Cardiopulmonary baroreflex is reset during dynamic exercise.

The purpose of this study was to examine the hypothesis that the operating point of the cardiopulmonary baroreflex resets to the higher cardiac filling pressure of exercise associated with the increased cardiac filling volumes. Eight men (age 26 +/- 1 yr; height 180 +/- 3 cm; weight 86 +/- 6 kg; means +/- SE) participated in the present study. Lower body negative pressure (LBNP) was applied at 8 and 16 Torr to decrease central venous pressure (CVP) at rest and during steady-state leg cycling at 50% peak oxygen uptake (104 +/- 20 W). Subsequently, two discrete infusions of 25% human serum albumin solution were administered until CVP was increased by 1.8 +/- 0.6 and 2.4 +/- 0.4 mmHg at rest and 2.9 +/- 0.9 and 4.6 +/- 0.9 mmHg during exercise. During all protocols, heart rate, arterial blood pressure, and CVP were recorded continuously. At each stage of LBNP or albumin infusion, forearm blood flow and cardiac output were measured. During exercise, forearm vascular conductance increased from 7.5 +/- 0.5 to 8.7 +/- 0.6 U (P = 0.024) and total systemic vascular conductance from 7.2 +/- 0.2 to 13.5 +/- 0.9 l.min(-1).mmHg(-1) (P < 0.001). However, there was no significant difference in the responses of both forearm vascular conductance and total systemic vascular conductance to LBNP and the infusion of albumin between rest and exercise. These data indicate that the cardiopulmonary baroreflex had been reset during exercise to the new operating point associated with the exercise-induced change in cardiac filling volume.     

Enzymatic properties of human aminopeptidase A. Regulation of its enzymatic activity by calcium and angiotensin IV

Aminopeptidase A (APA) is a type II membrane-bound protein implicated in the regulation of blood pressure in the brain renin-angiotensin system. In this study, a recombinant soluble form of APA was expressed in a baculovirus system, purified to homogeneity, and characterized. By using synthetic substrates, it was shown that although the enzyme has a rather broad substrate specificity in the absence of Ca2+, the preferential release of acidic amino acid residues was observed in the presence of Ca2+. Moreover, Ca2+ up- or down-regulated the enzymatic activity depending on the substrate. By searching for natural substrates of APA, we found that peptides having acidic amino acids at their N terminus (angiotensin II, neurokinin B, cholecystokinin-8, and chromogranin A) were cleaved by the enzyme efficiently in the presence but not in the absence of Ca2+. Moreover kallidin (Lys-bradykinin) was converted to bradykinin effectively only in the absence of Ca2+. These results suggest that Ca2+ increases the preference of the enzyme for the peptide substrates having N-terminal acidic amino acids. In addition, we found that angiotensin IV could bind to APA both in the presence and absence of Ca2+ and inhibited the enzymatic activity of APA competitively, suggesting that angiotensin IV acts as a negative regulator of the enzyme once generated from angiotensin II by the serial actions of aminopeptidases. Taken together, these results suggest that there exists a complex regulation of the enzymatic activity of APA, which may contribute to homeostasis such as regulation of blood pressure, maintenance of memory, and normal pregnancy by controlling the concentrations of peptide substrates.     

Prediction of operon-like gene clusters in the Arabidopsis thaliana genome based on co-expression analysis of neighboring genes

