Cigarette smoking and cervical cancer risk: an evaluation based on a systematic review and meta-analysis among Japanese women.

BACKGROUND: There is a body of evidence to suggest that cigarette smoking increases the risk of cervical cancer in women, but no study has examined the magnitude of the association in Japanese women. Here, we evaluated the association between cigarette smoking and the risk of cervical cancer in Japanese women based on a systematic review of epidemiological evidence. METHODS: Original data were obtained from a MEDLINE search using PubMed or from a search of the 'Ichushi' database, as well as by a manual search. Evaluation of associations was based on the strength of evidence and the magnitude of association, together with biological plausibility as evaluated previously by the International Agency for Research on Cancer. Meta-analysis of associations was also conducted to obtain a summarized overview of the data. RESULTS: We identified two cohort studies and three case-control studies. All five studies had indicated strong positive associations between cigarette smoking and the risk of cervical cancer. Our summary estimate indicated that the relative risk (RR) for individuals who had ever-smoked relative to never-smokers was 2.03 (95% confidence interval: 1.49-2.57). Four studies had also demonstrated dose-response relationships between cigarette smoking and the risk of cervical cancer. CONCLUSION: We conclude that there is convincing evidence that cigarette smoking increases the risk of cervical cancer among Japanese women.

Evidence-based cancer prevention recommendations for Japanese.

A comprehensive evidence-based cancer prevention recommendation for Japanese was developed. We evaluated the magnitude of the associations of lifestyle factors and infection with cancer through a systematic review of the literature, meta-analysis of published data, and pooled analysis of cohort studies in Japan. Then, we judged the strength of evidence based on the consistency of the associations between exposure and cancer and biological plausibility. Important factors were extracted and summarized as an evidence-based, current cancer prevention recommendation: 'Cancer Prevention Recommendation for Japanese'. The recommendation addresses six important domains related to exposure and cancer, including smoking, alcohol drinking, diet, physical activity, body weight and infection. The next step should focus on the development of effective behavior modification programs and their implementation and dissemination.

Validity of self-reported tooth counts and masticatory status study of a Japanese adult population.

Self-reported measures of oral health are often used to assess oral health in populations or groups, but their validity or reliability needs repeated confirmation. The objective of this cross-sectional study was to evaluate the validity of self-reported tooth counts and masticatory status, using data obtained from a sample of Japanese adults. A total of 2356 adults aged 40 to 75 years participated in a questionnaire survey and a clinical oral examination from 2013 through 2016. Self-reported measures were compared with clinically measured values. For tooth counts, mean clinical and self-reported tooth counts in all participants were 23.68 and 23.78 teeth, and no significant difference was detected. Spearman's, Pearson's and intra-class correlation coefficients between clinical and self-reported tooth counts were 0.771, 0.845 and 0.843, respectively. According to the Bland-Altman analysis, the mean difference between clinical and self-reported tooth counts was -0.098 (95% CI: -0.242, 0.047). The upper limit of agreement was 6.919 (95% CI: 6.669, 7.169), and the lower limit of agreement was -7.115 (95% CI: -7.365, -6.865). No significant fixed or proportional bias was observed. For masticatory status, the crude or age- and gender-adjusted mean numbers of total teeth, posterior teeth and 3 kinds of functional tooth units significantly decreased with the deterioration of masticatory status. This study indicated that self-reports were within an acceptable range of clinical measures. Therefore, self-reports were considered valid alternatives to clinical measures to estimate tooth counts and masticatory status in a current Japanese adult population.

Coping strategies and risk of cardiovascular disease incidence and mortality: the Japan Public Health Center-based prospective Study.

