Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB.

The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.

Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli.

This paper is a review of strategies to introduce protein into the liquid medium of Escherichia coli K-12 industrial production cells. The cell design strategies are generally based on one of two general mechanisms. The first strategy involves a two-stage translocation using active transporters in the cytoplasmic membrane followed by passive transport through the outer membrane. Passive transport is achieved through either external or internal destabilization of the E. coli structural components. The latter can be achieved by transplantation of destabilizing components (lysis proteins) that work by permeabilization of the outer membrane from the interior of the cell, or by using cells carrying mutations of structural components. Passive transport can also be achieved by a chemical, mechanical, or enzymatic permeabilization directed from outside the cell. The second strategy is realized through transplantation of proteins capable of active transport over one or both of the membranes. This involves the transplantation of secretion mechanisms into the K-12 cell from pathogenic E. coli as well as from other species. The process design strategies are dependent on environmental conditions and must take into account changes in physical parameters, medium design, and influx of limiting carbon source in fed-batch cultivation.

Growth rate-dependent changes in Escherichia coli membrane structure and protein leakage.

The phospholipid and fatty acid content of the Escherichia coli membrane were investigated during continuous cultivation. At low growth rates, there was an increase in cardiolipin produced at the expense of phosphatidylethanolamine. Phosphatidylglycerol had a maximum at a growth rate of 0.3 h(-1). The amount of cyclic fatty acids was markedly increased at lower growth rates, while there was an evident minimum at 0.3 h(-1). This was also the case for saturated fatty acids. At this point, the unsaturated fatty acids had a maximum depending mainly on changes in cis-vaccenic acid. The mechanical strength towards sonication and osmotic shock/enzymatic treatment showed that the cells were more rigid at low dilution rates. However, this was accompanied by a higher cell lysis, a reduced capacity for total and specific protein production and a lower yield of cells. The amount of lipid A in the medium (endotoxin) was constant and negligible at all growth rates. The leakage of periplasmic protein to the medium had an optimum at 0.3 h(-1), resulting in a transport of 20% of the total recombinant product. It is argued that this constitutes the point of highest membrane fluidity and thus an increase possibility for protein transport.

The prevalence of gentamicin resistance among clinical isolates of enterobacteria in a Danish region.

The susceptibility of enterobacteria to gentamicin was studied in the County of North Jutland during 1993-1998. A total of 378 patients had a first-time gentamicin-resistant isolate. The annual number rose from 34 patients in 1993 to 89 in 1998. The prevalence of resistance per 1000 patients with isolates examined was 13.5 for Escherichia coli, 15.8 for Klebsiella pneumoniae, 26.1 for Klebsiella oxytoca, and 150.4 for Citrobacter freundii. E. coli accounted for 67% of gentamicin-resistant isolates. K. oxytoca was probably associated with a nosocomial epidemic, whereas a source for C. freundii remained unresolved. An analysis confined to E. coli identified specimens other than urine as risk factors for gentamicin resistance. Likewise, resistance to sulfonamides and to fluoroquinolones were strong risk factors for gentamicin resistance among urinary isolates of E. coli. Thus, it is likely that aminoglycoside resistance may be promoted by other antibiotic groups due to co-resistance. The therapeutic and prophylactic uses of aminoglycosides have as yet not been undermined, but continuous population-based surveillance and vigilance are recommended.

Intensive rehabilitation of emotionally distressed patients after coronary by-pass grafting.

OBJECTIVES: To evaluate whether the status of emotionally distressed post coronary by-pass surgery patients can be improved by a comprehensive, in-patient rehabilitation programme. DESIGN: Cross-sectional. SETTING: Rehabilitation hospital. SUBJECTS: One hundred and fifty-two post coronary by-pass surgery patients referred to an intensive rehabilitation programme. The study group was divided into two subgroups, according to the level of emotional distress. Forty-three (30%) out of 142 patients who completed the Nottingham Health Profile were considered to be distressed. INTERVENTIONS: The comprehensive 4-week inpatient rehabilitation programme consisted of daily physical exercise, lectures about coronary disease and risk factors, psychological support and nutrition counselling. MAIN OUTCOME MEASURES: Physical fitness, blood lipids and quality of life questionnaires. RESULTS: The two-way analysis of variance showed that emotionally distressed patients achieved equally good results as those obtained by nondistressed patients in performance regarding the maximal exercise stress test, maximum rate pressure product achieved, or the level of resting heart rate. Significant improvement in psychological well-being as assessed by a seven-point rating scale was observed in both groups. Both groups of patients were also equally successful in weight reduction and lowering of total cholesterol and triglycerides. Of all the patients who were employed at admission, twice the number of patients in the distressed group were in receipt of a permanent disability pension and half the number of patients was employed, compared to the initially nondistressed group, at 1-year follow-up. CONCLUSIONS: Distressed patients were very successful in improving their functional status and reducing risk factors when offered an intensive rehabilitation programme but they were much less successful in returning to work. Secondly, patients who were emotionally distressed after surgery did not differ in regard to disease status or physical capacity from nondistressed patients. They did, however, experience more angina pectoris both in daily life and when exposed to a maximal exercise stress test. Finally, the presence of emotional distress was easily detected by any measure of psychological status.

