Effects of exercise and intermittent cold exposure on shivering and nonshivering thermogenesis in rats

The effects of both exercise training and intermittent cold acclimation on heat production (shivering and nonshivering thermogenesis (NST)) in rats were studied. Warm-acclimated rats (housed at 24 degrees C, WA) and intermittently cold-acclimated rats (exposed daily to -5 degrees C for 2 h, CA) were forced to run (25 m.min-1 for 1 h) every day (WA-T and CA-T). WA and CA left sedentary (WA-S and CA-S) served as controls. Norepinephrine (NE)-induced thermogenic capacity assessed from the increment of oxygen consumption (VO2) and colonic temperature (Tc) were measured 4 weeks after commencing acclimation and exercise training. The thermogenic capacity was greater in CA than in WA. However, in WA, WA-T responded to NE less than WA-S, whereas the response of CA-T and CA-S did not differ. Wet weight of interscapular brown adipose tissue (IBAT) and its protein (and dry matter--regarded to be highly representative of protein) content were larger in CA than in WA. Respective sedentary and exercised groups of rats had similar IBAT protein (and dry matter) content although tissue weight was lighter in WA-T than in WA-S. Lipid content of IBAT was also larger in CA than in WA. IBAT of WA-T had less lipid compared to that of WA-S while no difference was seen between CA-S and CA-T. Shivering activity during acute cold (4 degrees C) exposure was less in CA compared to WA and there was no difference between respective groups of exercised and sedentary rats. Propranolol, a blocker of NE-dependent NST, eliminated the difference in shivering among these four groups. When exposed to severe cold (-10 or -20 degrees C), the fall in Tc of rats fasted for 18 h was greater in WA than in CA. CA-T showed a greater decrease in Tc than CA-S during -20 degrees C exposure while it did not differ during -10 degrees C exposure. On the other hand, Tc of WA-T and WA-S did not differ significantly during either cold exposure period. These results suggest that exercise training in rats housed at 24 degrees C suppresses NE-dependent NST whereas another nonshivering thermogenic mechanism (NE-independent) may compensate this suppression. However, NE-dependent NST of WA-S and WA-T did not parallel protein (dry matter) content of IBAT; no difference existed in IBAT protein between these two groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

Noninvasive monitoring of hemoglobin. The effects of WBC counts on measurement

The efficacy of a noninvasive hemoglobin monitoring device (Astrin, Sysmex, Kobe, Japan) was evaluated for healthy volunteers and for patients with hematologic disorders. At the same time, the effects of WBC counts on noninvasive monitoring were studied by clinical evaluation and in ex vivo experiments. The hemoglobin levels determined by the device (Ast-Hb) and a conventional analyzer (T-Hb) were compared. The coefficient of correlation between findings with the Ast-Hb and the T-Hb for healthy volunteers was r = 0.626, whereas that for patients with hematologic disorders was r = 0.762. A comparison of the ratios of measurement errors in hemoglobin levels by Ast-Hb and T-Hb indicated that the number of WBCs had no effect on hemoglobin monitoring. Moreover, ex vivo studies using isolated WBCs and an optical model that imitates blood vessels and tissue in human fingers confirmed these results. Therefore, this new hemoglobin monitoring device can be expected to be useful for continuous hemoglobin monitoring.     

Inhibitory effect of sheep erythrocyte fragments on rosette formation of human T lymphocytes with sheep red blood cells.

The effect of sheep red blood cells (SRBC) fragments on rosette formation of human peripheral T lymphocytes with SRBC was evaluated on the active and total T-rosette tests. The rosetting capacity of active rosette-forming cells was selectively and nearly completely inhibited by the pretreatment of lymphocytes with SRBC fragments. The decrease in total rosettes by blocking with SRBC fragments was almost parallel to that of active rosettes. SRBC fragments had no inhibitory effect on the rosetting capacity of a lymphocyte population in which active rosette-forming cells were removed by gradient centrifugation. These results suggested that active rosette-forming cells in human T lymphocytes have the receptors of high affinity for SRBC and these receptors readily bind SRBC fragments, resulting in block of rosette formation.     