AIMS: Coping strategies may be significantly associated with health outcomes. This is the first study to investigate the association between baseline coping strategies and cardiovascular disease (CVD) incidence and mortality in a general population cohort. METHODS AND RESULTS: The Japan Public Health Center-based prospective Study asked questions on coping in its third follow-up survey (2000-04). Analyses on CVD incidence and mortality included 57 017 subjects aged 50-79 without a history of CVD and who provided complete answers on approach- and avoidance-oriented coping behaviours and strategies. Cox regression models, adjusted for confounders, were used to determine hazard ratios (HRs) according to coping style. Mean follow-up time was 7.9 years for incidence and 8.0 years for mortality.The premorbid use of an approach-oriented coping strategy was inversely associated with incidence of stroke (HR = 0.85; 95% CI, 0.73-1.00) and CVD mortality (HR = 0.74; 95% CI, 0.55-0.99). Stroke subtype analyses revealed an inverse association between the approach-oriented coping strategy and incidence of ischaemic stroke (HR = 0.79; 95% CI, 0.64-0.98) and a positive association between the combined coping strategy and incidence of intra-parenchymal haemorrhage (HR = 2.03; 95% CI, 1.01-4.10). Utilizing an avoidance coping strategy was associated with increased mortality from ischaemic heart disease (IHD) only in hypertensive individuals (HR = 3.46; 95% CI, 1.07-11.18). The coping behaviours fantasizing and positive reappraisal were associated with increased risk of CVD incidence (HR = 1.24; 95% CI, 1.03-1.50) and reduced risk of IHD mortality (HR = 0.63; 95% CI, 0.40-0.99), respectively. CONCLUSION: An approach-oriented coping strategy, i.e. proactively dealing with sources of stress, may be associated with significantly reduced stroke incidence and CVD mortality in a Japanese population-based cohort.

Soy food and isoflavone intake and endometrial cancer risk: the Japan Public Health Center-based prospective study.

OBJECTIVE: Compared with western populations, the consumption of soy foods among Japanese is very high and the incidence of endometrial cancer very low. We evaluated the association of soy food and isoflavone intake with endometrial cancer risk in Japanese women. DESIGN: Prospective cohort study. SETTING: Ten public health centre areas in Japan. POPULATION: Forty nine thousand one hundred and twenty-one women of age 45-74 years who responded to a 5-year follow-up survey questionnaire. METHODS: Intakes of soy foods as well as other covariates were assessed in 1995-1998 by a self-administered food frequency questionnaire. Cox proportional hazards regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI). MAIN OUTCOME MEASURE: Incidence of endometrial cancer. RESULTS: During an average of 12.1 years of follow up, 112 newly diagnosed endometrial cancer cases were identified. Energy-adjusted intakes of soy food and isoflavone were not associated with the risk of endometrial cancer. The multivariate-adjusted HR per 25 g/day increase in the intake of soy food was 1.02 (95% CI 0.94-1.10), and the corresponding value for isoflavone intake per 15 mg/day was 1.01 (95% CI 0.84-1.22). CONCLUSION: In this population-based prospective cohort study of Japanese women, we observed no evidence of a protective association between soy food or isoflavone intake and endometrial cancer risk.

Rice, bread, noodle and cereal intake and colorectal cancer in Japanese men and women: the Japan Public Health Center-based prospective Study (JPHC Study).

BACKGROUND: Colorectal cancer (CRC) incidence rate increased rapidly in Japan between the 1950s and 1990s. We examined the association between rice intake and CRC risk in comparison with bread, noodles and cereal among Japanese adults enrolled in the Japan Public Health Center-based prospective Study. METHODS: A total of 73,501 Japanese men and women were followed-up from 1995 to 1999 until the end of 2008 for an average of 11 years. During 801,937 person-years of follow-up, we identified 1276 incident cases of CRC. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) of CRC for rice, noodle, bread and cereal intake were calculated by Cox proportional hazards model. RESULTS: Overall, no significant association was observed for the highest quartile of rice intake compared with the lowest and the risk of CRC and its subsites in men (HR, 0.77; 95% CI, 0.56-1.07) and women (HR, 1.10; 95% CI, 0.71-1.68). However, a non-significant inverse trend was observed between rice intake and rectal cancer in men. No clear patterns of association were observed in bread, noodle and cereal intake. CONCLUSION: Our findings suggest that the consumption of rice does not have a substantial impact on the risk of CRC in the Japanese population.

