Swallow-induced esophageal shortening in patients without hiatal hernia is associated with gastroesophageal reflux.

Longitudinal esophageal body shortening with swallow-induced peristalsis has been reported in healthy individuals. Esophageal shortening is immediately followed by esophageal re-elongation, and the lower esophageal sphincter (LES) returns to the baseline position. High-resolution manometry (HRM) allows for objective assessment of extent of shortening and duration of shortening. In patients without hiatal hernia at rest, swallow-induced esophageal shortening can lead to transient hiatal hernia (tHH) which at times may persist after the completion of swallow. This manometric finding has not been investigated in the literature, but a question arises whether this swallow-induced transient herniation can effect on the likelihood of gastroesophageal reflux. This study aims to assess the relationship between gastroesophageal reflux and the subtypes of swallow-induced esophageal shortening, i.e. tHH and non-tHH, in patients without hiatal hernia at rest. After Institutional Review Board (IRB) approval, we queried a prospectively maintained database to identify patients who underwent HRM evaluation and 24-hour pH study between January to December 2015. Patients with type-I esophagogastric junction (EGJ) morphology (i.e. no hiatal hernia) according to the Chicago classification v3.0 were included. The patterns of the esophageal shortening with swallows were divided into two subtypes, i.e. tHH and non-tHH. tHH was defined as an EGJ double high-pressure zones (≥1 cm) at the second inspiration after the termination of swallow-induced esophageal body contraction. The number of episodes of tHH was counted per 10 swallows and tHH size was measured for each patient. In total, 41 patients with EGJ morphology Type-I met the inclusion criteria. The mean age was 47.2 years, 35 patients (85.4%) were women, and the mean body mass index was 33.9 kg/m2. The mean number of tHH episodes was 3 out of 10 swallows; mean maximal tHH size was 1.3 cm. Patients who had tHH in ≥3 out of 10 swallows (n = 16; 39.0%) were more likely to have abnormal DeMeester scores than patients with <3 swallows (56% vs. 28%; P = 0.070). Patients with maximal tHH ≥2 cm in at least 1 swallow (n = 17; 41.5%) were more likely to experience pathological reflux than patients with maximal tHH <2 cm (59% vs. 25%; P = 0.029). In conclusion, we showed that, in a subset of patients with Type-I EGJ morphology, swallowing induced transient EGJ double high-pressure zones (≥1 cm) after peristalsis. We have named this new manometric finding the swallow-induced tHH. A high prevalence of pathological reflux disease was observed in patients with maximal tHH ≥2 cm. The degree of swallow-induced tHH could be an early indicator of lower esophageal sphincter dysfunction in patients without manometric hiatal hernia.

Frequency-dependent selection acting on the widely fluctuating sex ratio of the aphid Prociphilus oriens.

Frequency-dependent selection is a fundamental principle of adaptive sex ratio evolution in all sex ratio theories but has rarely been detected in the wild. Through long-term censuses, we confirmed large fluctuations in the population sex ratio of the aphid Prociphilus oriens and detected frequency-dependent selection acting on these fluctuations. Fluctuations in the population sex ratio were partly attributable to climatic factors during the growing season. Climatic factors likely affected the growth conditions of host plants, which in turn led to yearly fluctuations in maternal conditions and sex ratios. In the process of frequency-dependent selection, female proportion higher or lower than ca. 60% was associated with a reduction or increase in female proportion, respectively, the next year. The rearing of aphid clones in the laboratory indicated that mothers of each clone produced an increasing number of females as maternal size increased. However, the mean male number was not related to maternal size, but varied largely among clones. Given genetic variance in the ability to produce males among clones, selection should favour clones that can produce more numerous males in years with a high female proportion. Population-level sex allocation to females was on average 71%-73% for three localities and more female-biased when maternal conditions were better. This tendency was accounted for by the hypothesis of competition among foundresses rather than the hypothesis of local mate competition. We conclude that despite consistent operation of frequency-dependent selection, the sex ratio continues to fluctuate because environmental conditions always push it away from equilibrium.

