Microbial diversity and iron oxidation at Okuoku-hachikurou Onsen, a Japanese hot spring analog of Precambrian iron formations.

Banded iron formations (BIFs) are rock deposits common in the Archean and Paleoproterozoic (and regionally Neoproterozoic) sedimentary successions. Multiple hypotheses for their deposition exist, principally invoking the precipitation of iron via the metabolic activities of oxygenic, photoferrotrophic, and/or aerobic iron-oxidizing bacteria. Some isolated environments support chemistry and mineralogy analogous to processes involved in BIF deposition, and their study can aid in untangling the factors that lead to iron precipitation. One such process analog system occurs at Okuoku-hachikurou (OHK) Onsen in Akita Prefecture, Japan. OHK is an iron- and CO2 -rich, circumneutral hot spring that produces a range of precipitated mineral textures containing fine laminae of aragonite and iron oxides that resemble BIF fabrics. Here, we have performed 16S rRNA gene amplicon sequencing of microbial communities across the range of microenvironments in OHK to describe the microbial diversity present and to gain insight into the cycling of iron, oxygen, and carbon in this ecosystem. These analyses suggest that productivity at OHK is based on aerobic iron-oxidizing Gallionellaceae. In contrast to other BIF analog sites, Cyanobacteria, anoxygenic phototrophs, and iron-reducing micro-organisms are present at only low abundances. These observations support a hypothesis where low growth yields and the high stoichiometry of iron oxidized per carbon fixed by aerobic iron-oxidizing chemoautotrophs like Gallionellaceae result in accumulation of iron oxide phases without stoichiometric buildup of organic matter. This system supports little dissimilatory iron reduction, further setting OHK apart from other process analog sites where iron oxidation is primarily driven by phototrophic organisms. This positions OHK as a study area where the controls on primary productivity in iron-rich environments can be further elucidated. When compared with geological data, the metabolisms and mineralogy at OHK are most similar to specific BIF occurrences deposited after the Great Oxygenation Event, and generally discordant with those that accumulated before it.

Binding of the La autoantigen to the 5' untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro.

Human translation elongation factor 1A (EF1A) is a member of a large class of mRNAs, including ribosomal proteins and other translation elongation factors, which are coordinately translationally regulated under various conditions. Each of these mRNAs contains a terminal oligopyrimidine tract (TOP) that is required for translational control. A human growth hormone (hGH) expression construct containing the promoter region and 5' untranslated region (UTR) of EF1A linked to the hGH coding region (EF1A/hGH) was translationally repressed following rapamycin treatment in similar fashion to endogenous EF1A in human B lymphocytes. Mutation of two nucleotides in the TOP motif abolished the translational regulation. Gel mobility shift assays showed that both La protein from human B lymphocyte cytoplasmic extracts as well as purified recombinant La protein specifically bind to an in vitro-synthesized RNA containing the 5' UTR of EF1A mRNA. Moreover, extracts prepared from rapamycin-treated cells showed increased binding activity to the EF1A 5' UTR RNA, which correlates with TOP mRNA translational repression. In an in vitro translation system, recombinant La dramatically decreased the expression of EF1A/hGH construct mRNA, but not mRNAs lacking an intact TOP element. These results indicate that TOP mRNA translation may be modulated through La binding to the TOP element.

Induction of differentiation of human myeloid leukemia cells by novel synthetic neurotrophic pyrimidine derivatives.