Association of vegetable and fruit intake with gastric cancer risk among Japanese: a pooled analysis of four cohort studies.

BACKGROUND: Prospective evidence is inconsistent regarding the association between vegetable/fruit intake and the risk of gastric cancer. METHODS: In an analysis of original data from four population-based prospective cohort studies encompassing 191 232 participants, we used Cox proportional hazards regression to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of gastric cancer incidence according to vegetable and fruit intake and conducted a meta-analysis of HRs derived from each study. RESULTS: During 2 094 428 person-years of follow-up, 2995 gastric cancer cases were identified. After adjustment for potential confounders, we found a marginally significant decrease in gastric cancer risk in relation to total vegetable intake but not total fruit intake: the multivariate-adjusted HR (95% CI; P for trend) for the highest versus the lowest quintile of total vegetable intake was 0.89 (0.77-1.03; P for trend = 0.13) among men and 0.83 (0.67-1.03; P for trend = 0.40) among women. For distal gastric cancer, the multivariate HR for the highest quintile of total vegetable intake was 0.78 (0.63-0.97; P for trend = 0.02) among men. CONCLUSIONS: This pooled analysis of data from large prospective studies in Japan suggests that vegetable intake reduces gastric cancer risk, especially the risk of distal gastric cancer among men.

Communicating by electrolarynx with a blind tetraplegic spinal cord injury patient on mechanical ventilation in the ICU.

STUDY DESIGN: Single-subject case. OBJECTIVES: To describe the atypical presentation of communication with a blind tetraplegic spinal cord injury patient on a respirator using an electrolarynx. SETTING: Critical care center in Osaka, Japan. METHODS: A 53-year-old blind man with tetraplegic spinal cord injury was admitted to our center. It was difficult for him to express his desires and to communicate with others about his severe condition and other details of his care. We began to use an electrolarynx to communicate with this patient because he could move his mouth. RESULTS: With use of the electrolarynx, the patient gradually became better able to speak fluently by electrolarynx on the first day of use. The electrolarynx allowed us and his family to communicate with him. He was pleased with the improvement in communication. CONCLUSION: An electrolarynx is a useful method for communicating with blind tetraplegic spinal cord injury patients on mechanical ventilation.

Attributable causes of cancer in Japan in 2005--systematic assessment to estimate current burden of cancer attributable to known preventable risk factors in Japan.

BACKGROUND: To contribute to evidence-based policy decision making for national cancer control, we conducted a systematic assessment to estimate the current burden of cancer attributable to known preventable risk factors in Japan in 2005. METHODS: We first estimated the population attributable fractions (PAFs) of each cancer attributable to known risk factors from relative risks derived primarily from Japanese pooled analyses and large-scale cohort studies and the prevalence of exposure in the period around 1990. Using nationwide vital statistics records and incidence estimates, we then estimated the attributable cancer incidence and mortality in 2005. RESULTS: In 2005, ≈ 55% of cancer among men was attributable to preventable risk factors in Japan. The corresponding figure was lower among women, but preventable risk factors still accounted for nearly 30% of cancer. In men, tobacco smoking had the highest PAF (30% for incidence and 35% for mortality, respectively) followed by infectious agents (23% and 23%). In women, in contrast, infectious agents had the highest PAF (18% and 19% for incidence and mortality, respectively) followed by tobacco smoking (6% and 8%). CONCLUSIONS: In Japan, tobacco smoking and infections are major causes of cancer. Further control of these factors will contribute to substantial reductions in cancer incidence and mortality in Japan.

Soymilk-fermented with Lactobacillus delbrueckii subsp. delbrueckii TUA4408L improves immune-health in pigs.