Antimicrobial Susceptibility Testing of Bilophila wadsworthia Isolates Submitted for Routine Laboratory Examination

MICs of antibiotics against Bilophila wadsworthia isolates were measured by agar and broth microdilution with pyruvic acid and by Etest. The inoculum size influenced greatly agar dilution. Despite discrepancies in MICs depending on the measurement method used, clindamycin consistently showed potent activity. Broth microdilution and Etest appear to be candidates for laboratory susceptibility testing.     

Expression of an endogenous and a heterologous gene in Candida maltosa by using a promoter of a newly-isolated phosphoglycerate kinase (PGK) gene

A gene encoding phosphoglycerate kinase (PGK) was isolated from the genomic library of C. maltosa to construct an expression vector for this yeast. The PGK gene had an open reading frame of 1251 base pairs encoding approximately 47-kDA polypeptide of 417 amino-acid residues. Expression of this gene assayed by Northern-blot analysis was significantly induced in cells grown on glucose but not in cells grown on n-tetradecane, n-tetradecanol, or oleic acid. By using the promoter region of this gene, an expression vector (termed pMEA1) for C. maltosa was constructed and expression of an endogenous gene (P450alkl encoding one of cytochrome P450s for n-alkane hydroxylation in C. maltosa) and a heterologous gene (LAC4 encoding Kluyveromyces lactis -galactosidase) was tested. Expression of P450alkl gene was confirmed at both mRNA and protein levels. LAC4 gene expression was confirmed by determining -galactosidase activity. The activity in cells grown on various carbon sources correlated very well with the expression levels of PGK mRNA in these cells.     

Microbiochemical studies on changes of phosphofructokinase and glucose-6-phosphate dehydrogenase activity in intima and media of aortic wall of rabbits in the course of cholesterol feeding.

The activities of phosphofructokinase (PFK), a key enzyme in the Embden-Meyerhof pathway, and glucose-6-phosphate dehydrogenase (G-6PDH), enzyme in the Warburg-Dickens pathway, were analyzed in the intima and media of aortic wall of rabbits by Lowry's quantitative histochemical method as modified by us. These rabbits were treated with a one-shot administration of cholesterol (1 g/kg PO) and epinephrine (1 μg/kg iv) or fed with 150 g pellets containing 1% of cholesterol for 1, 2, 5, and 15 wk.PFK in the intima exhibited a statistically significant decreased activity in one-shot treated rabbits, then restored to normal level in cholesterol-fed rabbits for 1 and 2 wk. A statistical significant decrease of this enzyme activity was observed in cholesterol-fed rabbits for more than 5 wk. The media showed the highest activity in the 1 wk cholesterol-fed rabbits (p < 0.05), then the gradual decrease towards cholesterol feeding. Decreased activity was also present in rabbits after 15 wk cholesterol feeding.On the other hand, G-6PDH showed an inclination toward the reverse change of PFK. One-shot treated rabbits showed an increased activity in both the media and intima of the aortic wall and rabbits fed with cholestrol for 1 and 2 wk revealed almost the same activity as those of rabbits fed with a regular diet. Gradual increase in this activity was observed in rabbits fed for more than 5 wk. However, there was not a statistically significant difference between the placebo control group and the treated group in the analysis of G-6PDH activity in both the intima and media of aortic wall.That change of these enzyme are possibly a protective reaction in the cells of the arterial wall against the atherogenic process.     

Discerning malignancy in resected adrenocortical neoplasms

Differential diagnosis between adenoma and carcinoma in resected human adrenocortical neoplasms may be one of the most problematic and difficult areas of surgical pathology practice. This is especially true in cases of relatively small adrenocortical tumors not associated with obvious signs of malignancy such as necrosis and/or hemorrhage. In addition, the numbers of these small adrenocortical neoplasms are increasing owing to the widespread application of sophisticated computed tomography and/or magnetic resonance imaging scans. No single parameter can be effective in this differential diagnosis of resected adrenocortical tumor. Histopathologic evaluation using a multivariate scoring system is considered most effective in discerning malignancy and biologic behavior of resected adrenocortical neoplasms. Molecular and cellular findings of adrenocortical carcinoma have been inconsistent except for the increased cell proliferation associated with adrenocortical malignancy. Therefore, an assessment of neoplastic cell proliferation using immunostains of cell cycle-associated nuclear antigen such as Ki-67 is the only useful auxilliary method of evaluating malignancy in resected adrenocortical neoplasms at present.     