OBJECTIVE: Some pyrimidine analogues have been found to induce differentiation of several human myeloid leukemia cells. Newly synthesized heterocyclic pyrimidine derivatives promote neurite outgrowth and survival in neuronal cell lines. In this study, the growth-inhibiting and differentiation-inducing effects of these pyrimidine derivatives on human myeloid leukemia cells were examined. MATERIALS AND METHODS: Several myeloid leukemia cells were cultured with novel heterocyclic pyrimidine derivatives. Cell differentiation was determined by nitroblue tetrazolium-reducing activity, morphologic changes, expression of CD11b, lysozyme activity, and hemoglobin production. RESULTS: MS-430 (2-piperidino-5,6-dihydro-7-methyl-6-oxo (7H) pyrrolo [2,3-d] pyrimidine maleate) effectively induced HL-60 cells into mature granulocytes. MS-430 activated the mitogen-activated protein kinase (MAPK) of the cells before causing granulocytic differentiation. MAPK activation was necessary for MS-430-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by MS-430. MS-430 also induced monocytic differentiation of THP-1, P39/Tsu, and P31/Fuj leukemia cells, but did not affect erythroid differentiation of K562 or HEL cells. CONCLUSIONS: MS-430 potently induces differentiation of some myelomonocytic leukemia cells. This novel synthesized pyrimidine compound shows promise as a therapeutic agent for treatment of leukemia and as a neurotrophic drug.

Expression of sodium/proton antiporter NhaA at various pH values in Escherichia coli.

It was reported that NhaA, one of sodium/proton antiporters in Escherichia coli, was expressed at alkaline pH [J. Biol. Chem. 266 (1991) 21753]. In disagreement with their results, expression of an nhaA-lacZ fusion gene was found to be very low in an E. coli strain derived from MC4100 within the wide pH range from 5 to 9. When nhaB was deleted, the fusion gene was expressed at pH values below 8, while the expression was observed at alkaline pH after chaA was deleted. The internal level of sodium ions was increased by deletion of nhaA in strains deficient in nhaB and chaA at low and high pH values, respectively. These results suggested that nhaA is induced only when a low level of internal sodium ions is not kept by NhaB and ChaA. Strains used in the previous study may have low active ChaA.

Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes.

We examined and compared enantioselectivity in the oxidation of propranolol (PL) by liver microsomes from humans and Japanese monkeys (Macaca fuscata). PL was oxidized at the naphthalene ring to 4-hydroxypropranolol, 5-hydroxypropranolol and side chain N-desisopropylpropranolol by human liver microsomes with enantioselectivity of [R(+)>S(-)] in PL oxidation rates at substrate concentrations of 10 microM and 1 mM. In contrast, reversed enantioselectivity [R(+)

[Interim report of JFMTC study no. 20 on the effectiveness of high dose CDDP plus 5-FU regimen as an adjuvant therapy for far-advanced cancer of the stomach].

This interim analysis of the JFMTC study as of May, 1998 covers 321 gastrectomized patients with far-advanced stomach cancer from 135 institutions between November, 1993 and March, 1996. The intensive therapy group (I-group) received CDDP i.p. administration on resective surgery with 70 mg/m2 followed by CDDP i.v. of 80 mg/m2 (day 1, i.v.), accompanying 5-FU of 350 mg/m2/day (day 1-5, c.v.i.) in the 4th, 8th and 12th weeks. The I-group was randomly compared with the standard therapy group (S-group) of MMC of 6 mg/m2 i.v. in the 4th, 8th and 12th weeks and UFT of 3-4 capsules daily for postoperative one year. The results obtained were that 1. adverse reactions were found more in the I-group than in the S-group, particularly notable in the decrease in blood cells, loss of appetite and nausea/vomiting, and incidence of grade 3 or more being 13% (neutrophile leukocytes), 26% and 21%, respectively; 2. there was no significant difference between I- and S-groups in terms of 3-year survival or disease-free survival rates. (JFMTC: Japanese Foundation of Multidisciplinary Treatment for Cancer).

Expression of outer membrane proteins in Escherichia coli growing at acid pH.

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.

[Interim report on JFMTC Study no. 21 on the effectiveness of UFT as an adjuvant therapy for semi-advanced cancer of the stomach].

This interim report, for findings as of May, 1998, covers data on 435 gastrectomized patients with semi-advanced stomach cancer collected from 144 institutions between November, 1993 and March, 1996. The active arm of the study involved CDDP i.p. administration of 70 mg/m2 at the time of resective surgery, followed by UFT oral administration for one year at 3-4 capsules daily. A randomized control involved no adjuvant therapy after CDDP i.p. administered as in the active arm. The results obtained indicated no significant difference between the groups in terms of 3 year survival or disease free survival rates. Reports appearing elsewhere have suggested that 3-4 capsules/day of UFT may be insufficient to reach the threshold of the effective tissue level, and that 6 capsules may be necessary to obtain the expected results. (JFMTC: Japanese Foundation of Multidisciplinary Treatment for Cancer).