Lactobacillus delbrueckii subsp. delbrueckii TUA4408L has the ability to grow and ferment soymilk and is able to modulate the innate immune response of intestinal epithelial cells in vitro. These two properties prompt us to evaluate whether the soymilk fermented with the TUA4408L strain can induce beneficial immunomodulatory effects in vivo. For this purpose, pigs were selected as a preclinical model. The studies performed here demonstrated that the L. delbrueckii subsp. delbrueckii TUA4408L-fermented soymilk (TUA4408L FSM) reduced blood markers of inflammation and differentially regulated the expression of inflammatory and regulatory cytokines in the intestinal mucosa. These immunological changes induced by the TUA4408L FSM were associated to an enhanced resistance to pathogenic Escherichia coli and an improved grow performance and meat quality of pigs. The experiments and analysis in our study indicate that the immunobiotic TUA4408L FSM could be an interesting non-dairy functional food to beneficially modulate the intestinal immune system, improve protection against pathogens and reduce inflammatory damage. The preclinical study carried out here in pigs could have a better correlation in humans, compared to a rodent model. However, the clinical relevance of these findings still needs to be confirmed by further research, for example, in controlled human challenge studies.

Green tea consumption and gastric cancer in Japanese: a pooled analysis of six cohort studies.

BACKGROUND: Previous experimental studies have suggested many possible anti-cancer mechanisms for green tea, but epidemiological evidence for the effect of green tea consumption on gastric cancer risk is conflicting. OBJECTIVE: To examine the association between green tea consumption and gastric cancer. METHODS: We analysed original data from six cohort studies that measured green tea consumption using validated questionnaires at baseline. Hazard ratios (HRs) in the individual studies were calculated, with adjustment for a common set of variables, and combined using a random-effects model. RESULTS: During 2 285 968 person-years of follow-up for a total of 219 080 subjects, 3577 cases of gastric cancer were identified. Compared with those drinking <1 cup/day, no significant risk reduction for gastric cancer was observed with increased green tea consumption in men, even in stratified analyses by smoking status and subsite. In women, however, a significantly decreased risk was observed for those with consumption of > or =5 cups/day (multivariate-adjusted pooled HR = 0.79, 95% confidence interval (CI) = 0.65 to 0.96). This decrease was also significant for the distal subsite (HR = 0.70, 95% CI = 0.50 to 0.96). In contrast, a lack of association for proximal gastric cancer was consistently seen in both men and women. CONCLUSIONS: Green tea may decrease the risk of distal gastric cancer in women.

Increase in body mass index category since age 20 years and all-cause mortality: a prospective cohort study (the Ohsaki Study).

BACKGROUND: It is still unclear whether weight gain from early to late adulthood affects longevity. Furthermore, no study has addressed its association with all-cause and cause-specific mortality in an Asian population. METHODS: We prospectively assessed the association between an increase in body mass index (BMI) category since age 20 years and risk of all-cause, cardiovascular disease (CVD) and cancer mortality. Self-reported information pertaining to BMI was collected from 38 080 Japanese men and women aged 40-79 years at study entry in 1994 after exclusion of participants with a BMI of <18.5 kg/m(2) at age 20 years or at study entry. We defined six patterns of increase in BMI category at age 20 years and study entry: stable normal, overweight and obese, normal to overweight or obese, and overweight to obese. RESULTS: During 7 years of follow-up, 2617 participants died. After adjustment for potential confounders, we observed a significantly increased risk of all-cause mortality for the pattern of normal weight at age 20 years and obese at study entry and of stable obese compared with stable normal in BMI category, the multivariate HRs (95% confidence interval (CI)) being 1.42 (1.08-1.88) and 2.26 (1.45-3.51), respectively. For the pattern of overweight at age 20 years and obese at study entry, the multivariate hazard ratio (95% CI) was 1.35 (0.92-1.98). In contrast, we did not observe an increased risk of all-cause mortality for normal weight at age 20 years and overweight at study entry, and stable overweight. For CVD and cancer mortality, these results were consistently observed. CONCLUSION: We observed an increased risk of all-cause mortality both among participants who had been persistently obese since early adulthood and participants who showed an increase in BMI category from normal to obese, compared with participants with a stable normal BMI category.