Classification for esophagogastric junction (EGJ) complex based on physiology.

We propose a new classification for esophagogastric junction (EGJ) incorporating both physiologic and morphologic characteristics. Additionally, we contrast it with the Chicago v 3.0 EGJ classification. With Institutional Review Board (IRB) approval, prospectively maintained database was queried to identify patients who underwent high-resolution manometry (HRM) and pH-study between October 2011 and October 2015. Patients with prior foregut intervention, pH study on acid suppression, esophageal dysmotility, or lower esophageal sphincter-crural diaphragm separation of >5 cm were excluded. We classified patients into three groups-Type-A: Complete overlap of lower esophageal sphincter-crural diaphragm (single high-pressure zone); Type-B: Double high-pressure zone with pressure inversion point (PIP) at or above lower esophageal sphincter; Type-C: Double high-pressure zone with PIP below lower esophageal sphincter. A total of 214 included patients were divided into Type-A (n = 101), Type-B (n = 32), and Type-C (n = 81). Abdominal lower esophageal sphincter length (AL), lower esophageal sphincter pressure (LESP), and lower esophageal sphincter pressure integral (LESPI) were significantly lower in Type-C than both Type-A and Type-B [AL(cm): 0.2 vs. 2(P < 0.001) vs. 1.6(P <0.001); LESP(mmHg): 20.1 vs. 32.1(P < 0.001) vs. 29.2(P < 0.001); LESPI(mmHg.cm.s): 187 vs. 412(P < 0.001) vs. 343(P < 0.05)] while overall lower esophageal sphincter length(OL) and Integrated Relaxation Pressure (IRP) were significantly lower in Type-C than Type-A [OL(cm): 2.9 vs. 3.6(P < 0.001); IRP(mmHg): 8.2 vs. 9.6(P < 0.05)]. Type-C patients had significantly higher positive pH score (>14.7) than Type-A and Type-B [72% vs. 47% (P < 0.05) vs. 41% (P < 0.001)]. In Type-C morphology, there is both anatomical and physiological deterioration, weakest lower esophageal sphincter function (abdominal length, lower esophageal sphincter pressure, and lower esophageal sphincter pressure integral) and is most likely to be associated with pathological reflux. This proposed classification incorporates both physiological and morphological derangements in a graded fashion.

Spatiotemporal fluctuations in natural selection acting on the gall-parasitic aphid Tetraneura sorini.

The measurement of the selection gradient is crucial for understanding the magnitude of selection acting directly on a trait and predicting the evolutionary trajectory of that trait. This study evaluated the selection gradient acting on the morphology of the gall-parasitic aphid Tetraneura sorini during the galling process and compared the strength among populations. Gall formers (first instars) frequently fight with conspecifics or heterospecifics for usurping incipient galls using their well-developed hind legs. First instars that successfully acquired galls were found within galls, whereas those that failed were found dead on leaf surfaces. Selection gradients were estimated using logistic stepwise regression and partial least square (PLS) regression. Calculated selection differentials indicated that first instars that secured galls were larger in body size than failed individuals through all populations. However, selection gradients on weapon traits varied largely among populations or among years in the same population. We confirmed microevolutionary changes in the relationship between traits, which accorded with the expectation from changes in the selection gradients. When gall formers were transferred onto developing buds individually, individuals that successfully induced galls had smaller body size than failed individuals. Available evidence suggests that the selection gradient on body size becomes higher with an increasing proportion of T. sorini in the Tetraneura species community. Thus, we concluded that more intense fighting with conspecifics leads to stronger selective pressure on body size, but that selective pressure for each trait is variable depending on differences in the tactics and species composition among populations.

High-resolution manometry findings in symptomatic post-Nissen fundoplication patients with normal endoscopic configuration.