[The history of surgical treatment for esophageal carcinoma in the 20th century in Japan].

In the 20th century surgical results in the treatment of esophageal carcinomas significantly improved in Japan. For the reason, I review the trends in esophageal cancer surgery in Western countries and discuss the reasons for the choice of surgical procedures. In Japan, esophageal cancer surgery was initiated with the reports of surgical results presented by Professors Seo and Osawa at the Congress of the Japan Surgical Society held in 1933. Subsequently, the results in esophageal cancer patients undergoing surgical resection were improved with the progress in anesthesia. Subsequent to the establishment of the Japan Society of Esophageal Diseases in 1965, any institutes performed esophagectomy for esophageal carcinomas, and the procedure accounted for less than 2% of all operative deaths in Japan. The main reason for this reduction in the number of surgical deaths was improved perioperative care for major complications (cardiopulmonary failure and anastomotic leakage). In particular, the surgical procedure for curative lymphadenectomy called three-field dissection significantly improved surgical prognosis. In the last 10 years, the relationship between the number of positive lymph nodes and surgical prognosis was demonstrated in advanced esophageal cancers, while endoscopic mucosal resection was also performed for early esophageal cancers as a curative treatment. In the next century, better treatment should be designed for individual patients based on fundamental studies and clinical trials.

Study of the functional share of lysosomal cathepsins by the development of specific inhibitors.

To analyze the functional share of individual cathepsins, we developed powerful and specific inhibitors for individual cathepsins using computer graphics of substrate binding pockets based on X-ray crystallography. These new inhibitors were named CLIK group. Epoxy succinate peptide derivatives, CLIK-066, 088, 112, 121, 148, 181, 185 and 187, are typical specific inhibitors for cathepsin L. Aldehyde derivatives CLIK-060 and CLIK-164 showed specific inhibition against cathepsin S and cathepsin K, respectively. We found that pyridoxal phosphate (PLP), a coenzyme form of vitamin B6, inhibits all cathepsins and also new artificially synthesized pyridoxal derivatives, CLIK-071 and -072, in which the phosphate esters of PLP were replaced by propionic acid, exhibited strong inhibition for cathepsins. Furthermore, CLIK-071 was easy to incorporate into cells and showed powerful inhibition for intracellular cathepsins. Using these selective inhibitors, the allotment of individual cathepsin functions in cells has been studied as follows. Cathepsin L and/or K participate in bone resorption based on bone type-1 collagen degradation and the L-type protease inhibitors suppressed the bone resorption. Cathepsins B and S participate in antigen presentations based on antigen processing and invariant chain degradation, respectively. Also cathepsin L participates in cell apoptosis mediated by caspase III activation.

Cation movements at alkaline pH in bacteria growing without respiration.

The respiratory chain has a central role in energy metabolism as a generator of a proton motive force in aerobic bacteria. In contrast, Enterococcus hirae (formerly Streptococcus faecalis), which lacks the respiratory chain, generates this proton gradient via a F-type H+ ATPase, but it works only at a pH below 8; no significant proton motive force is generated at a pH above 8. An Escherichia coli mutant deficient in both the respiratory chain and the H+ ATPase grew with a negligible proton motive force within the wide range of medium pH. It has been suggested that both E. hirae and E. coli are able to grow even when the cytoplasm is alkalinized beyond pH 8. These observations lead to the conclusion that bacteria can survive without operating cation transport systems driven by a proton motive force at alkaline pH. The activity of any transport system with optimum pH around neutrality should decline when both the outside and inside of cells are alkalinized. Thus, changes in transport systems as well as cellular metabolism may be essential for bacterial adaptation to changes in environmental pH.

Total esophagectomy versus proximal esophagectomy for esophageal cancer at the cervicothoracic junction.

