Metal-ion-induced expression of gene fragments from subseafloor micro-organisms in the Kumano forearc basin, Nankai Trough.

AIMS: Using substrate-induced gene-expression (SIGEX) screening on subseafloor sediment samples from the Nankai Trough, Japan, we identified gene fragments showing an induction response to metal ions. METHODS AND RESULTS: Environmental DNA libraries in Escherichia coli host cells were tested by the addition of metal ions (Ni2+ , Co2+ , Ga3+ or Mo6+ ), followed by cell sorting of clones exhibiting green fluorescence upon co-expression of green fluorescence protein downstream of the inserted gene fragments. One clone displayed Ni2+ -specific induction, three clones displayed Ga3+ -specific induction and three clones displayed an induction response to multiple metal ions. DNA sequence analysis showed that a variety of genes showed induction responses in the screened clones. CONCLUSIONS: Using the SIGEX approach, we retrieved gene fragments with no previously identified response to metal ions that exhibited metal-ion-induced expression. This method has the potential to promote exploration of gene function through gene-induction response. SIGNIFICANCE AND IMPACT OF THE STUDY: We successfully linked gene-induction response with sequence information for gene fragments of previously unknown function. The SIGEX-based approach exhibited the potential to identify genetic function in unknown gene pools from the deep subseafloor biosphere, as well as novel genetic components for future biotechnological applications.

Post-challenge hyperglycaemia is an independent risk factor for arterial stiffness in Japanese men.

AIMS: Arterial stiffness is an independent predictor of cardiovascular disease, but the impact of post-challenge hyperglycaemia on arterial stiffness is unknown. To investigate the association between arterial stiffness and post-challenge hyperglycaemia, we measured the second derivative of photoplethysmogram as an indicator of arterial stiffness. METHODS: This study was done in 159 asymptomatic Japanese men aged 50.7 +/- 13.0 years. All subjects underwent a 75-g oral glucose tolerance test and measurement of the second derivative of photoplethysmogram. RESULTS: According to the World Health Organization criteria (1998), 110 subjects had normal glucose tolerance, 10 had impaired fasting glucose, 30 had impaired glucose tolerance, and nine had diabetes. The b/a ratio (an index of arterial stiffness) showed a significant relationship with age (r = 0.58, P < 0.0001), height (r = -0.33, P < 0.0001), 2-h post-challenge glucose (r = 0.32, P < 0.0001), systolic blood pressure (r = 0.22, P = 0.006), and diastolic blood pressure (r = 0.21, P = 0.009). After adjustment for age and height, there were significant correlations between the b/a ratio and diastolic blood pressure (r = 0.18, P = 0.02), fasting glucose (r = 0.16, P = 0.049), and 2-h post-challenge glucose (r = 0.21, P = 0.009). Stepwise multiple regression analysis showed that only age (beta= 0.006, SE = 0.0007, P < 0.001) and 2-h post-challenge glucose (beta = 0.0005, SE = 0.0002, P < 0.05) contributed significantly to the b/a ratio (adjusted R(2) = 0.38). CONCLUSIONS: These results indicate that post-challenge hyperglycaemia is an independent risk factor for arterial stiffness.

[The effects of intravenous nicardipine on jugular venous oxygen saturation].

We investigated the effects of intravenous nicardipine on jugular venous oxygen saturation (SjO2) in patients undergoing superficial temporal artery-middle cerebral artery anastomosis. Anesthesia was induced with intravenous thiamylal and fentanyl, and maintained by isoflurane and nitrous oxide in oxygen. In the presence of stable condition after the induction, nicardipine 0.5 microgram.kg-1.min-1 was administered intravenously for 180 minutes. Mean arterial pressure, heart rate, SjO2 and bladder temperature were recorded. Three hours after continuous infusion of nicardipine, plasma levels of nicardipine in artery and jugular vein were measured by liquid chromatography. The arterial concentration of nicardipine reached 23.9 +/- 8.06 ng.ml-1. The area under the curve between the arterial and jugular venous nicardipine during infusion was significantly different. Mean arterial pressure was reduced in response to administration of nicardipine. There were no significant changes in heart rate, bladder temperature and SjO2. We concluded that the current dose of nicardipine reached effective concentration for systemic hypotension without influencing SjO2.