Operon-like arrangements of genes occur in eukaryotes ranging from yeasts and filamentous fungi to nematodes, plants, and mammals. In plants, several examples of operon-like gene clusters involved in metabolic pathways have recently been characterized, e.g. the cyclic hydroxamic acid pathways in maize, the avenacin biosynthesis gene clusters in oat, the thalianol pathway in Arabidopsis thaliana, and the diterpenoid momilactone cluster in rice. Such operon-like gene clusters are defined by their co-regulation or neighboring positions within immediate vicinity of chromosomal regions. A comprehensive analysis of the expression of neighboring genes therefore accounts a crucial step to reveal the complete set of operon-like gene clusters within a genome. Genome-wide prediction of operon-like gene clusters should contribute to functional annotation efforts and provide novel insight into evolutionary aspects acquiring certain biological functions as well. We predicted co-expressed gene clusters by comparing the Pearson correlation coefficient of neighboring genes and randomly selected gene pairs, based on a statistical method that takes false discovery rate (FDR) into consideration for 1469 microarray gene expression datasets of A. thaliana. We estimated that A. thaliana contains 100 operon-like gene clusters in total. We predicted 34 statistically significant gene clusters consisting of 3 to 22 genes each, based on a stringent FDR threshold of 0.1. Functional relationships among genes in individual clusters were estimated by sequence similarity and functional annotation of genes. Duplicated gene pairs (determined based on BLAST with a cutoff of E<10(-5)) are included in 27 clusters. Five clusters are associated with metabolism, containing P450 genes restricted to the Brassica family and predicted to be involved in secondary metabolism. Operon-like clusters tend to include genes encoding bio-machinery associated with ribosomes, the ubiquitin/proteasome system, secondary metabolic pathways, lipid and fatty-acid metabolism, and the lipid transfer system.     

KNApSAcK family databases: integrated metabolite-plant species databases for multifaceted plant research

A database (DB) describing the relationships between species and their metabolites would be useful for metabolomics research, because it targets systematic analysis of enormous numbers of organic compounds with known or unknown structures in metabolomics. We constructed an extensive species-metabolite DB for plants, the KNApSAcK Core DB, which contains 101,500 species-metabolite relationships encompassing 20,741 species and 50,048 metabolites. We also developed a search engine within the KNApSAcK Core DB for use in metabolomics research, making it possible to search for metabolites based on an accurate mass, molecular formula, metabolite name or mass spectra in several ionization modes. We also have developed databases for retrieving metabolites related to plants used for a range of purposes. In our multifaceted plant usage DB, medicinal/edible plants are related to the geographic zones (GZs) where the plants are used, their biological activities, and formulae of Japanese and Indonesian traditional medicines (Kampo and Jamu, respectively). These data are connected to the species-metabolites relationship DB within the KNApSAcK Core DB, keyed via the species names. All databases can be accessed via the website http://kanaya.naist.jp/KNApSAcK_Family/. KNApSAcK WorldMap DB comprises 41,548 GZ-plant pair entries, including 222 GZs and 15,240 medicinal/edible plants. The KAMPO DB consists of 336 formulae encompassing 278 medicinal plants; the JAMU DB consists of 5,310 formulae encompassing 550 medicinal plants. The Biological Activity DB consists of 2,418 biological activities and 33,706 pairwise relationships between medicinal plants and their biological activities. Current statistics of the binary relationships between individual databases were characterized by the degree distribution analysis, leading to a prediction of at least 1,060,000 metabolites within all plants. In the future, the study of metabolomics will need to take this huge number of metabolites into consideration.     

Cerebral hypoperfusion during hypoxic exercise following two different hypoxic exposures: independence from changes in dynamic autoregulation and reactivity

We examined the effects of exposure to 10-12 days intermittent hypercapnia [IHC: 5:5-min hypercapnia (inspired fraction of CO(2) 0.05)-to-normoxia for 90 min (n = 10)], intermittent hypoxia [IH: 5:5-min hypoxia-to-normoxia for 90 min (n = 11)] or 12 days of continuous hypoxia [CH: 1,560 m (n = 7)], or both IH followed by CH on cardiorespiratory and cerebrovascular function during steady-state cycling exercise with and without hypoxia (inspired fraction of oxygen, 0.14). Cerebrovascular reactivity to CO(2) was also monitored. During all procedures, ventilation, end-tidal gases, blood pressure, muscle and cerebral oxygenation (near-infrared spectroscopy), and middle cerebral artery blood flow velocity (MCAv) were measured continuously. Dynamic cerebral autoregulation (CA) was assessed using transfer-function analysis. Hypoxic exercise resulted in increases in ventilation, hypocapnia, heart rate, and cardiac output when compared with normoxic exercise (P < 0.05); these responses were unchanged following IHC but were elevated following the IH and CH exposure (P < 0.05) with no between-intervention differences. Following IH and/or CH exposure, the greater hypocapnia during hypoxic exercise provoked a decrease in MCAv (P < 0.05 vs. preexposure) that was related to lowered cerebral oxygenation (r = 0.54; P < 0.05). Following any intervention, during hypoxic exercise, the apparent impairment in CA, reflected in lowered low-frequency phase between MCAv and BP, and MCAv-CO(2) reactivity, were unaltered. Conversely, during hypoxic exercise following both IH and/or CH, there was less of a decrease in muscle oxygenation (P < 0.05 vs. preexposure). Thus IH or CH induces some adaptation at the muscle level and lowers MCAv and cerebral oxygenation during hypoxic exercise, potentially mediated by the greater hypocapnia, rather than a compromise in CA or MCAv reactivity.     