To investigate the adequate extent of esophagectomy and lymphadenectomy for an esophageal cancer localized at the cervicothoracic junction, the mortality and morbidity rates, survival rates, and patterns of recurrence were retrospectively analyzed in two groups-14 patients who underwent total esophagectomy with or without laryngectomy and 15 patients who underwent proximal esophagectomy with or without laryngectomy-at Kurume University Hospital from 1981 to 1996. Proximal esophagectomy with or without laryngectomy resulted in a lower hospital mortality rate and better overall survival for patients who underwent curative esophagectomy compared with total esophagectomy with or without laryngectomy. Multivariate analysis indicated that the extent of esophagectomy (total esophagectomy versus proximal esophagectomy) was not a prognostic factor. The incidence of recurrence was not different between the two groups. Lymph node metastasis or recurrence from such esophageal cancers was localized to the neck and upper mediastinum. For an esophageal cancer localized at the cervicothoracic junction, therefore, proximal esophagectomy with or without laryngectomy and with cervical and upper mediastinal lymphadenectomy could be better indicated for preselected patients.

Enzyme level of enterococcal F1Fo-ATPase is regulated by pH at the step of assembly.

The amount of F1Fo-ATPase in Enterococcus hirae (formerly Streptococcus faecalis) increases when the cytoplasmic pH is lowered below 7.6, and protons are extruded to maintain the cytoplasmic pH at around 7.6. In the present study, we found that the transcriptional activity of the F1Fo-ATPase operon was not regulated by pH. The synthesis of F1 subunits was increased 1.65 +/- 0.12-fold by the acidification of medium from pH 8.0 to pH 5.3. Western-blot analysis showed that there were F1 subunits in the cytoplasm, and the number of alpha plus beta subunits in the cytoplasm was 50% of the total number of the subunits in cells growing at pH 8.0. This decreased to 22% after shifting the medium pH to 5.3, with a concomitant 5.1-fold increase in the level of membrane-bound F1Fo-ATPase. The cytoplasmic F1 subunits were shown to be degraded, and Fo subunits not assembled into the intact F1Fo complex were suggested to be digested. These data suggest that regulation of the enzyme level of F1Fo-ATPase by the intracellular pH takes place mainly at the step of enzyme assembly from its subunits.

Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor.

In the search for an endogenous ligand of the orphan G protein-coupled receptor APJ, the presence of the ligand in various tissue extracts was examined by measuring the increase in extracellular acidification rate of the cells expressing the APJ receptor as a specific signal induced by the interaction of the receptor and ligand. By monitoring this activity, we isolated an APJ receptor ligand, designated apelin, from bovine stomach extracts. The structures of bovine and human apelin preproproteins were deduced from the sequences of the corresponding cDNAs. The preproproteins consisted of 77 amino acid residues, and the apelin sequence was encoded in the C-terminal regions. Synthetic peptides derived from the C-terminal amino acid sequence of bovine preproapelin were capable of specifically promoting the acidification rate in the cells expressing the APJ receptor in a range from 10(-7) to 10(-10) M, indicating that apelin is an endogenous ligand for the APJ receptor.

Treatment of superficial cancer of the esophagus: a summary of responses to a questionnaire on superficial cancer of the esophagus in Japan.

BACKGROUND: Histopathologic characteristics and optimal treatment modality for superficial esophageal cancer were reevaluated on the basis of 2418 patients from 143 institutions through a nationwide questionnaire to the members of the Japanese Society for Esophageal Diseases. METHODS: A questionnaire was designed for patients with preoperatively untreated superficial cancer of the esophagus who had undergone either surgical or endoscopic treatment between January 1, 1990, and December 30, 1994. Mucosal cancer and submucosal cancer were divided into three subclasses according to the criteria formulated by the Society. RESULTS: The incidence of positive lymphatic invasion or lymph node metastases tended to increase markedly as cancer infiltrates reached the lamina muscularis mucosa. The majority of the cases with 0-I or 0-III components were submucosal cancer. The indication of endoscopic mucosal resection (EMR) was limited to mucosal 1 and mucosal 2 superficial cancer in 76% of the institutions surveyed. Tumors measuring 2 cm or more in diameter were resected piecemeal in 94% of patients. Complications of EMR, including perforation, stenosis, and hemorrhage, were observed in approximately 6.8% of patients. Almost all patients with mucosal 1 or mucosal 2 cancer are still alive. There was no significant difference in prognosis between mucosal 3 cancer and mucosal 1 or mucosal 2 cancer, but submucosal 1 cancer showed worse prognosis than mucosal cancer. CONCLUSIONS: Local resection of cancer lesions is regarded as the treatment of choice against the superficial esophageal cancers limited to the lamina propria mucosae. Further study is advocated to define the treatment strategy against mucosal 3 or submucosal 1 cancer.