Safety evaluation of yeast glutaminase.

The consumption of soy and soy products (including soy sauce) has been increasing in Western countries due to purported health benefits of soy (cancer protective, estrogenic effects). In addition to providing soy proteins and isoflavones, soy sauce also functions as a flavor enhancer and is able to impart a "umami" taste. Glutaminases are used in the production of soy sauce and enzymatically hydrolyzed protein. The glutaminases described herein were produced from the cultured broth of Cryptococcus albidus (ATCC-20293) which is designated as CK, a mutant of C. albidus (ATCC-20293) which is designated as CK-D10 and the newly isolated Cryptococcus sp. NISL-3771 which is designated as TK. All three preparations (CK, CK-D10 and TK) were evaluated for pathogenicity and virulence in mice and were found to be non-pathogenic. The acute LD(50)s for CK in male mice was greater than 4.8 g/kg body weight and for female mice was greater than 6.5 g/kg body weight. Acute LD(50)s for CK and CK-D10 in male and female rats was greater than 7.5 g/kg body weight, and that for TK was greater than 10 g/kg body weight. Subchronic (90-day) feeding studies (wherein the glutaminases were presented as dietary admixtures) were conducted in mice and rats. The NOAEL for CK in mice was 7.5 g/kg body weight/day. The NOAELs in rats were as follows: for CK, 9 g/kg body weight/day; for CK-D10, 1.2 g/kg body weight/day, and for TK, 8 g/kg body weight/day. Mice received CK as a dietary admixture at levels of 0, 1.0 and 10.0% for 1 year. The NOAEL was 13 g/kg body weight/day. The glutaminases from C. albidus described herein demonstrate very low toxicity.

[Micro-canaliculi in juxtaglomerular cells of two-kidney Goldblatt hypertensive rats].

Juxtaglomerular (JG) cells in two-kidney Goldblatt hypertensive (2KGH) rats were examined ultrastructurally. In JG cells of the clipped kidneys of 2KGH rats, micro-canaliculi branched off from channel-like invaginations of plasma membrane and connected with the secretory renin granules. These findings suggest that micro-canaliculi may play important role in renin secretion from JG cells in 2KGH rats.

Immunohistochemical findings of juxtaglomerular cells on acute phase in two-kidney Goldblatt hypertensive rats and spontaneously hypertensive rats.

Juxtaglomerular (JG) cells on the acute phase in two-kidney Goldblatt hypertensive (2KGH) rats and spontaneously hypertensive rats (SHR) were examined immunohistochemically. JG cells in 2KGH rats and SHR were positively stained with anti-renin serum and anti-angiotensin II (A II) serum. In 2KGH rats, the number of renin and AII immunoreactive JG cells in the clipped kidneys increased throughout the observation period. The number of renin and AII immunoreactive JG cells in the unclipped kidneys was almost the same as that in control rats, although immunoreactivity of these cells was weak and they were small in size. These changes in the unclipped kidneys became obvious with the time course after operation. We did not see any changes in these cells in SHR. In 2KGH rats treated with captopril, the number of renin immunoreactive JG cells in the clipped kidneys increased, whereas that of AII immunoreactive JG cells in the bilateral kidney decreased. When captopril was administered to SHR, the number of renin immunoreactive JG cells in the bilateral kidney increased, whereas that of AII immunoreactive JG cells in the bilateral kidney decreased. These results suggested that the JG cell in the bilateral kidney was closely related to the development of hypertension in 2KGH rats, but not in SHR. The increase of renin immunoreactive JG cells in 2KGH rats and SHR treated with captopril was probably due to the removal of negative feedback inhibition of AII on JG cells.

[Effects of captopril on acute phase in two-kidney Goldblatt hypertensive rats and spontaneously hypertensive rats].

