[Competencies expected of labor and social security attorneys in harmonizing work with diseases treatment].

OBJECTIVES: Labor and social security attorneys (LSSAs) are involved in various positions in harmonizing work with disease treatment; however, their qualification requirements do not include knowledge about the same. Expectations of their involvement in harmonizing work with disease treatment are insufficient. This study aimed to identify the competencies expected of the labor and social security LSSAs in harmonizing work with disease treatment. METHODS: In step 1, semi-structured interviews were conducted with LSSAs in this field. In step 2, a draft competency list was created based on the interview results. In step 3, the Delphi method was used to conduct a questionnaire survey among LSSAs who had over 10 consultation cases on harmonizing work with disease treatment, and they were asked about the level of importance (how important they thought it was to promote harmonizing work with disease treatment) and level of achievement (how much they had achieved). We also asked them about the competencies they considered necessary and added them as additional items in the draft. In step 4, the results of the previous step were presented to the participants who had given valid answers in step 3, and they were asked whether they would adopt the items as competencies. Items with an agreement rate of 80% or higher were considered competency items. Additionally, we asked them about the level of importance and level of achievement of the additional items created in step 3. RESULTS: In step 1, 24 LSSAs participated, and in step 2, a draft competency list of six major items, 18 medium items, and 71 minor items was created. In step 3, 49 LSSAs participated and 41 cooperated (response rate: 83.6%). Five items were selected for the draft competency list to be newly added. In step 4, 30 LSSAs cooperated (response rate: 73.1%). None of the items had an agreement rate of less than 80%, and over 40% of the items had an agreement rate of 100%. As a result, six major items, 18 medium items, and 76 minor items were selected for the competency list. CONCLUSIONS: This study identified the competencies expected of labor and social security LSSAs in harmonizing work with disease treatment. The results of this study can be used as a reference for developing a systematic training curriculum for LSSAs in this field in the future.

Effects of Alcohol Consumption and Smoking on the Onset of Hypertension in a Long-Term Longitudinal Study in a Male Workers' Cohort.

AIM: To determine the effects of alcohol consumption and smoking on the onset of hypertension in a long-term longitudinal study. METHODS: 7511 non-hypertensive male workers were enrolled. This cohort study was performed over an 8-year period using the results of the annual workers-health screening. The end-point was defined as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or use of antihypertensive drugs. For alcohol consumption, weekly alcohol intake (g ethanol/week) was estimated (1 "gou" = 22 g ethanol). Annual survey data were analyzed by pooled logistic regression that included alcohol consumption, smoking, age, body mass index, job schedule types, habitual exercise, and blood test measurements into the statistical model. RESULTS: A significant positive dose-response relationship between alcohol consumption and onset of hypertension was observed, with synergistic health effects present. Compared with abstainers and nonsmokers, the adjusted odds ratios (95% confidence interval) for the onset of hypertension were: 1.51 (1.27-1.79) for 154 g ethanol/week and nonsmokers, and 1.81 (1.54-2.11) for 154 g ethanol/week and smokers. An interaction between alcohol and smoking was confirmed. CONCLUSIONS: This study provided information useful to the prevention of hypertension. By reducing alcohol consumption and smoking simultaneously, the risk of hypertension may be considerably lowered.

Association between High-Sensitive C-Reactive Protein and the Development of Liver Damage in Japanese Male Workers.

BACKGROUND: The aim of this study was to determine whether a causative relationship exists between the development of liver damage and increased high-sensitivity C-reactive protein (HsCRP) levels by long-term follow-up in Japanese workers. METHODS: The target participants comprised 7830 male workers in a Japanese steel company. The prospective cohort study was performed over a 6-year period, and annual health screening information was analyzed by pooled logistic regression. The endpoint, regarded as the development of liver damage, was defined as aspartate aminotransferase (AST) ≥ 40 IU/L. RESULTS: A significant relationship between the development of liver damage and increased HsCRP levels was observed after adjusting for confounding factors such as various physiological and blood chemistry parameters and lifestyle factors. The odds ratio of a 1.5-fold increase in HsCRP was 1.07 (95% confidence interval: 1.03-1.10, p < 0.001). CONCLUSIONS: The results suggested that an increase of HsCRP is associated with the development of liver damage.

[Examining competencies for labor and social security attorneys in the field of occupational mental health].