The aim of this study was to investigate high-resolution manometry (HRM) findings in symptomatic post-fundoplication patients with normal endoscopic configuration. A retrospective review of a prospectively maintained database was conducted to identify patients who underwent evaluation with HRM and endoscopy for symptom evaluation after previous fundoplication. Study period extends from September 2008 to December 2012. Only patients with complete 360° fundoplication (Nissen) were included, and patients with partial fundoplication were excluded. Patients with endoscopic abnormality or patients who underwent Collis procedure were also excluded. Additionally, contrast study and 24-hour pH study if done were reviewed. Symptoms were graded using a standard questionnaire with symptoms graded on a scale of 0-3. Symptom grade 2 or 3 was considered a significant symptom. One hundred seventy-nine symptomatic patients with previous Nissen fundoplication underwent HRM and endoscopy during the study period. Of these, 136 patients were excluded (51 had recurrent hiatal hernia, 2 had disrupted fundoplication, 68 had slipped fundoplication, 10 had twisted fundoplication, 2 had esophageal stricture, and 3 had Collis procedure). Remaining forty-three patients met inclusion criteria (mean age of 56.0 ± 14.8, 32 females).The most common symptom was dysphagia (67%). Patients with dysphagia had a significantly longer length of distal esophageal high pressure zone (HPZ) and a higher integrated relaxation pressure (IRP) than patients without dysphagia (P = 0.020, 0.049). Especially, patients who had shorter HPZ (≤2 cm) were less likely to have significant dysphagia. Twenty-three patients (53%) had heartburn. There was no significant difference in HRM findings between patients with and without heartburn. Only 4 of 28 patients with concomitant pH study showed abnormal DeMeester score (>14.7), and there was no correlation between results of pH study and lower esophageal sphincter pressure/length and IRP. Longer HPZ complex length and higher IRP as measured with HRM is associated with post-Nissen fundoplication dysphagia in patients with normal endoscopic configuration. No HRM parameters are associated with reported heartburn or a positive pH score.

Effect of endurance training supplemented with green tea extract on substrate metabolism during exercise in humans.

Endurance training and ingestion of green tea extract (GTE), composed mainly of tea catechins (TC), are well known to enhance fat metabolism. However, their synergistic effects remain to be fully elucidated. We tested the hypothesis that endurance training supplemented with GTE would further accelerate whole-body fat utilization during exercise, compared with training alone, in humans. Twelve healthy male subjects [peak oxygen consumption (VO2peak), 50.7 ± 1.3 (SEM) mL/kg/min] were divided into two groups: GTE and placebo (PLA) groups. Subjects in both groups performed a cycle ergometer exercise at 60% of VO2peak for 60 min/day, 3 days/week, and daily ingested 572.8 or 0 mg TC in GTE and PLA groups for 10 weeks, respectively. Before and after training, respiratory gas exchange was measured during 90-min exercise at pre-training ∼55% of VO2peak. After training, the average respiratory exchange ratio during exercise remained unchanged in the PLA group (post-training: 0.834 ± 0.008 vs pre-training: 0.841 ± 0.004), whereas it was lower in the GTE group (post-training: 0.816 ± 0.006 vs pre-training: 0.844 ± 0.005, P<0.05). These results suggest that habitual GTE ingestion, in combination with moderate-intense exercise, was beneficial to increase the proportion of whole-body fat utilization during exercise.

Distribution and species composition of juvenile and adult scombropids (Teleostei, Scombropidae) in Japanese coastal waters.

Two scombropid fishes, Scombrops boops and Scombrops gilberti, are closely related and commercially important species in Japan. These species are often confused in commercial markets because of their morphological similarity. In this study, scombropid specimens collected from various Japanese coastal waters were subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and phylogenetic analysis of the 16S rRNA gene in mitochondrial DNA. These analyses showed that all the scombropid specimens collected from localities in the Sea of Japan were identified as S. boops, whereas those from the Pacific Ocean included two species, S. boops and S. gilberti. Almost all juvenile (<200 mm standard body length, S(L)) S. gilberti originated from the Pacific coastal waters of the northern Japan, whereas adults (>400 mm S(L)) were found only in deep water off the Izu Peninsula to the Izu Islands. This suggests that S. gilberti might migrate extensively during its life cycle. In addition, differences in the number of specimens and the distribution between the two species suggest that S. gilberti is less abundant than S. boops in Japanese waters.

