was significantly longer (P = 0.0002) than for the steps out of hypoxia (105.5 +/- 10.1 s). The time constants for the increase and decrease in ventilation after step changes in the central arterial PCO2 found in a previous study (J. Appl. Physiol. 66: 2168-2172, 1989) were not significantly different (P greater than 0.2) from the corresponding time constants in this study (for 7 cats common to both studies). Theories of the mechanisms behind hypoxic ventilatory decline need to account for the long latency, the similarity between the time constants for the ventilatory response to O2 and CO2, and the differences between the time constants for increasing and decreasing ventilation.     

THE GENUS PIKEA (DUMONTIACEAE,RHODOPHYTA) IN ENGLAND AND THE NORTH PACIFIC: COMPARATIVE MORPHOLOGICAL,LIFE HISTORY,AND MOLECULAR STUDIES1

A Pikea species attributed to Pikea californica Harvey has been established in England since at least 1967. Previously, this species was believed to occur only in Japan and Pacific North America. Comparative morphological studies on field-collected material and cultured isolates from England, California, and Japan and analysis of organellar DNA restriction fragment length polymorphisms, detected using labeled organellar DNA as a non-radioactive probe, showed that English Pikea is conspecific with P. californica from California. Both populations consist of dioecious gametophytes with heteromorphic life histories involving crustose tetrasporophytes; 96% of organellar DNA bands were shared between interoceanic samples. A second dioecious species of Pikea, P. pinnata Setchell in Collins, Holden et Setchell, grows sympatrically with P. californica near San Francisco but can be distinguished by softer texture, more regular branching pattern, and elongate cystocarpic axes. Pikea pinnata and P. californica samples shared 49–50% of organellar DNA bands, consistent with their being distinct species. Herbarium specimens of P. robusta Abbott resemble P. pinnata in some morphological features but axes are much wider; P. robusta may represent a further, strictly sub-tidal species but fertile material is unknown. Pikea thalli from Japan, previously attributed to P. californica and described here as Pikea yoshizakii sp. nov., are monoecious and show a strikingly different type of life history. After fertilization, gonimoblast filaments grow outward through the cortex and form tetrasporangial nemathecia; released tetraspores develop directly into erect thalli. Tetrasporoblastic life histories are characteristic of certain members of the Phyllophoraceae but were previously unknown in the Dumontiaceae. Japanese P. yoshizakii shared 55 and 56% of organellar DNA bands with P. californica and P. pinnata, respectively; phylogenetic analysis indicated equally distant relationships to both species. Pikea yoshizakii or a closely similar species with the same life history occurs in southern California and Mexico.     

Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts

A recent study determined that cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts degraded angiotensins and kinins via neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). Due to the possible importance of other peptides to skeletal muscle blood flow and function, the present study looked specifically at the metabolism of the neurokinins substance P (SP) and neurokinin A (NKA) by skeletal muscle peptidases. The results show that SP is degraded not only by NEP-24.11, but also sequentially by dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5)/APN. NKA is unaffected by DAP IV but is metabolized by NEP-24.11 and APN. NEP-24.11 was inhibited by phosphoramidon (IC₅₀ = 80 nM), thiorphan and ZINCOV, DAP IV by diprotin A (IC₅₀ = 8 μM), and APN by amastatin (IC₅₀ = 50 nM) and bestatin (IC₅₀ = 100 μM). Skeletal muscle myocyte and fibroblast metabolism of SP and NKA may regulate local skeletal muscle vascular and extravascular functions including SP- and NKA-mediated nerve-induced vasodilation. Inhibition of both NEP-24.11 and DAP IV/APN may increase skeletal muscle blood flow and decrease peripheral vascular resistance via potentiation of local neurokinin levels.     

