Validity of NIR spectroscopy for quantitatively measuring muscle oxidative metabolic rate in exercise

The purpose of this studywas to examine the validity of the quantitative measurement of muscleoxidative metabolism in exercise by near-infrared continuous-wavespectroscopy (NIRcws). Twelve male subjects performed two bouts ofdynamic handgrip exercise, once for the NIRcws measurement and once forthe ³¹P-magnetic resonance spectroscopy (MRS) measurementas a standard measure. The resting muscle metabolic rate (RMRmus) wasindependently measured by ³¹P-MRS during 15 min of arterialocclusion at rest. During the first exercise bout, the quantitativevalue of muscle oxidative metabolic rate at 30 s postexercisewas evaluated from the ratio of the rate of oxyhemoglobin/myoglobindecline measured by NIRcws during arterial occlusion 30 s afterexercise and the rate at rest. Therefore, the absolute values of muscleoxidative metabolic rate at 30 s after exercise[O_2NIR(30)] wascalculated from this ratio multiplied by RMRmus. During the secondexercise bout, creatine phosphate (PCr) resynthesis rate was measuredby ³¹P-MRS at 30 s postexercise[Q₍₃₀₎] under the same conditions but without arterial occlusion postexercise. To determine the validity ofNIRcws, O_2NIR(30) wascompared with Q₍₃₀₎. There was a significant correlation betweenO_2NIR(30), which rangedbetween 0.018 and 0.187 mM ATP/s, and Q₍₃₀₎,which ranged between 0.041 and 0.209 mM ATP/s (r = 0.965, P < 0.001). This result supports theapplication of NIRcws to quantitatively evaluate muscle oxidativemetabolic rate in exercise.

     

Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells

The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme.     

The leukocyte common antigen (CD45) of the Pacific hagfish,Eptatretus stoutii: implications for the primordial function of CD45

CD45, originally known as the leukocyte common antigen, is a prototypical transmembrane protein tyrosine phosphatase that plays a critical role in signal transduction through T-cell and B-cell receptors, as well as in T-cell and B-cell development. In the present study, we show that the Pacific hagfish, widely believed to lack the adaptive immune system, has CD45. The presence of CD45 in jawless fish is consistent with the recent discovery that CD45 also plays a crucial role in innate immunity via the regulation of signaling through type I and type II cytokine receptors. It is likely that CD45 was recruited to activate lymphocytes through antigen receptors encoded by rearranging genes in jawed vertebrates.     

Spatial distribution of total, ammonia-oxidizing, and denitrifying bacteria in biological wastewater treatment reactors for bioregenerative life support

Bioregenerative life support systems may be necessary for long-term space missions due to the high cost of lifting supplies and equipment into orbit. In this study, we investigated two biological wastewater treatment reactors designed to recover potable water for a spacefaring crew being tested at Johnson Space Center. The experiment (Lunar-Mars Life Support Test Project-Phase III) consisted of four crew members confined in a test chamber for 91 days. In order to recycle all water during the experiment, an immobilized cell bioreactor (ICB) was employed for organic carbon removal and a trickling filter bioreactor (TFB) was utilized for ammonia removal, followed by physical-chemical treatment. In this study, the spatial distribution of various microorganisms within each bioreactor was analyzed by using biofilm samples taken from four locations in the ICB and three locations in the TFB. Three target genes were used for characterization of bacteria: the 16S rRNA gene for the total bacterial community, the ammonia monooxygenase (amoA) gene for ammonia-oxidizing bacteria, and the nitrous oxide reductase (nosZ) gene for denitrifying bacteria. A combination of terminal restriction fragment length polymorphism (T-RFLP), sequence, and phylogenetic analyses indicated that the microbial community composition in the ICB and the TFB consisted mainly of Proteobacteria, low-G+C gram-positive bacteria, and a Cytophaga-Flexibacter-Bacteroides group. Fifty-seven novel 16S rRNA genes, 8 novel amoA genes, and 12 new nosZ genes were identified in this study. Temporal shifts in the species composition of total bacteria in both the ICB and the TFB and ammonia-oxidizing and denitrifying bacteria in the TFB were also detected when the biofilms were compared with the inocula after 91 days. This result suggests that specific microbial populations were either brought in by the crew or enriched in the reactors during the course of operation.     

Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display-positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5-trisphosphate (IP(3)) 5-phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large-type Kenyon cells, suggesting that IP(3)-mediated Ca(2+) signaling is enhanced in these neurons.     

Association of plasma viral RNA load with prognosis in cats naturally infected with feline immunodeficiency virus

We measured the quantity of plasma feline immunodeficiency virus (FIV) RNA using a real-time sequence detecting system. Plasma viral RNA load was shown to correlate with the clinical stage, survival time, and disease progression in naturally FIV-infected cats. The present study indicates that the plasma viral RNA load can be used as a clinical marker representing the impairment of the immune system and predicting the clinical outcome in FIV-infected cats.     

Effect of carnosine and related compounds on the inactivation of human Cu,Zn-superoxide dismutase by modification of fructose and glycolaldehyde

Glycolaldehyde, an intermediate of the Maillard reaction, and fructose, which is mainly derived from the polyol pathway, rapidly inactivate human Cu,Zn-superoxide dismutase (SOD) at the physiological concentration. We employed this inactivation with these carbonyl compounds as a model glycation reaction to investigate whether carnosine and its related compounds could protect the enzyme from inactivation. Of eight derivatives examined, histidine, Gly-His, carnosine and Ala-His inhibited the inactivation of the enzyme by fructose (p<0.001), and Gly-His, Ala-His, anserine, carnosine, and homocarnosine exhibited a marked protective effect against the inactivation by glycolaldehyde (p<0.001). The carnosine-related compounds that showed this highly protective effect against the inactivation by glycolaldehyde had high reactivity with glycolaldehyde and high scavenging activity toward the hydroxyl radical as common properties. On the other hand, the carnosine-related compounds that had a protective effect against the inactivation by fructose showed significant hydroxyl radical-scavenging ability. These results indicate that carnosine and such related compounds as Gly-His and Ala-His are effective anti-glycating agents for human Cu,Zn-SOD and that the effectiveness is based not only on high reactivity with carbonyl compounds but also on hydroxyl radical scavenging activity.     

Identification of a toxic mechanism of the plasticizers,phtahlic acid esters,which are putative endocrine disrupters: time-dependent increase in quinolinic acid and its metabolites in rats fed di(2-ethylhexyl)phthalate

We have reported that the administration of di(2-ethylhexyl)phthalate (DEHP) increased the formations of quinolinic acid (QA) and its lower metabolites on the tryptophan-niacin pathway. To discover the mechanism involved in disruption of the tryptophan-niacin pathway by DEHP, we assessed the daily urinary excretion of QA and its lower metabolites, and enzyme activities on the tryptophan-niacin pathway. Rats were fed with a niacin-free, 20% casein diet or the same diet supplemented with 0.1% DEHP or 0.043% phthalic acid and 0.067% 2-ethylhexanol added for 21 days. Feeding of DEHP increased the urinary excretions of QA and its lower metabolites in a time-dependent manner, and the increase of these excretions reached a peak at 11 days, but feeding of phthalic acid and 2-ethylhexanol had no effect. Feeding of DEHP, however, did not affect any enzyme activity including alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), affecting the formation of QA, on the tryptophan-niacin pathway.     

Enzyme inhibitors to increase poly-3-hydroxybutyrate production by transgenic tobacco

Chemical regulation of secondary-metabolite synthesis was investigated through the improvement of poly-3-hydroxybutyrate (PHB) production in transgenic tobacco plants by the use of enzyme inhibitors. Two tobacco lines, BC3 and rCAB8, that produce PHB in both the cytosol and plastids were used. An acetyl-CoA carboxylase inhibitor, D-(+)-Quizalofop-ethyl, increased PHB accumulation in both lines 2-fold. The accumulation rate of plastidial PHB in the rCAB8 line was 2.5-fold higher than that of cytosolic PHB in the BC3 line. A specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, mevastatin, also increased PHB accumulation but only in the BC3 line. These results indicated that chemical regulation of the native metabolic flows by the specific enzyme inhibitors increased secondary-metabolite production in the transgenic tobacco plants we used.