Crystallization of some lunar mafic magmas and generation of rhyolitic liquid.

During crystallization of lunar crystalline rocks 10022 and 10024, augite changes in composition almost continuously from titanaugite (Ca(36)Mg(47) Fe, (17) with TiO(2) 3 percent) to a very iron-rich variety (Ca(9)Mg(5)Fe(86)), pigeonite changes from Ca(9)Mg(66)Fe(25) to Ca(1O)Mg(51)Fe(39), and olivine changes (in 10022) from Mg(71)Fe(29) to Mg(41)Fe(59), whereas plagioclase stays as bytownite. These compositional variations, as well as the textural relations, may be explained by rapid crystallization of undercooled magmas. The residual liquids found as mesostasis are rhyolitic, which suggests that fractional crystallization of some lunar mafic magmas can generate rhyolitic magmas. Melting experiments were made on crystalline rocks to determine liquidus temperatures and crystallizing phases.

Modified spinel, Beta-manganous orthogermanate: stability and crystal structure.

A new high-pressure polymorph with a modified spinel structure, beta-Mn(2)GeO(4), is stable in a pressure range intermediate between the field of the polymorph with the olivine structure and that of another high-pressure polymorph. Oxygen atoms are located approximately in cubic close packing with manganese and germanium atoms in octahedral and tetrahedral interstices, respectively, as in the spinel structure; however, germanium atoms form Ge(2)O(7) groups instead of isolated GeO(4) groups.

Relationship between ethanol consumption and TBL2 rs17145738 on LDL-C concentration in Japanese adults: a four season 3-day weighed diet record study.

BACKGROUND: LDL cholesterol (LDL-C) concentration is modified by dietary and genetic factors; however, little is known about the details of this relationship. Our aim was to investigate the associations taking into account dietary assessment methods, seasonal effects and missing values. METHODS: Study subjects completed food frequency questionnaires (FFQ) and supplied 3-day weighed dietary records (WDRs) and blood samples in four seasons. Approximately 660,000 single nucleotide polymorphisms (SNPs) were measured. Candidate SNPs related to LDL-C concentration were systematically selected. Multiple imputation was applied for missing values. A total of 312 repeated measures data were used for analyses. After adjusting for season and subjects as fixed and random effects, effects of nutrient intake and SNPs on LDL-C concentration were assessed according to three dietary assessment methods: the FFQ and first and four season 3-day WDRs (4 s-3d WDRs). RESULTS: For LDL-C concentration, ethanol consumption derived from all three dietary assessment methods was consistently associated (P < 0.09 for all). Positive and negative relationships were consistently shown with rs651007 and rs1160985 in the first and four seasons; but the latter remained after adjusting for total dietary fiber intake derived from the FFQ and 4 s-3d WDRs (P < 0.05, excepting the first 3-day WDRs). rs599839 was negatively associated after cholesterol intakes derived from the first and 4 s-3d WDRs were considered (P < 0.05 and 0.07, respectively). Each rs17145738 and ethanol consumption based on the 4 s-3d WDRs was related to LDL-C concentration (P < 0.05). Seasonal variations of LDL-C concentration were observed only in summer. CONCLUSIONS: In contrast to nutrient intake, ethanol consumption was shown to be comprehensively related to LDL-C concentration, regardless of dietary assessment methods. Taking into account seasonal effects, critical relationships with LDL-C concentration for some SNPs, after adjustment for specific nutrients, were revealed. Our findings can be used to help to interpret the relationships between dietary and genetic factors on LDL-C concentration in large-scale epidemiological studies.(10/10 keywords).

Distribution of gene segments of the pandemic A(H1N1) 2009 virus lineage in pig populations.