AIM: Labor and social security attorneys (LSSAs) are involved in the field of occupational mental health. However, little attention has been paid to the involvement of LSSAs in this field. This study investigated the occupational mental health competencies that are expected of LSSAs. SUBJECTS AND METHODS: Our investigation utilized the Delphi method. In Step 1, we conducted semi-structured interviews with LSSAs and then created an initial list of competencies based on the interviews and a previous investigation. In Step 2, we recruited LSSAs with 10 or more cases related to occupational mental health. They completed a questionnaire assessing the importance of their work (how important they felt it was to conduct work related to mental health) and level of achievement (how much they felt they had achieved). The respondents were also asked to provide additional competencies (not listed on the questionnaire) if they regarded them as necessary for their work, and these were later added to the list of proposed competencies. In Step 3, we presented the results of Step 2 to the same respondents and asked them to rate their agreement with the proposed competencies. Items with agreement of 80% or higher were set as competencies. We also asked LSSAs about the level of importance of their work and their perceived level of achievement with regard to the additional items created in Step 2. Items for which the level of achievement fell below the median were extracted even if the level of importance of the work fell at or above the median. RESULTS: We recruited 8 LSSAs in Step 1 and created a list of 68 preliminary competencies in 20 fields. We recruited 57 LSSAs in Step 2, and 45 LSSAs completed the survey (response rate: 78.9%). Seven competencies were added to the list as a result. We recruited 34 LSSAs in Step 3 (response rate: 75.6%) . Two items with an agreement rate of less than 80% were removed, resulting in 73 competencies in 20 fields. One of the items with an agreement rate of 100% was "The plan is based on the merits and disadvantages (risks) for both labor and management." CONCLUSIONS: This study identified the competencies required of LSSAs in the field of occupational mental health. Our findings suggest that specifying these competencies will enable efficient training of LSSAs.

[Examination of factors for promoting cooperation using documents between occupational health physicians and psychiatrists].

OBJECTIVES: There is little specific information concerning the method and the efficacy of sharing information between occupational health physicians and psychiatrists regarding the employment status and medical history of their patients with mental illnesses. To promote cooperation between occupational health physicians and psychiatrists, we examined the points necessary to be included on medical information request forms exchanged between them. METHODS: We conducted focused group discussion (FGD) to identify the points that need to be described on the request form and the concerns in cooperation between occupational health physicians and psychiatrists. We conducted FGDs twice, with two different groups of nine psychiatrists participating in each round. We extracted and organized FGD results and determined the necessary request form points. Next, we assumed two different cases of workers with mental illnesses and created three request form templates with differing item descriptions and lengths. We also conducted a questionnaire survey among clinical psychiatrists to determine their impression of the templates. We performed logistic regression analysis on the obtained results. RESULTS: On the basis of the FGD results we extracted the situation in the workplace, clarification of points to be confirmed, representation of the occupational health physician's position, and handling of information provided by the doctor as points required for the request form. On the basis of these results and the opinions of occupational health specialists, we created a new request form using these points. Additionally, the results from the questionnaire survey about the prescribed items revealed the proportion of favorable answers regarding sufficient information written on the request form and a feeling of security for information provision increased (p < 0.01). Conversely, the proportion of favorable responses for readability decreased. CONCLUSIONS: Psychiatrists are concerned about the possibility that their patient may be at a disadvantageous situation by providing their personal medical information and believe the clinical information required by the occupational health physicians is unclear. This suggests that there are factors impeding the cooperation between the occupational health physicians and psychiatrists. When an occupational health physician writes a request form, cooperation with psychiatrists may be promoted by enriching the request form contents and by including the representation of the occupational health physician's position and the intended purpose of the provided information by paying attention to the volume of sentences.

Dietary Modification Trial in Community-Dwelling Japanese Elderly: A Pilot Study.

