Delta15N of soil N and plants in a N-saturated, subtropical forest of southern China.

We investigated the delta(15)N profile of N (extractable NH(4)(+), NO(3)(-), and organic N (EON)) in the soil of a N-saturated subtropical forest. The order of delta(15)N in the soil was EON > NH(4)(+) > NO(3)(-). Although the delta(15)N of EON had been expected to be similar to that of bulk soil N, it was higher than that of bulk soil N by 5 per thousand. The difference in delta(15)N between bulk soil N and EON (Delta(15)N(bulk-EON)) was correlated significantly with the soil C/N ratio. This correlation implies that carbon availability, which determines the balance between N assimilation and dissimilation of soil microbes, is responsible for the high delta(15)N of EON, as in the case of soil microbial biomass delta(15)N. A thorough delta(15)N survey of available N (NH(4)(+), NO(3)(-), and EON) in the soil profiles from the organic layer to 100 cm depth revealed that the delta(15)N of the available N forms did not fully overlap with the delta(15)N of plants. This mismatch in delta(15)N between that of available N and that of plants reflects apparent isotopic fractionation during N uptake by plants, emphasizing the high N availability in this N-saturated forest.

Genetic diversity of gamma-hexachlorocyclohexane-degrading sphingomonads isolated from a single experimental field.

AIMS: To isolate gamma-hexachlorocyclohexane (gamma-HCH)-degrading bacteria from a single field and to examine their genetic diversity. METHODS AND RESULTS: Gamma-HCH-degrading bacteria were screened from a long-term experimental field in which gamma-HCH has been continuously applied to, and a gamma-HCH-degrading sphingomonad strain SS86 was isolated from in 1986. As the result, five strains of sphingomonads were newly isolated. The sequences of several housekeeping genes separated the six strains, including SS86, into two genotypes. Among the genes involved in gamma-HCH degradation, the sequences of linC, linD and linE were identical among all six strains, that of linA was identical among five strains, and that of linB was diverse. CONCLUSIONS: We calculated that the gamma-HCH-degrading populations of the two genotypes arose independently. Not just one but diverse sphingomonads that degrade a particular xenobiotic compound possibly tend to arise and/or accumulate in fields, where that compound has been applied. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the potential usefulness of a long-term continuous application of xenobiotic compounds to an experimental field in that it would potentially generate diverse micro-organisms able to degrade the compounds.

Effect of MDR1 C3435T polymorphism on cure rates of Helicobacter pylori infection by triple therapy with lansoprazole, amoxicillin and clarithromycin in relation to CYP 2C19 genotypes and 23S rRNA genotypes of H. pylori.

BACKGROUND: Polymorphism in MDR1 is associated with variation in the plasma level of a proton pump inhibitor. AIM: To investigate whether MDR1 polymorphism is associated with eradication rates of Helicobacter pylori by a triple therapy with lansoprazole, amoxicillin and clarithromycin in relation to CYP2C19 genotype status and bacterial susceptibility to clarithromycin. METHODS: A total of 313 patients infected with H. pylori completed the treatment with lansoprazole 30 mg b.d., clarithromycin 200 mg b.d. and amoxicillin 750 mg b.d. for 1 week. MDR1 C3435T polymorphism and CYP2C19 genotypes of patients and sensitivity of H. pylori to clarithromycin were determined. RESULTS: Logistic regression analysis revealed that the MDR1 polymorphism as well as CYP2C19 genotypes of patients and clarithromycin-resistance of H. pylori were significantly associated with successful eradication. Eradication rates for H. pylori were 82% (83/101: 95% CI = 73-89), 81% (112/139: CI = 73-87), and 67% (44/73: CI = 48-72) in patients with the MDR1 3435 C/C, C/T and T/T genotype, respectively (P = 0.001). CONCLUSIONS: Polymorphism of MDR1 is one of the determinants of successful eradication of H. pylori by the triple therapy with lansoprazole, amoxicillin and clarithromycin, together with CYP2C19 genotype and bacterial susceptibility to clarithromycin.

Guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase.

The guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase (PCL) was studied at pH 7 by monitoring the changes in the fluorescence and circular dichroism of the enzyme. The denaturation was irreversible as a whole, and the addition of Ca2+ ions decreased the velocity of the denaturation. The denaturation process was well explained consistently by a two-step mechanism, as follows: [see equation in text] where N is the native state of PCL, D(I) an intermediate denatured-state which can be refolded into the native state, and D(F) the final denatured-state that can not be renatured. Ethanol (10%) increased the denaturation velocity by decreasing the refolding step, D(I) + Ca2+ --> N x Ca2+, which would be caused by the stabilization of D(I) by ethanol.

Molecular mechanisms of glucocorticoid inhibition of human proopiomelanocortin gene transcription.