Swine influenza viruses (SIVs) are important not only for pig farming, but also for public health. In fact, pandemic A(H1N1) 2009 viruses [A(H1N1)pdm09] were derived from SIVs. Therefore, timely characterization of locally circulating SIVs is necessary for understanding the global status of SIVs. To genetically characterize SIVs circulating in Japanese pig populations, we isolated 24 SIVs of three subtypes (17 H1N1, four H1N2 and three H3N2 strains) from 14 pig farms in Japan from 2013 to 2016. Genetic analyses revealed that the haemagglutinin (HA) and neuraminidase (NA) genes of the 17 H1N1 and the HA gene of one H1N2, A/swine/Aichi/02/2016 (H1N2), SIVs belonged to the A(H1N1)pdm09 lineage. More importantly, all of the remaining six gene segments (i.e., PB1, PB1, PA, NP, M and NS) of the 24 SIVs, regardless of the HA and NA subtype, were also classified as belonging to the A(H1N1)pdm09 lineage. These results indicate that gene segments of A(H1N1)pdm09 lineage are widely distributed in SIVs circulating in Japanese pig populations In addition, the NA gene of A/swine/Aichi/02/2016 (H1N2) shared less than 88.5% nucleotide identity with that of the closest relative A/swine/Miyagi/5/2003 (H1N2), which was isolated in Japan in 2003. These results indicate the sustained circulation of classical H1N2-derived SIVs with remarkable diversity in the NA genes in Japanese pig populations. These findings highlight the necessity of both intensive biosecurity systems and active SIV surveillance in pig populations worldwide for both animal and public health.

Laminar shear stress inhibits vascular endothelial cell proliferation by inducing cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1)

Alterations in the functions of vascular endothelial cells (ECs) induced by fluid shear stress may play a pivotal role in both the development and prevention of vascular diseases. We found that DNA synthesis of bovine aortic and human umbilical vein ECs, determined by [(3)H]thymidine incorporation, was inhibited by steady laminar shear stress (5 and 30 dyne/cm(2)). This growth inhibition due to shear stress was associated with suppression of cell transition from the G(1) to S phase of the cell cycle. Therefore, we studied G(1)-phase events to find the molecules responsible for this cell cycle arrest. Shear stress inhibited the phosphorylation of a retinoblastoma protein (pRb) and the activity of cyclin-dependent kinase (cdk) 2 and cdk4, which phosphorylate pRb. The level of cdk inhibitor p21(Sdi1/Cip1/Waf1) protein, but not that of p27(Kip1), increased as a result of shear stress, and the amount of p21 protein associated with cdk2 also increased, although the protein level of cdk2 was unchanged. Shear stress markedly elevated the mRNA level of p21, and this elevation in mRNA faded after the release of cells from shear stress, concomitant with a recovery of DNA synthesis. These results suggest that steady laminar shear stress induces cell cycle arrest by upregulating p21. Derangement of the steady laminar flow may release cells from this inhibition and induce cell proliferation, which, in turn, may cause atherosclerosis through the induction of EC stability disruption.

Water chemistry gradient in a degraded bog area.

Surface and ground water was sampled in a degraded bog area 36 times during 1993 - 2003 at Five representative points: point E (natural area with Sphagnum as the main vegetal cover), point W (boundary between the natural and degraded areas), point W' (area installed with vinyl sheeting), point WW (area where Sasa thrives), and point NC (area with naturally formed ditches). Analysis of variance (ANOVA) was conducted for parameters measured in surface water and ground water at 0.5, 1.0, 1.5, and 2.0 m depths. "Sampling point" (i.e. locations along the degradation gradient) accounted for most of the variation in surface and ground water chemistry. It accounted for 30-80% of the total variation in pH, electrical conductivity, ammonia, dissolved nitrogen, major cations (Na+, K+, Ca2+, Mg2+), alkalinity and dissolved organic carbon. "Year" accounted for more variation in nitrate, nitrite, chloride, and sulfate than the sampling point did, but the variation in dissolved reactive phosphorus and dissolved phosphorus concentrations was not based on any of the calculated variables.

Progression and selection in heterogeneous tumor composed of androgen-responsive Shionogi carcinoma 115 and its autonomous subline (Chiba subline 2).