BACKGROUND: This study examined the effects of 6-month nutrition education programs for community-dwelling elderly. METHODS: This study enrolled 50 community-dwelling elderly who regularly visit outpatient clinics. The programs had three goals: salt reduction, increase in dietary fiber, and adequate protein intake. Since it would be difficult for elderly to achieve all goals concurrently, a single goal was chosen by participants themselves. Anthropometric measurements, blood sampling, and assessment of dietary intake were performed at baseline, 3 months, and 6 months. RESULTS: The nutrition education program for salt reduction was well accepted by the participants and the amount of daily salt intake showed median value of 9.6 g at baseline, 8.0 g at 3 months and 8.1 g at 6 months (P = 0.005). The amount of dietary fiber intake only slightly increased after taking the nutrition program (median value of 13.4 g at baseline, 15.3 g at 3 months and 15.5 g at 6 months; P = 0.695), because of difficulties in introducing new food options to the diet. After taking the adequate protein intake program, participants showed small decreases in protein (a modification from 1.24 g/kg IBW to 1.20 g/kg IBW) and salt intake (8.2 to 7.3 g) at 3 months, but the effects were not sustained at 6 months. CONCLUSION: This nutrition education program focusing on a single nutrient may serve as a strategy to successfully reduce salt intake and improve systolic blood pressure control in community-dwelling elderly individuals who regularly visit outpatient clinics. In our view, dietary and lifestyle habits should be taken into account as much as possible in nutrition education for elderly individuals.

[Development of a Tool for Training and Evaluation of the Competencies in Occupational Mental Health Necessary for Labor and Social Security Attorneys].

Labor and Social Security Attorneys (LSSAs) advise their clients about occupational mental health, but the competencies necessary in this field are not clear to them. We standardized the necessary competencies as a counseling guide for LSSAs, and we also designed a related discussion training program. These competencies were summarized in a brainstorming session at a research conference comprised of physicians, an occupational health nurse, LSSAs, an instructional design expert, and a management consultant, and then a training program (lasting 9 hours 30 minutes) was developed. Nineteen trainees who were introduced by members of the research conference collectively completed a seven-question written test, both before and after the training, in order to assess its effectiveness. Sixteen trainees who completed the training were surveyed, with a recovery rate of 100%. The necessary competencies that they identified were: information about circular notices from the Ministry of Health, Labor and Welfare; behavior such as the gathering of information; and dealing with the reinstatement of employees. The scores were subjected to the Wilcoxon signed-rank test in order to evaluate the training, and the answers from the pre-training were compared with those from the post-training. A significant difference (P < 0.05) was seen for each question. These results show the effectiveness of the developed training program for the learning of the competencies necessary for LSSAs.

Dose-response relationship between tobacco or alcohol consumption and the development of diabetes mellitus in Japanese male workers.

BACKGROUND: The aim of this study was to examine the dose-response relationships between tobacco or alcohol consumption and the development of diabetes mellitus. METHODS: An 8-year prospective cohort study was conducted in 8423 male workers who received annual health check-ups between 2002 and 2010 at a Japanese steel company. The endpoints were defined as an HbA(lc)≥6.1% or taking any anti-diabetic medication. The dose-response relationships of tobacco or alcohol consumption were investigated using a proportional hazards regression with time-dependent covariates selected from baseline age, body mass index, mean arterial pressure, total serum cholesterol, aspartate aminotransferase, creatinine and uric acid, shift work or day work, and habitual exercise by stepwise selection method. RESULTS: A positive dose-response relationship between tobacco consumption and the development of diabetes mellitus was observed, with a significantly higher hazard ratio (HR) seen with higher tobacco consumption (11-20 cigarettes/day, HR 1.26 [95% confidence interval (CI), 1.00-1.59], ≥21 cigarettes/day, HR 1.54 [95%CI, 1.20-1.97]). In contrast, we observed a negative dose-response relationship between alcohol consumption and the development of diabetes mellitus, with a significantly lower HR with higher weekly alcohol consumption (7.0-13.9 gou/week [154-307 g/week], HR 0.73 [95% CI, 0.55-0.97], ≥14.0 gou/week [308 g/week], HR 0.75 [95% CI, 0.57-0.98]). CONCLUSIONS: The results indicated that decreasing tobacco consumption will achieve significant prevention of diabetes mellitus. On the other hand, we observed a significant, negative dose-response relationship between alcohol consumption and the development of diabetes mellitus, in contrast to previous studies that reported a positive relationship in the Japanese population.

Estimation of the benchmark duration of shiftwork associated with weight gain in male Japanese workers.