Regional vulnerability to chronic hypoxia and chronic hypoperfusion in the rat brain

The purpose of this study was to compare the pathological findings of injury induced by chronic hypoperfusion and by chronic hypoxia in rat brain. Adult male Wistar rats were divided into three groups: chronic hypoperfusion (n=5), chronic hypoxia (n=5), and normal control groups (n=5). Hypoperfusion was induced by ligation of the bilateral carotid arteries under 2.5% halothane anesthesia. Chronic hypoxia was induced by keeping the animals in a chamber with an atmosphere of 10% O₂ in N₂ for 3 weeks. Twelve weeks later (chronic hypoperfusion group) and 3 weeks later (chronic hypoxia group), the animals were sacrificed and perfused through the femoral artery with a fixative containing 4% paraformaldehyde. Hematoxylin and eosin staining was done in all sections in the three groups, and the number of normal-appearing cells was counted. Normal-appearing cells in CA3 were significantly decreased in the chronic hypoperfusion group compared with those in the chronic hypoxia group, although neurons in CA1, CA2 and CA4 in both groups were equally damaged. We concluded that the CA3 hippocampus shows different vulnerabilities to chronic hypoperfusion and chronic hypoxia, possibly owing to a difference in the kinds of glutaminergic receptors.     

Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, ade1, ura3) as a useful host for the genetic engineering of Candida maltosa

The C-URA3 gene of the n-alkane assimilating-yeast Candida maltosa was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae. The nucleotide sequence of C-URA3 and its deduced amino-acid sequence showed significant homology to those of the orotidine 5-phosphate decarboxylases of other fungal species. To construct a useful host for genetic engineering of C. maltosa using C-URA3 as a marker, one allele of C-URA3 in a double auxotroph (his5, ade1) was disrupted by C-ADE1, and subsequently two kinds of ura3 mutants were isolated by selecting for spontaneous 5-fluoro-orotic acid (5FOA) resistance. One of the mutants was homozygous for the disruption (ura3::C-ADE1/ura3::C-ADE1); the other was heterozygous (ura3::C-ADE1/ura3). The ura3::C-ADE1 allele in the latter strain was re-substituted by C-URA3 to rescue the adenine auxotroph (his5, ade1, C-URA3/ura3). Finally, by selecting a 5FOA-resistant mutant, a triple auxotroph (his5, ade1, ura3/ura3) was isolated.     

Unconventional NK1.1(-) intermediate TCR cells as major T lymphocytes expanding in chronic graft-versus-host disease

Chronic graft-versus-host disease (GVHD) accompanying autoimmune disease was induced in (C57BL/6xDBA/2) F(1) mice (H-2(b/d)) by an injection of splenic T cells of parental DBA/2 origin (H-2(d)). In parallel with the onset of proteinuria, an expansion of lymphocytes was induced in the liver and kidney, showing a peak at 2 weeks after the onset of disease. The majority of lymphocytes were of recipient origin (H-2(b/d)). The main lymphocyte subset among T cells at the pre-onset stage and after the onset of disease was CD8(+) NK1.1(-) CD3(int) cells (of extrathymic, hepatic origin) in both the liver and kidney. NK1.1(-) CD3(int) cells confer primarily neither NK-like nor NKT-like cytotoxicity. No induction of these types of cytotoxicity was observed in these mice with the expansion of NK1.1(-) CD3(int) cells. This raised the possibility that granulocytes induced in the liver and kidney might be associated with tissue damage. The present results suggest that, similarly to the case of autoimmune-prone mice with genetic background (e.g. MRL-lpr/lpr mice and BXSB mice), NK1.1(-) CD3(int) cells of extrathymic, hepatic origin might be crucial lymphocytes involved in the induction of the autoimmune-like disease in mice with chronic GVHD, in conjunction with Bcells (e.g. B-1 cells).