Shionogi Carcinoma 115 (SC 115) is an androgen-dependent mouse tumor, and Chiba subline 2 (CS 2) is its androgen-independent subline which differs from SC 115 in cell size, amount of androgen receptors, and karyotype. To shed light on the mechanism of clonal selection of androgen-independent tumors, mixed tumors with SC 115 and CS 2 were prepared, and growth of these tumors was examined in vivo and in vitro. When the mixed tumor was transplanted in mice, CS 2 showed a predominant growth over SC 115. In a culture of mixed tumor cells, however, CS 2 showed no selective growth advantage. The suppressive interaction which occurred in vivo was due neither to transferable substances, nor to some immunological factor(s). It may be, at least partially, attributable to necrosis formation in SC 115, which developed with an increase in the size of the tumor.

Alteration of beta-catenin expression in colonic epithelial cells of familial adenomatous polyposis patients.

It has been found that beta-catenin, a key regulator of the cadherin-mediated cell adhesion system, forms complexes with adenomatous polyposis coli (APC) tumor suppressor protein, and beta-catenin expression levels are affected by exogenously induced APC protein. The effects of intrinsic APC protein alteration on beta-catenin expression levels and its subcellular localization were examined in colonic epithelia of eight patients with familial adenomatous polyposis. In all eight patients, beta-catenin was immunostained at the membranes of the cell-to-cell borders in normal epithelial cells, whereas the nuclei and cytoplasms stained intensely in addition to the membranes in both adenoma and cancer cells. beta-Catenin expression levels in tumor tissues were over three times higher than those in corresponding normal mucosae of all of the three patients, whose resected specimens were available for quantitative immunoblot analysis. In these three patients, mutant truncated APC proteins were detected and shown to have lost the central region, including a known beta-catenin binding domain. beta-Catenin was not coimmunoprecipitated with these mutant APC proteins in tumor tissues but was able to be coprecipitated with glutathione S-transferase-fused APC protein containing a beta-catenin binding domain. These results suggest that the absence of wild type APC protein affects the subcellular localization and expression levels of beta-catenin in human tissues.

A truncated beta-catenin disrupts the interaction between E-cadherin and alpha-catenin: a cause of loss of intercellular adhesiveness in human cancer cell lines.

Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and are considered to work as an invasion suppressor system of cancer cells. They form a molecular complex with catenins, a group of cytoplasmic proteins including alpha- and beta-catenins. While alpha-catenin has been demonstrated to be crucial for cadherin function, the role of beta-catenin is not yet fully understood. In this study, we analyzed the cadherin-catenin system in two human cell lines, HSC-39 and its putative subline HSC-40A, derived from a signet ring cell carcinoma of stomach. These cells grow as loose aggregates or single cells, suggesting that their cadherin system is not functional. In these cell lines, an identical 321-base pair in-frame mRNA deletion of beta-catenin was identified; this led to a 107-amino-acid deletion in the NH2-terminal region of the protein. Southern blot analysis disclosed a homozygous deletion in part of the beta-catenin gene. On the other hand, these cells expressed E-cadherin, alpha-catenin, and plakoglobin of normal size. Immunoprecipitation analyses showed that E-cadherin was coprecipitated with the mutated beta-catenin but not with alpha-catenin, and antibodies against beta-catenin did not copurify alpha-catenin. However, the recombinant fusion protein containing wild-type beta-catenin precipitated alpha-catenin from these cells. These results suggest that the dysfunction of E-cadherin in these cell lines is due primarily to its failure to interact with alpha-catenin, and that this defect results from the mutation in beta-catenin. Thus, it is most likely that the association between E-cadherin and alpha-catenin is mediated by beta-catenin, and that this process is blocked by NH2-terminal deletion in beta-catenin. These findings indicate that genetic abnormality of beta-catenin is one of the mechanisms responsible for loosening of cell-cell contact, and may be involved in enhancement of tumor invasion in human cancers.

Dominant negative inhibition of the association between beta-catenin and c-erbB-2 by N-terminally deleted beta-catenin suppresses the invasion and metastasis of cancer cells.