Glycogen synthetase activity in liver: regulation by the autonomic nerves.

Electrical stimulation of the vagus nerve in intact or pancreatectomized rabbits resulted in a marked increase, within 5 minutes of the onset of stimulation, in the total activity of liver glycogen synthetase. The effect was completely counteracted by simultaneous stimulation of the splanchnic nerve, although stimulation of the splanchnic nerve alone had little effect on the enzyme. Injection of insulin caused an increase in both the total activity of glycogen synthetase and the activity that was independent of glucose-6-phosphate. The response of the enzyme to vagal stimulation was much faster than to administration of insulin.

Cholinergic stimulation of the rat hypothalamus: effects of liver glycogen synthesis.

Cholinergic stimulation of the lateral hypothalamic neurons with intrahypothalamic microinjections of acetylcholine or carbachol caused a marked increase in the content of the active form of glycogen (starch) synthase in the liver. Total activity of the enzyme (active plus inactive forms) was not increased significantly. The lowest effective dose of acetylcholine was 5 X 10(-10) mole, and the optimum dose was 5 X 10(-9) mole. Similar applications of other neurotransmitters, such as norepinephrine, dopamine, serotonin, and gamma-aminobutyric acid, did not affect the enzyme's activity.

Comparison of neurological outcomes following witnessed out-of-hospital ventricular fibrillation defibrillated with either biphasic or monophasic automated external defibrillators.

BACKGROUND: Biphasic waveform defibrillation results in higher rates of termination of fibrillation than monophasic waveform defibrillation but has not been shown to improve survival outcomes. OBJECTIVE: To compare the effectiveness of a biphasic automated external defibrillator (AED) with a monophasic AED for witnessed out-of-hospital cardiac arrest (OHCA) due to ventricular fibrillation (VF). METHODS: In a prospective population-based cohort study, adults with witnessed VF OHCA were treated with either monophasic or biphasic waveform AED shocks. The primary outcome measure was neurologically favourable 1-month survival, defined as a Cerebral Performance Categories score of 1 or 2. RESULTS: Of 366 adults with witnessed OHCA of presumed cardiac aetiology, 74 (20%) had VF. Termination of VF with the first shock tended to occur more frequently after biphasic AED shocks (36/44 (82%) vs 20/30 (67%), p = 0.14). Return of spontaneous circulation (ROSC) occurred more frequently after biphasic AED shocks (29/44 (66%) vs 8/30 (27%), p = 0.001). Neurologically favourable 1-month survival was also more frequent in the biphasic group (10/44 (23%) vs 1/30 (3%), p = 0.04). The median time interval from the first shock to the second shock was 67 s in the monophasic group and 24 s in the biphasic group (p = 0.001). CONCLUSIONS: Treatment with biphasic AED shocks improved the likelihood of ROSC and neurologically favourable 1-month survival after witnessed VF compared with monophasic AED shocks. In addition to waveform differences, a shorter time interval from the first shock to the second shock could account for the better outcomes with biphasic AED.

Regulation of SV40 large T-antigen stability by reversible acetylation.