The gene encoding proopiomelanocortin(POMC) offers an interesting model for negative regulation of gene transcription by glucocorticoids. A fragment of human genomic DNA containing the entire POMC gene, together with the neo marker gene, was introduced by transfection into the ACTH-producing mouse pituitary tumor cell line, AtT-20, and the mouse fibroblast L cell line. In the transformed AtT-20 cells the human POMC gene was transcribed correctly and the transcript was spliced faithfully. Furthermore, the addition of dexamethasone to the transformed AtT-20 cells resulted in a 40% reduction of the human POMC mRNA levels. Deletion analysis demonstrated that no more than 417 bp in the 5'-flanking region of the human POMC gene are required for transcriptional repression by glucocorticoid. This region was also responsible for the transcription induction of the human POMC gene by cyclic AMP (cAMP). In the transformed L cells, however, most of the transcripts of the human POMC gene were not correctly initiated. The addition of dexamethasone to the transformed L cells did not significantly affect the content of human POMC mRNA, although these cells expressed glucocorticoid receptor(GR). However, the increase of the transcripts by forskolin, a post-receptor adenylate cyclase-activating agent, was partially but significantly suppressed by dexamethasone in the transformed L cells. These results suggest that binding of GR to the negative glucocorticoid response element (nGRE) could lead to steric occlusion of positive transcription factors, such as cAMP-response element binding protein and tissue specific factors or that GR bound to nGRE could interact with DNA-bound positive factors in such a way as to prevent their transcriptional stimulatory activity.

Basic subsite theory assumptions may not be applicable to hydrolysis of cellooligosaccharides by almond beta-glucosidase.

Steady-state kinetic parameters for the hydrolysis of cellooligosaccharides by almond beta-glucosidase were evaluated at pH 5.0 and 25 degrees C in relation to the subsite theory (K. Hiromi, Biochem. Biophys. Res. Commun., 40, 1-6, 1970). The value of k0/Km decreased monotonously with increasing degree of polymerization (DP) of the substrates (DP = 2-6). Also, the Km and k0 values for cellotriose were smaller than those for cellobiose. These DP dependencies differ from those of most amylases and glucosidases studied so far, to which the subsite theory has been successfully applied. The subsite parameters could not be consistently obtained, which suggests that one or both of the two basic assumptions of the subsite theory might not be applicable to the hydrolysis of cellooligosaccharides by the enzyme. That is, the intrinsic rate of the hydrolysis may depend on the DP and/or there may be interaction between subsites for binding the glucose residues of a substrate.

Multiple tolerance of Rhodotorula glutinis R-1 to acid, aluminum ion and manganese ion, and its unusual ability of neutralizing acidic medium.

A red yeast isolated from the acidic water of Kusatsu hot spring could grow in an acidic medium of pH 1.5 and was identified as Rhodotorula glutinis. Electron microscope observations (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]) showed that cell envelope became wrinkled and thick as the pH values of media became lower. The cell membrane grown at pH 1.5 was about four times as thick as that grown at pH 6.0. It was suggested that the change of cell envelope plays an important role in the acid tolerance. Cellular proteins at pH 1.5 appeared to be different from those at pH 6.0 and the amounts of phospholipids and non-phospholipids increased and decreased under low pH conditions, respectively. The acid-tolerant yeast also showed strong resistance to both aluminum and manganese ions. An acidic medium (pH 3.0) containing these ions (100 mM) was shifted to neutral pH by long-term cultivation of the red yeast, suggesting the potential of using this yeast in the bioremediation of acidic soil containing these ions at a high level.

[Study of organisms isolated from non-infected patients with pneumoconiosis].

Studies on the sputum organisms and their seasonal incidences were conducted on non-infected patients with pneumoconiosis. A total of 3318 organisms were isolated from 1427 sputum examinations, an average of 4 examinations per patient. alpha-Streptococcus, GPC, Neisseria and GNC were isolated in 74.1, 22.1, 64.8, 21.3% of the patients respectively. In addition, organisms to cause respiratory infection were isolated in the non-infected phase. S. pneumoniae, S. aureus, B. catarrhalis, H. influenzae, E. coli, K. pneumoniae and P. aeruginosa were isolated in 1.5, 5.1, 2.5, 3.3, 2.9, 6.4, 2.8% of the patients respectively. Studies of the seasonal incidences in these organisms showed that H. influenzae, B. catarrhalis and S. pneumoniae were isolated mostly in winter, S. aureus mostly in spring, E. coli and K. pneumoniae mostly in summer. On the other hand, P. aeruginosa showed no seasonal incidence. In relation to the causing organisms of respiratory infection with pneumoconiosis, it is very interesting that many organisms were isolated in the non-infected phase, and seasonal incidences were observed.

Possibility for discriminating between two representative non two-state thermal unfolding models of proteins by DSC.