Aberrant tyrosine phosphorylation of beta-catenin inactivates the E-cadherin-mediated cell adhesion and invasion suppressor system in cancer cells. Elucidation of the association between beta-catenin and c-erbB-2 protein prompted us to investigate whether interference with this interaction can change the invasive phenotype. In a human gastric cancer cell line, TMK-1, N-terminally deleted beta-catenin, which binds to c-erbB-2 but not to cadherin, inhibited the association between endogenous beta-catenin and c-erbB-2 protein, and suppressed the tyrosine phosphorylation of beta-catenin. Cells expressing truncated beta-catenin exhibited markedly reduced invasiveness in vitro and peritoneal metastasis in vivo, and developed an epithelial morphology. These results suggest that tyrosine phosphorylation of beta-catenin regulated by c-erbB-2 protein may play an important role in the invasion, metastasis and morphogenesis of cancer cells and that inhibition of the aberrant tyrosine phosphorylation of beta-catenin effectively prevents invasion and metastasis of cancer cells.

The pnd gene in E. coli plasmid R16: nucleotide sequence and gene expression leading to cell Mg2+ release and stable RNA degradation.

The pnd gene promotes the degradation of stable RNA in the presence of rifampicin at 42 degrees C, but is repressed during normal growth (Ohnishi, Y. and Akimoto, S. (1980) J. Bacteriol. 144, 833-835). We have determined the sequence of a third srnB-pnd-type gene, and have analyzed the effects of its expression from an inducible promoter. The nucleotide sequence of the pnd gene of the R16 plasmid exhibits an open reading frame for a polypeptide with 50 amino-acid residues, with high sequence homology to the pnd gene of a plasmid (R483) of a different incompatibility group. A possible base-paired stem and loop structure, which may participate in the regulation of gene expression, was detected between the promoter and the initiation codon, analogous to that in two comparable genes, srnB in the F and pnd in the R483 plasmid. When bacterial cells containing a lac-pnd fusion plasmid were incubated with a lac inducer at 30 degrees C, magnesium was released from the cells in bulk, and spheroplasts of the cells lysed even in hypertonic solution. Furthermore, when Mg2+ efflux was inhibited in the medium containing 5 mM Mg2+ or in Tris-HCl buffer, the degradation of stable RNA at 42 degrees C was inhibited. These results suggest that expression of the pnd gene effects a release of cellular magnesium by a membrane alterations, resulting in the stable RNA degradation at a higher temperature.

Expression of srnB gene of F plasmid by altered RNA polymerase in Escherichia coli.

Degradation of otherwise stable rRNA and tRNA takes place in the presence of rifampin, dependent on the F plasmid srnB gene. We have reported that a protein newly synthesized in the presence of rifampin might be a product of the srnB gene required for stable RNA degradation (Ito, R. and Ohnishi, Y. (1983) Biochim. Biophys. Acta 739, 27-34). Here we have further studied the mechanism of srnB expression. Among eighteen mutants with altered RNA polymerase, two (TJ2470 (rpoC4) and TJ302 (rpoC56)) showed RNA degradation at high temperature (42 degrees C) when the srnB gene was present. Labeling proteins at 42 degrees C in strain TJ2470 indicated that a protein of molecular weight 12 000 was a product of the srnB gene, and that expression of the srnB gene provoked RNA degradation. Using plasmid pTK4, in which the srnB gene is inserted downstream of the promoter of lacZ, lac promoter-dependent expression of the srnB gene, with production of the putative protein product, also induced RNA degradation at 42 degrees C, with no requirement for added rifampin or altered RNA polymerase. RNA degradation in these conditions was quite similar to that in the case of the addition of rifampin; e.g., it showed some responses to Mg2+, temperature and RNAase I content of the cells. Expression of the srnB gene dependent on lac promoter was also observed in minicells. Thus, it is inferred that the srnB gene is probably repressed under normal conditions with its own promoter; its expression initiates RNA turnover.

Plasmid genes increase membrane permeability in Escherichia coli.

The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids. The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin. These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively. srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product. In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain. Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation. These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.