The authors estimated the benchmark durations (BMDs) and their 95% lower confidence limit (BMDL) for the reference duration of shiftwork for weight gain. A 14-yr prospective cohort study was conducted in male workers at a Japanese steel company (n = 7254) who had received annual health check-ups between 1991 and 2005. The endpoints in the study were either a 5%, 7.5%, or 10% increase in body mass index (BMI) during the period of observation, compared to the BMI at entry. The association between the duration of shiftwork and weight gain was investigated using multivariate pooled logistic regression analyses with stepwise selection of covariates, including age, BMI measured during the study, drinking and smoking habits, and habitual exercise. The BMDL/BMD for shiftwork in subjects aged in their 40s or ≥50 yrs was estimated using benchmark responses (BMRs) of 5% or 10% and parameters for the duration of shiftwork and other covariates. For workers aged in their 40s, the BMDL/BMD for shiftwork with a BMR of 5% was 18.6/23.0 yrs (≥7.5%) and 16.9/19.4 yrs (≥10%). For workers aged ≥50 yrs, the BMDL/BMD with a BMR of 5% was 22.9/28.2 yrs (≥7.5%) and 20.6/23.6 yrs (≥10%). The reference duration of shiftwork that associated with weight gain was shown to be at least 17 yrs in middle-aged workers. Special attention should be paid to prevent weight gain at an earlier stage and not when this increase in weight has become apparent.

Calculation of the benchmark duration of shift work associated with the development of impaired glucose metabolism: a 14-year cohort study on 7104 male workers.

OBJECTIVES: The aim of this study was to calculate the benchmark doses (BMD) and their 95% lower confidence boundary (BMDL) for the threshold number of years of shift work associated with a relative increase in haemoglobin A1c (HbA(1c)), an index of glucose metabolism. METHODS: A 14-year prospective cohort study was conducted in male workers at a Japanese steel company (n=7104) who had received annual health check-ups between 1991 and 2005. The endpoints were either a 10%, 15%, 20%, 25% or 30% increase in HbA(1c) levels during the observation period, compared to HbA(1c) at entry to the study. The associations between years of shift work and increases in HbA(1c) were investigated using pooled logistic regression, adjusted for age, body mass index, mean arterial pressure, total serum cholesterol, creatinine, alanine aminotransferase, gamma-glutamyl transpeptidase, uric acid, drinking habits, smoking habits and habitual exercise. RESULTS: The BMDL/BMD for years of shift work were calculated using benchmark responses (BMRs) of 5% or 10% and parameters for duration of shift work and other covariates. Assuming a mean age of 53 years in workers aged 50 years or older, the BMDL/BMD for years of shift work with a BMR of 5% were 17.8/23.9 (> or = 15%), 15.7/18.7 (> or = 20%), 18.9/22.7 (> or = 25%) and 25.2/31.7 (> or = 30%). With a BMR of 10%, the respective values were 29.5/39.7 (> or = 15%), 24.3/28.9 (> or = 20%), 27.3/32.7 (> or = 25%) and 34.1/42.9 (> or = 30%). CONCLUSION: These results suggest that special attention should be paid to middle-aged workers whose years of shift work exceeds these threshold times.

Longitudinal effect of smoking cessation on physical and laboratory findings.

BACKGROUND: Detailed information on the expected physiologic changes after smoking cessation is practically useful to encourage people to stop smoking. Furthermore, weight increase after cessation may affect such physiologic changes. PURPOSE: This article aims to evaluate the effect of smoking cessation on annual changes in body weight, blood pressure, and blood biochemistry. METHODS: This study analyzed the results of annual health examinations from 1991 to 2005 in male Japanese workers in 2009. Subjects classified as stopping smoking (n=445) responded initially as smokers in a self-administered questionnaire (baseline year) and then answered consistently as nonsmokers for 3 subsequent years. Of the 2672 smokers identified in the study, 2403 subjects who had data available for at least 4 successive years were selected as controls. The time course of physiologic and laboratory data was analyzed using a linear mixed model. RESULTS: Data adjusted for age, type of job schedule, drinking and physical activity showed that subjects who stopped smoking had significantly greater increases in weight, BMI, systolic and diastolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, and uric acid and a greater decrease in hemoglobin in the 3 years following smoking cessation than continuing smokers. Additional adjustment for change in BMI from baseline negated the significant deterioration in systolic and diastolic blood pressure and total cholesterol that occurred following smoking cessation. CONCLUSIONS: Increase in body weight, blood pressure, and blood biochemistry can continue for at least 3 years after smoking cessation. This study also indicated that these increases were related to the weight increase that occurred after smoking cessation.

Estimation of the benchmark duration of alternating shift work associated with increased total cholesterol levels among male Japanese workers.