Nitrification and denitrification: Probing the nitrogen cycle in aquatic environments

Methods designed to detect microorganisms involved in the biogeochemistry of nitrogen in the marine environment are rapidly being developed and deployed in ecological investigations. Probes based on phylogenetic sequences (usually rRNA) and those based on the sequences of functional genes or proteins have both been demonstrated in the nitrogen cycle. The most progress has been made for ammonia oxidizers; several sets of PCR primers have been described and their specificity may be optimized to allow detection of genetically and ecologically meaningful groups. For denitrifying bacteria, functional probes based on nitrite reductase show most promise. These approaches should complement the more familiar, but no less sophisticated, methods that focus on quantification of in situ transformation rates. Both approaches in combination will be useful in understanding regulation and environmental control of biogeochemical processes. Correspondence to: B.B. Ward     

Type IIS restriction enzyme footprinting I. Measurement of a triple helix dissociation constant with Eco57I at 25 degrees C.

A method is described to measure triple helix dissociation constants by inhibiting the cleavage of a plasmid constructed to contain a target sequence for the triplex forming oligonucleotide (TFO) dT20 by the type IIS restriction enzyme Eco57I. The method relies upon the TFO's ability to block the cleavage reaction by occupying the enzymes cleavage site but not its specific binding sequence. Using this protocol, the dissociation constant for dT20 bound to its target was 0.16 +/- 0.01 microM at 25 degrees C. The accuracy of this experiment was demonstrated by measuring the Kd of an affinity cleavage TFO using Eco57I and Quantitative Affinity Cleavage Titration. Type IIS restriction endonuclease footprinting should be useful for the qualitative and quantitative investigation of ligand-DNA interactions.     

Cultivation of aerobic chemoorganotrophic proteobacteria and gram-positive bacteria from a hot spring microbial mat.

The diversity of aerobic chemoorganotrophic bacteria inhabiting the Octopus Spring cyanobacterial mat community (Yellowstone National Park) was examined by using serial-dilution enrichment culture and a variety of enrichment conditions to cultivate the numerically significant microbial populations. The most abundant bacterial populations cultivated from dilutions to extinction were obtained from enrichment flasks which contained 9.0 x 10(2) primary producer (Synechococcus spp.) cells in the inoculum. Two isolates exhibited 16S rRNA nucleotide sequences typical of beta-proteobacteria. One of these isolates contained a 16S rRNA sequence identical to a sequence type previously observed in the mat by molecular retrieval techniques. Both are distantly related to a new sequence directly retrieved from the mat and contributed by a beta-proteobacterial community member. Phenotypically diverse gram-positive isolates genetically similar to Bacillus flavothermus were obtained from a variety of dilutions and enrichment types. These isolates exhibited identical 16S rRNA nucleotide sequences through a variable region of the molecule. Of the three unique sequences observed, only one had been previously retrieved from the mat, illustrating both the inability of the cultivation methods to describe the composition of a microbial community and the limitations of the ability of molecular retrieval techniques to describe populations which may be less abundant in microbial communities.     

Photosynthate Partitioning and Fermentation in Hot Spring Microbial Mat Communities

Patterns of (sup14)CO(inf2) incorporation into molecular components of the thermophilic cyanobacterial mat communities inhabiting hot springs located in Yellowstone National Park and Synechococcus sp. strain C1 were investigated. Exponentially growing Synechococcus sp. strain C1 partitioned the majority of incorporated (sup14)CO(inf2) into protein, low-molecular-weight metabolites, and lipid fractions (45, 22, and 18% of total incorporated carbon, respectively). In contrast, mat cores from various hot springs predominantly accumulated polyglucose during periods of illumination (between 77 and 85% of total incorporated (sup14)CO(inf2)). Although photosynthetically active, mat photoautotrophs do not appear to be rapidly growing, since we also detected only limited synthesis of macromolecules associated with growth (i.e., protein and rRNA). To test the hypothesis that polysaccharide reserves are fermented in situ under the dark anaerobic conditions cyanobacterial mats experience at night, mat cores were prelabeled with (sup14)CO(inf2) under illuminated conditions and then transferred to dark anaerobic conditions. Radiolabel in the polysaccharide fraction decreased by 74.7% after 12 h, of which 58.5% was recovered as radiolabeled acetate, CO(inf2), and propionate. These results indicate tightly coupled carbon fixation and fermentative processes and the potential for significant transfer of carbon from primary producers to heterotrophic members of these cyanobacterial mat communities.