Reversible acetylation on protein lysine residues has been shown to regulate the function of both nuclear proteins such as histones and p53 and cytoplasmic proteins such as alpha-tubulin. To identify novel acetylated proteins, we purified several proteins by the affinity to an anti-acetylated-lysine antibody from cells treated with trichostatin A (TSA). Among the proteins identified, here we report acetylation of the SV40 large T antigen (T-Ag). The acetylation site was determined to be lysine-697, which is located adjacent to the C-terminal Cdc4 phospho-degron (CPD). Overexpression of the CBP acetyltransferase acetylated T-Ag, whereas HDAC1, HDAC3 and SIRT1 bound and deacetylated T-Ag. The acetylation and deacetylation occurred independently of p53, a binding partner of T-Ag, but the acetylation was enhanced in the presence of p53. T-Ag in the cells treated with TSA and NA or the acetylation mimic mutant (K697Q) became unstable in COS-7 cells, suggesting that acetylation regulates stability of T-Ag. Indeed, NIH3T3 cells stably expressing K697Q showed decreased anchorage-independent growth compared with those expressing wild type or the K697R mutant. These results demonstrate that acetylation destabilizes T-Ag and regulates the transforming activity of T-Ag in NIH3T3 cells.

Nucleolin is involved in interferon regulatory factor-2-dependent transcriptional activation.

We have previously shown that interferon regulatory factor-2 (IRF-2) is acetylated in a cell growth-dependent manner, which enables it to contribute to the transcription of cell growth-regulated promoters. To clarify the function of acetylation of IRF-2, we investigated the proteins that associate with acetylated IRF-2. In 293T cells, the transfection of p300/CBP-associated factor (PCAF) enhanced the acetylation of IRF-2. In cells transfected with both IRF-2 and PCAF, IRF-2 associated with endogenous nucleolin, while in contrast, minimal association was observed when IRF-2 was transfected with a PCAF histone acetyl transferase (HAT) deletion mutant. In a pull-down experiment using stable transfectants, acetylation-defective mutant IRF-2 (IRF-2K75R) recruited nucleolin to a much lesser extent than wild-type IRF-2, suggesting that nucleolin preferentially associates with acetylated IRF-2. Nucleolin in the presence of PCAF enhanced IRF-2-dependent H4 promoter activity in NIH3T3 cells. Nucleolin knock-down using siRNA reduced the IRF-2/PCAF-mediated promoter activity. Chromatin immunoprecipitation analysis indicated that PCAF transfection increased nucleolin binding to IRF-2 bound to the H4 promoter. We conclude that nucleolin is recruited to acetylated IRF-2, thereby contributing to gene regulation crucial for the control of cell growth.

Plasma 25-hydroxy vitamin D and subsequent prostate cancer risk in a nested Case-Control study in Japan: The JPHC study.

BACKGROUND/OBJECTIVES: Although vitamin D has been experimentally reported to inhibit tumorigenesis, cell growth and prostate cancer invasion, epidemiologic data regarding prostate cancer risk are inconsistent, and some studies have suggested positive but nonsignificant associations. Further, the impact of vitamin D on prostate cancer between Western and Japanese populations may differ due to different plasma vitamin D levels. SUBJECTS/METHODS: We performed a nested case-control study within the Japan Public Health Center-based Prospective (JPHC) Study in 14,203 men (40-69 years) who answered a self-administered questionnaire at baseline (1990-1994) and gave blood samples, and were followed until 2005. We identified 201 prostate cancers which are newly diagnosed during follow-up (mean 12.8 years). We selected two matched controls for each case from the cohort. We used a conditional logistic regression model to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for prostate cancer with respect to levels of 25-hydroxy vitamin D (25(OH)D) in plasma. RESULTS: We did not observe statistically significant association between 25(OH)D level and total prostate cancer (multivariate OR=1.13 (95%CI=0.66-1.94, Ptrend=0.94) for the highest versus lowest tertile) However, 25(OH) levels were slightly positively associated with advanced cancer. The results remained substantially unchanged after stratification by intake of fish or calcium intake. CONCLUSIONS: 25(OH)D level showed no association with overall prostate cancer among Japanese men in this large cohort.

Regulation of glycogen metabolism in liver by the autonomic nervous system. VI. Possible mechanism of phosphorylase activation by the splanchnic nerve.