OBJECTIVE: The aim of this study was to estimate the benchmark doses (BMD) and their 95% lower confidence limits (BMDL) for the threshold number of years of alternating shift work associated with a relative increase in serum total cholesterol level (T-Cho), as an index of lipid metabolism. METHODS: We conducted a 14-year prospective cohort study among male workers (N=6886) at a Japanese steel company who had received annual health check-ups between 1991 and 2005. The endpoints were either a 20, 25, 30, 35, 40 or 45% increase in T-Cho levels during the observation period, compared to T-Cho at baseline. We investigated the associations between the years of alternating shift work and the relative increases in T-Cho using pooled logistic regression, adjusted for other potential covariates. RESULTS: We estimated the BMDL and BMD for years of alternating shift work among 40-, 50-, or >50-year old subjects using benchmark responses (BMR) of 5 or 10% and parameters for the duration of alternating shift work and other covariates. Assuming a mean age of 44 years among workers in their 40s, the BMDL/BMD for years of alternating shift work with a BMR of 5% were 21.0/28.0 (> or =20%), 21.3/26.1 (> or =25%), 24.1/28.8 (> or =30%), 25.6/29.8 (> or =35%), 27.1/31.5 (> or =40%), and 27.7/32.1 (> or =45%). CONCLUSIONS: The threshold number of years of alternating shift work that caused a 5% increase in T-Cho was shown to be > or =21 years among middle-aged workers. Special attention should be paid to influence the process at an earlier stage and not when the risk has already materialized.

Longitudinal evaluation of the effect of smoking initiation on body weight, blood pressure, and blood biochemistry.

OBJECTIVE: To evaluate the effect of smoking initiation on annual changes in body weight, blood pressure, and blood biochemistry. METHODS: This study analyzed the results of annual health examinations from 1991 to 2005 in male Japanese workers. Subjects who started smoking (n=214) initially responded as non-smokers in a self-administered questionnaire (baseline year) and then answered consistently as smokers for 3 subsequent years. Out of 2547 non-smokers, we selected 1589 controls who had data available for at least four successive years. The time course of physiological and laboratory data was analyzed using a linear mixed model. RESULTS: A significant temporal decrease from baseline in body mass index (first year, -0.1 kg/m(2)), diastolic blood pressure (second year, -1.5 mm Hg) and gamma-glutamyl transpeptidase (second year, -3.5 IU/L) was observed for subjects who started smoking. An opposite pattern was observed in non-smokers. On average, those who started smoking had significantly lower body mass index (first year, -0.2 kg/m(2); second year, -0.2 kg/m(2)), systolic blood pressure (second year, -2.1 mm Hg), diastolic blood pressure (second year, -2.0 mm Hg), and gamma-glutamyl transpeptidase (second year, -4.5 IU/L) than non-smokers. CONCLUSION: In this study, smoking initiation did not yield clinically significant long-term benefits with respect to physiological or biochemical outcomes. These results are important because few studies have tracked these types of changes longitudinally from initiation through 3 years of follow-up.

Novel mechanisms of pH sensitivity in tuna hemoglobin: a structural explanation of the root effect.

The crystal structure of hemoglobin has been known for several decades, yet various features of the molecule remain unexplained or controversial. Several animal hemoglobins have properties that cannot be readily explained in terms of their amino acid sequence and known atomic models of hemoglobin. Among these, fish hemoglobins are well known for their widely varying interactions with heterotropic effector molecules and pH sensitivity. Some fish hemoglobins are almost completely insensitive to pH (within physiological limits), whereas others show extremely low oxygen affinity under acid conditions, a phenomenon called the Root effect. X-ray crystal structures of Root effect hemoglobins have not, to date, provided convincing explanations of this effect. Sequence alignments have signally failed to pinpoint the residues involved, and site-directed mutagenesis has not yielded a human hemoglobin variant with this property. We have solved the crystal structure of tuna hemoglobin in the deoxy form at low and moderate pH and in the presence of carbon monoxide at high pH. A comparison of these models shows clear evidence for novel mechanisms of pH-dependent control of ligand affinity.

Oxygen equilibrium and EPR studies on alpha1beta1 hemoglobin dimer.

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [alpha(N)(Fe-NO)beta(M)(Mg)] hybrid showed some alpha1beta1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.

Peripheral blood mononuclear cells are possible extrahepatic replication sites for hepatitis C virus.