Possible differences between two representative non two-state thermal unfolding mechanisms of protein are discussed concerning differential scanning calorimetry. Numerical simulations showed that, by DSC measurement, it is hard to discriminate between the independent model, which assumes independent unfolding domains in a protein, and the sequential model, which assumes intermediate(s) between native and denatured states, especially when values of molecular weight, denaturation enthalpy, and difference in denaturation temperature of each denaturation process are large. DSC curve analysis of Aspergillus niger glucoamylase based on these two models gave essentially the same thermodynamic parameters.

Cloning and sequence analysis of czc genes in Alcaligenes sp. strain CT14.

We have isolated 14 cadmium (Cd)-resistant, soil-borne bacteria. Among those, strain CT14, which was identified as an Alcaligenes sp., has a czc (cadmium, zinc, and cobalt divalent cation resistant determinant) system. Here we report the nucleotide sequence of 4 genes (czcCBAD) of the system. CzcCBA showed over 98% identity with those of A. eutrophus CH34, however, CzcD, the distal gene product, was 117 amino acids longer than that (199 amino acids) of A. eutrophus CH34, and had considerable similarity to the members of the CDF (cation diffusion facilitator) family proteins all over the region.

[Balloon-occluded arterial infusion chemotherapy in treatment of the patients with locally recurrent carcinoma of the cervix who previously received radiation therapy].

Nine patients with locally recurrent carcinoma of the cervix were treated with balloon-occluded arterial infusion chemotherapy (BOAI) in order to secure high concentrations of antitumor agents. All the patients had previously received radiation therapy for squamous cell carcinoma of the cervix. Recurrence was diagnosed by cytology and/or biopsy, or CT. Either cisplatin 100 mg/body and doxorubicin hydrochloride 40 mg/body or cisplatin 50-100 mg/body and pirarubicin 40-60 mg/body were infused after the bilateral internal iliac arteries had been occluded using balloon catheters. As the largest diameter of the tumors on CT increased, the mean survival after BOAI decreased. The mean survival of 4 patients with no detectable masses on CT was 45 +/- 30.7 months. In 5 patients, neurological complications, subcutaneous and/ or skin reactions of the buttock, or necrosis of the uterus developed. The neurological complications were damage to the sciatic nerve at the level of S1 or S2. Our study suggests that BOAI therapy may lead to a high complication rate in patients with locally recurrent carcinoma of the cervix who previously received radiation therapy, although long-term survival can be expected in patients with no detectable masses on CT.

Thermal unfolding of the starch binding domain of Aspergillus niger glucoamylase.

A fragment of the starch-binding domain (SBDF) of Aspergillus niger glucoamylase was prepared using recombinant DNA techniques, and its thermal unfolding was investigated by high-sensitivity differential scanning calorimetry (DSC). Thermal unfolding of SBDF was found to be reversible at pH 7 as expected from a DSC study of the whole enzyme molecule [Tanaka A. et al., J. Biochem., 117, 1024-1028 (1995)] but not reversible at acidic region. Numerical analysis of the DSC curves showed that the denaturation was two-state, and some of the SBDF molecules were oligomeric (average degree of oligomerization was 1.2) at pH 7. It was suggested that the denaturation temperature of SBDF was lower than that of the starch-binding domain in the whole enzyme molecule by about 4.5 degrees (decrease in the Gibbs energy change was 5.3 kJ mol-1) indicating a possibility that the starch-binding domain is stabilized by glycosylation of the domain itself, or by the highly glycosylated linker region.

Steady-state inhibitory kinetic studies on the ligand binding modes of Aspergillus niger glucoamylase.

Inhibitory activities of 1-deoxynojirimycin and gluconolactone on Aspergillus niger glucoamylase were studied in relation to the subsite structure of the enzyme. Although both of these inhibitors are considered to bind at subsite 1 of the enzyme active site, 1-deoxynojirimycin showed competitive type inhibition but gluconolactone was a mixed type (or noncompetitive type) inhibitor for the hydrolysis of p-nitrophenyl alpha-D-glucoside. The former type of inhibition suggested that the main binding mode of the substrate was productive, but the latter, nonproductive. A possible way of explaining these apparent inconsistent results is to assume that the main binding mode of the substrate is productive and gluconolactone forms a nonproductive ternary complex with the enzyme and the substrate.

Usefulness of the sensitivity-resistance index to estimate the toxicity of copper on bacteria in copper-contaminated soils.