The effects of autonomic-nerve stimulation on the activities of phosphorylase (EC 2.4.1.1), dephospho-phosphorylase kinase (EC 2.7.1.38) and phosphorylase phosphatase (EC 3.1.3.17), and on the concentration of adenosine 3', 5'-monophosphate in rabbit liver were investiaged. Results were compared with the effects of epinephrine and glucagon on these enzymes. 1. The acitivity of liver phosphorylase increased rapidly and markedly on electrical stimulation of the splanchnic nerve, or after intraportal administration of epinephrine or glucagon. The activity was not affected by vagal stimulation. 2. The activity of dephospho-phosphorylase kinase increased about 2--3-fold 1 min after injections of epinephrine and glucagon, glucagon causing more activation than epinephrine. The enzyme activity was not altered by splanchnic-nerve, or vagal stimulation. 3. Injections of epinephrine and glucagon caused marked elevation of liver adenosine 3', 5'-monophosphate within a few minutes. With epinephrine, the nucleotide concentration rose to a maximum after 1 min and amounted to about 3-fold increase, while with glucagon the maximum increase of approximately 8-fold increase was observed after 2 min. Stimulation of the splanchnic nerve for 10 min did not affect the adenosine 3', 5'-monophosphate level in the liver. Vagal stimulation also had no effect on the level. 4. The activity of phosphorylase phosphatase decreased promptly (within 30 s) and markedly on splanchnic-nerve stimulation, but did not change significantly on administration of epinephrine of glucagon. A small but insignificant increase in phosphatase activity wasobserved upon vagal stimulation. 5. The effect of Ca-2+ on purified dephospho-phosphorylase kinase was studied. The activity was found to depend partially on free Ca-2+ at low Ca-2+ concentrations (1-10-minus 7--1-10-minus 5 M). 6. These results suggest that the rise in hepatic phosphorylase content upon splanchnic-nerve stimulation, unlike that induced by epinephrine and glucagon, is not mediated by adenosine 3', 5'-monophosphate and subsequent activation of dephospho-phosphorylase kinase, but rather by inactivation of phosphorylase phosphatase. The possible existence of a new factor in this mechanism is discussed.

Two regions of the Mn-stabilizing protein from Synechococcus elongatus that are involved in binding to photosystem II complexes.

Limited proteolysis of the Mn-stabilizing protein (MSP) from the thermophilic cyanobacterium Synechococcus elongatus with chymotrypsin, trypsin or lysylendopeptidase that yielded four major polypeptides of 26 kDa, 22 kDa, 15 kDa and 11 kDa on denaturing gel electrophoresis resulted in total loss of the binding capacity of the protein to PSII complexes. Analyses of electrophoretic patterns and amino-terminal sequences of the proteolytic products revealed that the three proteases specifically cleaved the protein at a site between Phe156 and Gly163 or between Arg184 and Ser191. Site-directed mutagenesis was used to construct two mutant MSPs that had a nick between Phe156 and Leu157, a chymotrypsin-cleavage site, and Met before Leu157 or in place of Leu157. The two mutant proteins failed to bind to PSII complexes, although they largely retained ordered secondary structure and comigrated with the wild-type proteins in non-denaturing gel electrophoresis. The loss of the protein binding can be ascribed to introduction of a nick because a mutant protein that had Met in place of Leu157 but no nick was able to specifically bind to the functional site of PSII complexes and restore the oxygen-evolving activity as effectively as the wild-type protein. In contrast, a mutant MSP with Met inserted between Phe156 and Leu157 bound only weakly and non-specifically to PSII complexes and failed to reactivate oxygen evolution. Thus, the binding of the protein to the functional site of the PSII complex was highly sensitive to a small structural change that was caused by cleavage or insertion of a single amino acid residue between Phe156 and Leu157. The results suggest that the Phe156-Gly163 and Arg184-Ser191 sequences of the cyanobacterial MSP are regions for interaction with PSII complexes.