BACKGROUND/AIMS: Hepatitis C virus is a major causative agent of chronic liver disease and hepatocellular carcinoma and is considered to be a hepatotropic virus. It remains controversial whether hepatitis C virus exists in peripheral blood mononuclear cells and replicates there. In order to resolve this issue, we performed nested RT-PCR (reverse transcription polymerase chain reaction) and RT-PCR in situ hybridization in peripheral blood mononuclear cells of patients with chronic hepatitis C. METHODOLOGY: We collected peripheral blood mononuclear cells from patients with chronic hepatitis C, extracted total RNA from the samples, and performed nested RT-PCR to detect hepatitis C virus RNA in the peripheral blood mononuclear cells lysates. We also fixed peripheral blood mononuclear cells of the patients in 4% paraformaldehyde and performed RT-PCR in situ hybridization with a digoxigenin-labeled RNA probe to detect hepatitis C virus RNA in the cells. RESULTS: Using these methods, we detected both positive- and negative-stranded hepatitis C virus RNA in peripheral blood mononuclear cells of hepatitis C patients. To determine in which cell population of peripheral blood mononuclear cells hepatitis C virus is present, we performed PCR in situ hybridization after incubation with fluorescent latex microbeads which could be phagocytozed by monocytes. We obtained positive signals of the replicative hepatitis C virus genome not only in lymphocytes but also in monocytes. CONCLUSIONS: RT-PCR in situ hybridization with a nonradioactive probe was found to be useful for in situ detection of hepatitis C virus RNA. Our findings suggest that peripheral blood mononuclear cells may be extrahepatic replication sites for hepatitis C virus.

Coordination geometry of Cu-porphyrin in Cu(II)-Fe(II) hybrid hemoglobins studied by Q-band EPR and resonance Raman spectroscopies.

Cu(II)-Fe(II) hybrid hemoglobins were investigated by UV-vis, Q-band (35 GHz) EPR and resonance Raman spectroscopies. EPR results indicated that Cu-porphyrin in alpha-subunit within hybrid hemoglobin had either 5- or 4-coordination geometry depending on the pH conditions, while Cu-porphyrin in beta-subunit had only 5-coordination geometry at high and low pH values. These results were consistent with UV-vis absorption results. A new resonance Raman band appeared around 190 cm(-1), which was present whenever 5-coordinated Cu-porphyrin existed in Cu(II)-Fe(II) hybrid hemoglobins irrespective of the coordination number in Fe(II) subunit. This Raman band might be assigned to Cu-N(epsilon) (His) stretching mode. These results are direct demonstration of the existence of coordination changes of Cu-porphyrin in alpha-subunit within hybrid hemoglobin by shifting the molecular conformation from fully unliganded state to intermediately liganded state.

Crystal structures of deoxy- and carbonmonoxyhemoglobin F1 from the hagfish Eptatretus burgeri.

Hagfish are extremely primitive jawless fish of disputed ancestry. Although generally classed with lampreys as cyclostomes ("round mouths"), it is clear that they diverged from them several hundred million years ago. The crystal structures of the deoxy and CO forms of hemoglobin from a hagfish (Eptatretus burgeri) have been solved at 1.6 and 2.1 A, respectively. The deoxy crystal contains one dimer and two monomers in a unit cell, with the dimer being similar to that found in lamprey deoxy-Hb, but with a larger interface and different relative orientation of the partner chains. Ile(E11) and Gln(E7) obstruct ligand binding in the deoxy form and make room for ligands in the CO form, but no interaction path between the two hemes could be identified. The BGH core structure, which forms the alpha1beta1 interface of all vertebrate alpha2beta2 tetrameric Hbs, is conserved in hagfish and lamprey Hbs. It was shown previously that human and cartilaginous fish Hbs have independently evolved stereochemical mechanisms other than the movement of the proximal histidine to regulate ligand binding at the hemes. Our results therefore suggest that the formation of the alpha2beta2 tetramer using the BGH core and the mechanism of quaternary structure change evolved between the branching points of hagfish and lampreys from other vertebrates.

The contribution of the asymmetric alpha 1beta 1 half-oxygenated intermediate to human hemoglobin cooperativity.

Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O(2)] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O(2))beta(Fe-O(2))][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O(2) binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O(2) equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O(2)-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)]( 2) and [alpha(Zn)beta(Fe)]( 2) were measured by using an Imai apparatus. It was shown that the first and second O(2) equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg(-1), respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O(2) binding study.