pH dependence of the function of sodium ion extrusion systems in Escherichia coli.

Escherichia coli has three systems for sodium ion extrusion, NhaA, NhaB and ChaA. In this study, we examined the effect of pH on the function of these transporters using mutants having one of them, and found that (1) a mutant having NhaB excreted sodium ions at pH 7.5 but not at pH 8.5, (2) the efflux of sodium ions from mutant cells having ChaA was observed at both pH 7.5 and 8.5, but the activity was lower at pH 7.5, and (3) sodium ions were excreted from mutant cells having NhaA at pH 6.5 to 8.5. The extrusion activity of cells having NhaA was higher than that of cells having NhaB or ChaA. These results indicate that NhaB functions at a pH below 8, and ChaA extrudes sodium ions mainly at an alkaline pH above 8. It was also suggested that the activity of NhaB and ChaA is not enough to maintain a low level of internal sodium ions when the external concentration of sodium ions is high, and NhaA is induced within a wide range of medium pH under such conditions.

Postoperative adjuvant chemotherapy with 1-hexylcarbamoyl-5-fluorouracil in patients with colorectal cancer and at a high risk for recurrence.

We examined the effects of postoperative 5-fluorouracil (5-FU) infusions and oral treatment with 1-hexylcarbamoyl-5-fluorouracil (HCFU) on patients curatively resected for stages II-IV colorectal cancer. The study was prospectively randomized, and 251 (93.3%) of 269 patients were valid candidates for statistical assessment. The inductive regimen for group A included 5-FU 10 mg intravenous (i.v.) injections on days 0, 1, 2, 7, 8 and 9, postoperatively. For maintenance therapy, group A received HCFU 300 mg orally and daily for 52 weeks beginning 2 weeks after surgery. The regimen for group B included only 5-FU injections. The effects of this chemotherapy were also retrospectively analyzed for groups at a high risk for recurrence, stages III-IV, transmural invasion-positive and lymph node metastasis-positive cases. There was no statistical difference in survival time between the groups for 251 eligible cases (p = 0.079). In group A given 5-FU plus HCFU, there was a reduction in the recurrence rate for patients with stages III-IV or lymph node metastasis-positive colorectal cancers (p < 0.05) and prolongation of the survival time for patients with stage III-IV, transmural invasion-positive or lymph node-positive colorectal cancers (p < 0.05). Our findings show that the combination of 5-FU infusion and the continuous administration of HCFU is effective in treating patients with surgically resected colorectal cancer who are at high risk for a recurrence.

Growth of an Escherichia coli mutant deficient in respiration.

An Escherichia coli mutant deficient in genes for heme biosynthesis grew in medium of initial pH 8 containing 1% tryptone and glucose under aerobic growth conditions, and its doubling time was approximately 60 min at 37 degrees C. The growth rate was not increased under O2-limiting conditions. When the mutant was grown in medium of initial pH 6, growth stopped at the middle of the exponential growth phase. This could be overcome and the growth yield increased by the addition of 20 mM lysine to the growth medium. Lysine did not prevent the decrease in the medium pH as growth proceeded, making it unlikely that lysine decarboxylation stimulates growth by the alkalinization of the medium. These results indicate that respiration is not obligatory for growth under aerobic conditions, but growth without respiration at low pH requires a large amount of lysine.