Examination was made of the fluctuations of numbers of the total bacteria and copper (Cu)-resistant bacteria with soluble/exchangeable Cu (Ex-Cu) fraction in three types of soils spiked with Cu at four concentrations. Drastic increase in Cu-resistant bacteria was observed in three soils spiked with 20 mmol Cu kg(-1) after 2 weeks of incubation, indicating the strong selection of individuals originally resistant to Cu. Adaptation and proliferation of bacteria were also observed in the soil environment under the long-term exposure to extremely high concentration of Cu (800 mg kg(-1) soil of Ex-Cu), deriving from the development of Cu resistance. These bacterial fluctuations and the toxic effects of Cu depended on soil types, due to the chemical forms in which Cu occurs. It was also found that the ratio of Cu-resistant bacterial number to total bacteria was significantly correlated with the amount of Ex-Cu in the soils. This sensitivity-resistance index seems to be useful for evaluating the toxic effects of Cu on the soil bacterial community. Whereas the toxicity of Cu depended on the soil properties, they also changed with time. This phenomenon can be explained by the decrease in the most labile Cu phase, Ex-Cu, with time in the soils.

Isolation of two different phenotypes of mycorrhizal mutants in the model legume plant Lotus japonicus after EMS-treatment.

Lotus japonicus has been proposed as a model plant for the molecular genetic study of plant-microbe interaction including Mesorhizobium loti and arbuscular mycorrhizal (AM) fungi. Non-mycorrhizal mutants of Lotus japonicus were screened from a collection of 12 mutants showing non-nodulating (Nod-), ineffectively nodulating (Fix-) and hypernodulating (Nod++) phenotypes with monogenic recessive inheritance induced by EMS (ethylmethane sulfonate) mutagenesis. Three mycorrhizal mutant lines showing highly reduced arbuscular mycorrhizal colonization were obtained. All of them were derived from Nod- phenotypes. In Ljsym72, the root colonization by Glomus sp. R-10 is characterized by poor development of the external mycelium, formation of extremely branched appressoria, and the blocking of hyphal penetration at the root epidermis. Neither arbuscules nor vesicles were formed in Ljsym72 roots. Fungal recognition on the root surface was strongly affected by the mutation in the LjSym72 gene. Unique characteristics in mutant lines Ljsym71-1 and Ljsym71-2 were the overproduction of deformed appressoria and arrested hyphal penetration of the exodermis. Small amounts of internal colonization including degenerated arbuscule formation occurred infrequently in these types of mutants. Not only fungal development on the root surface but also that in the root exodermis and cortex was affected by the mutation in LjSym71 gene. These mutants represent a key advance in molecular research on the AM symbiosis.

Pro-opiomelanocortin messenger RNA levels in anterior pituitaries of female Wistar fatty rats.

We have studied pro-opiomelanocortin (POMC) gene expression by quantifying the POMC mRNA contents in anterior pituitaries of female Wistar fatty rats, a strain obtained by transfer of the fa gene in Zucker rat to Wistar Kyoto rat, in an attempt to understand the role of ACTH synthesis in altered ACTH and corticosterone secretion in these rats. Five- and 12-week-old female Wistar fatty rats and their lean littermates were examined. Plasma ACTH levels were significantly higher in fatty rats than in lean rats (5 weeks: 114.5 +/- 17.5 pg/ml vs. 54.3 +/- 12.4 pg/ml; 12 weeks: 83.8 +/- 12.3 pg/ml vs. 51.7 +/- 6.8 pg/ml; P < 0.01). There was no significant difference between 5-week-old fatty rats and lean rats in POMC mRNA contents nor in POMC/beta-actin ratios in anterior pituitaries. Twelve-week-old fatty rats, however, had significantly higher POMC mRNA contents and also higher POMC/beta-actin ratios in anterior pituitaries than those in lean littermates (P < 0.01, approximately three-fold difference between lean and obese rats). These data suggest that the difference in POMC mRNA contents between lean and obese rats becomes apparent as they grow and develop obesity and the elevated POMC mRNA levels are at least partly responsible for the increased ACTH secretion in 12-week-old fatty rats.

AT motif binding factor 1-A (ATBF1-A) negatively regulates transcription of the aminopeptidase N gene in the crypt-villus axis of small intestine.

This is the first study to demonstrate that the AT motif binding factor 1-A (ATBF1-A) is expressed in the crypts and the bases of villi of the small intestine and negatively regulates transcription of brush-border enzyme gene, aminopeptidase-N (APN). In situ hybridization visualized a limited ATBF1-A mRNA expression in the crypts and the bases of villi. Transient transfection and dual luciferase-reporter assay demonstrated that ATBF1-A suppressed the activity of APN promoter, but did not that of AT motif deleted promoter. These results imply that ATBF1-A inhibits the transcription of APN gene through its direct binding to the AT motif element. Furthermore, butyrate-induced differentiation of Caco-2 cells, retaining the enterocytic phenotypes such as a villus structure and the expression of brush-border enzymes, leads to a reduced expression of ATBF1-A mRNA. We proposed that ATBF1-A regulating APN gene expression in the crypt-villus axis of the small intestine is a landmark of enterocyte differentiation and maturation.