Genetic Population Structure of Chum Salmon in the Pacific Rim Inferred from Mitochondrial DNA Sequence Variation

We examined the genetic population structure of chum salmon, Oncorhynchus keta, in the Pacific Rim using mitochondrial (mt) DNA analysis. Nucleotide sequence analysis of about 500 bp in the variable portion of the 5′ end of the mtDNA control region revealed 20 variable nucleotide sites, which defined 30 haplotypes of three genealogical clades (A, B, and C), in more than 2,100 individuals of 48 populations from Japan (16), Korea (1), Russia (10), and North America (21 from Alaska, British Columbia, and Washington). The observed haplotypes were mostly associated with geographic regions, in that clade A and C haplotypes characterized Asian populations and clade B haplotypes distinguished North American populations. The haplotype diversity was highest in the Japanese populations, suggesting a greater genetic variation in the populations of Japan than those of Russia and North America. The analysis of molecular variance and contingency χ² tests demonstrated strong structuring among the three geographic groups of populations and weak to moderate structuring within Japanese and North American populations. These results suggest that the observed geographic pattern might be influenced primarily by historic expansions or colonizations and secondarily by low or restricted gene flow between local groups within regions. In addition to the analysis of population structure, mtDNA data may be useful for constructing a baseline for stock identification of mixed populations of high seas chum salmon.     

Cellobiose Transport by Mixed Ruminal Bacteria from a Cow

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 μM for the K_m and 10 nmol/min/mg of protein for the V_max. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher V_max values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.     

Molecular dynamics and forces of a motile cell simultaneously visualized by TIRF and force microscopies

Cells must exert traction forces onto the substratum for continuous migration. Molecular dynamics such as actin polymerization at the front of the cell and myosin II accumulation at the rear should play important roles in the exertion of forces required for migration. Therefore, it is important to reveal the relationships between the traction forces and molecular dynamics. Traction forces can be calculated from the deformation of the elastic substratum under a migrating cell. A transparent and colorless elastic substratum with a high refractive index (1.40) and a low Young's modulus (1.0 kPa) were made from a pair of platinum-catalyzed silicones. We used this substratum to develop a new method for simultaneous recording of molecular dynamics and traction forces under a migrating cell in which total internal refractive fluorescence (TIRF) and force microscopies were combined. This new method allows the detection of the spatiotemporal distribution of traction forces produced by individual filopodia in migrating Dictyostelium cells, as well as simultaneous visualization of these traction forces and the dynamics of filamentous myosin II.     

Carotid baroreflex control of leg vascular conductance at rest and during exercise.

We sought to test the hypothesis that the carotid baroreflex (CBR) alters mean leg blood flow (LBF) and leg vascular conductance (LVC) at rest and during exercise. In seven men and one woman, 25 +/- 2 (SE) yr of age, CBR control of LBF and LVC was determined at rest and during steady-state one-legged knee extension exercise at approximately 65% peak O(2) uptake. The application of 5-s pulses of +40 Torr neck pressure and -60 Torr neck suction significantly altered mean arterial pressure (MAP) and LVC both at rest and during exercise. CBR-mediated changes in MAP were similar between rest and exercise (P > 0.05). However, CBR-mediated decreases in LVC (%change) to neck pressure were attenuated in the exercising leg (16.4 +/- 1.6%) compared with rest (33 +/- 2.1%) and the nonexercising leg (23.7 +/- 1.9%) (P < 0.01). These data suggest CBR control of blood pressure is partially mediated by changes in leg vascular tone both at rest and during exercise. Furthermore, despite alterations in CBR-induced changes in LVC during exercise, CBR control of blood pressure was well maintained.