Effects of continuous oral administration of captopril were investigated on acute phase in two-kidney Goldblatt hypertensive (2 KGH) rats and spontaneously hypertensive rats (SHR). Systolic blood pressure gradually rose throughout the experimental period of 7, 14, 21 and 28 days in both 2 KGH rats and SHR. These gradual increases of systolic blood pressure were reduced by administration of captopril in both rats. Plasma renin activity were markedly increased throughout the experimental period in both rats treated with captopril, and were modestly increased in 2 KGH rats. In contrast, those changes in plasma renin activity were not obvious in SHR. In 2 KGH rats, juxtaglomerular index (JGI) and juxtaglomerular cell count (JGCC) of the clipped kidneys increased whereas JGI of the opposite kidneys decreased. In contrast, those changes in JGI and JGCC were not obvius in SHR. On the other hand, JGI and JGCC of the clipped kidneys increased in 2 KGH rats treated with captopril and those of the both kidneys increased in SHR treated with captopril. These results suggested that juxtaglomerular cells were related to the development of hypertension in 2 KGH rats, but were not clear in SHR. And these results were found that captopril showed antihypertensive effects, in spite of rises in JGI and JGCC of both 2 KGH rats and SHR.

[Clinical and pathological findings on acute phase in two-kidney Goldblatt hypertensive rats].

Clinical and pathological changes were investigated on acute phase in two-kidney Goldblatt hypertensive rats. Systolic blood pressure, plasma renin activity, plasma aldosterone and juxtaglomerular cell count (JGCC) increased slightly as early as 7 days after operation. Systolic blood pressure and plasma renin activity rose in the course of time after operation. Juxtaglomerular index (JGI) and JGCC of the clipped kidneys increased and JGI of the opposite kidneys decreased with the increase in systolic blood pressure. These results suggested that juxtaglomerular cells had an important role on acute phase in two-kidney Goldblatt hypertensive rats.

Investigation of soy sauce treated with nitrite in the chromosomal aberration test in vitro and the micronucleus test in vivo.

Soy sauce pretreated with 2300 ppm nitrite caused no more aberrations than did untreated soy sauce in the chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line with or without S9 mixture. The aberration induction by soy sauce is likely to be caused by the 17% sodium chloride it contains. Soy sauce with or without pretreatment with 2300 ppm nitrite was orally given to ICR mice at a dose of 14 ml/kg body weight once or 6 ml/kg body weight/day for 5 consecutive days. This oral administration did not induce any significant increase in micronuclei in the micronucleus test in vivo.

Cytotoxic effect of 8-amino adenosine 3',5'-cyclic monophosphate on FM3A mouse mammary tumor cells.

Derivatives of adenosine 3',5'-cyclic monophosphate (cAMP) with modifications at the 8-position were synthesized and examined for their cytotoxic effects on FM3A mouse mammary tumor cells and ZR-75 human mammary tumor cells. On in vitro tests of these derivatives, 8-amino (8-NH2) cAMP was the most effective against both cell lines. This compound showed the dose-dependent inhibition of FM3A cell growth in the concentration of over 2.5 microM with the ID50 value of 4 microM. Furthermore, antitumor activity of 8-NH2 cAMP was also tested against FM3A cells in vivo. T/C% values of 8-NH2 cAMP were respectively 162% and 138% in response to doses of 30 and 10 mg/kg by intraperitoneal injections of 8-NH2 cAMP for 5 d. 8-NH2 cAMP was converted to 8-NH2 adenosine via 8-NH2 adenosine 5'-monophosphate by some enzymes in the fetal bovine serum and the cytotoxic effect of 8-NH2 cAMP on FM3A cells was actually stemmed from 8-NH2 adenosine.

Relation of nitrite concentration to mutagen formation in soy sauce.

When soy sauce was mixed with nitrite solutions at pH 1.0 and 3.0, only mixtures containing nitrite concentrations above 250 ppm were mutagenic in the Salmonella/mammalian microsome test. In buffered aqueous solution (pH 3) the mutagen precursor, (-)-(1S,3S)-1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid [(1S,3S)-MTCA] and its stereoisomer ( - )-(1R,3S)-MTCA reacted with 10 ppm or more of sodium nitrite; but at least 250 ppm nitrite was required for them to react in soy sauce (pH 3). Similarly, another mutagen precursor, tyramine, did not react even with 2300 ppm nitrite in soy sauce (pH 1), although pure tyramine in aqueous solution (pH 1) reacted with as little as 50 ppm nitrite. Nitrite concentration in human saliva and gastric juice does not generally exceed 50 ppm. Therefore, the most probable source of mutagens--nitrosation of MTCAs and tyramine--is likely to be very restricted in vivo and soy sauce is unlikely to be significantly mutagenic.