Systemic and deep-seated infections caused by Arcanobacterium haemolyticum.

Arcanobacterium haemolyticum has been implicated mainly in non-streptococcal pharyngitis and wound infections. Rarely, it has been reported to cause systemic infection, often in combination with other pathogens. Two cases of systemic and deep-seated infections caused by Arcanobacterium haemolyticum are reported, and the literature is reviewed. Sixteen cases of bacteremia and seven cases of non-bacteremic deep-seated have been published previously. Eight of the bacteremic and two of the non-bacteremic cases occurred in younger, apparently healthy immunocompetent patients. Six patients had infections of the central nervous system. The optimal treatment of infections caused by Arcanobacterium haemolyticum is not known. Although in vitro susceptibility tests have demonstrated tolerance of Arcanobacterium haemolyticum to penicillin, penicillins with or without aminoglycosides have been the most widely used antibiotics, in most cases with success.

Lung cancer and mesothelioma in the pleura and peritoneum among Swedish insulation workers.

OBJECTIVES: To estimate the risk of cancer and death in Swedish insulation workers some years after their exposure to asbestos had stopped. One hypothesis was that the risk of lung cancer would tend to decrease some years after the exposure had ended. METHODS: In a cohort study the cancer morbidity and cause of death was investigated in 248 insulation workers and compared with the corresponding morbidity and mortality in the general population. Due to stringent regulations, exposure to asbestos of all types had almost ended in Sweden in the mid-1970s. Through a questionnaire, surviving insulation workers were asked about their exposure to asbestos and their smoking habits. RESULTS: Between 1970 and 1994 there were 86 deaths compared with the 46.0 expected (standardised incidence ratio (SIR) 1.9; 95% confidence interval (95% CI) 1.5 to 2.3), the increase was mainly due to an increased cancer mortality. The morbidity was increased for lung cancer (11 cases v 2.5 expected (SIR 4.4; 95% CI 2.2 to 7.9)), peritoneal mesothelioma (seven cases; no expected incidence could be calculated as the occurrence is too rare in the general population), cancer in pancreas (five cases v 0.7 expected (SIR 7.1; 95% CI 2.3 to 16.7)). No cases of pleural mesothelioma were found. The risk of lung cancer did not tend to approach that of the general population after the exposure to asbestos decreased. CONCLUSIONS: In the 1980s and the early 1990s, Swedish insulation workers still have a highly increased risk of diseases related to asbestos. The attributable risk for death and cancer was about 50%. The study also confirms the previous finding that mesothelioma in insulation workers seems to be situated in the peritoneum more often than in the pleura.

Calcium in patients on hemodialysis.

Analyzing 415 measurements from 33 patients on chronic maintenance hemodialysis we confirm that 50% of total calcium in the range 1.59 to 3.33 mmol/l gives a valuable approximation to ionized calcium in the range 0.79 to 1.62 mmol/l. However, a small but significant effect of albumin and phosphate on ionized calcium can also be demonstrated. Conventional albumin correction of total calcium using the formula: total calcium + (43 g/l-Albumin g/l).0.017 mmol/g is of poor value in these patients on maintenance hemodialysis.

Ultradian variation in serum phosphate concentration in patients on haemodialysis.

To study how much the serum phosphate concentration could vary on the day before scheduled dialysis we obtained 24-37 timed measurements during 24 h in nine patients on maintenance haemodialysis. During the observation there was an increase in serum phosphate concentration of 0.239 +/- 0.022, P < 0.01). Surprisingly six of the nine patients exhibited nine statistically and clinically significant nadirs with decrements of 0.21 +/- 0.04 mmol/l against an intra-assay standard deviation of about 0.05 mmol/l. Five of the nadirs occurred at 11.40-14.50, mean 12.59, and three at 19.00-21.30, mean 20.00. We found no coinciding changes in the serum calcium concentration. These findings indicate that serum phosphate concentrations do not always reflect phosphorus balance or intake.

The importance of lung function, non-malignant diseases associated with asbestos, and symptoms as predictors of ischaemic heart disease in shipyard workers exposed to asbestos.

The mortality from ischaemic heart disease was studied in a prospective cohort of 1725 shipyard workers exposed to asbestos. The analyses were stratified for age and smoking habits and restricted to men. In agreement with other findings, men with impaired lung function had a significantly higher risk (relative risk (RR) = 3.5) of dying from ischaemic heart disease than men with normal lung function. Men with asbestosis or suspected asbestosis had a significantly higher risk (RR = 3.1) of dying from ischaemic heart disease than men without asbestosis. Thus asbestosis or suspected asbestosis also seemed to be a risk factor for ischaemic heart disease. This finding was independent of respiratory function. There was no increased risk for ischaemic heart disease in men with compared with men without pleural plaques. Men with production of phlegm or sputum and wheezing or whistling had no increased risk for ischaemic heart disease compared with men without these symptoms. In the group with normal lung function men with dyspnoea had a significantly higher risk of dying from ischaemic heart disease than men without dyspnoea. The findings for men with asbestosis or suspected asbestosis indicated a further risk factor besides impaired lung function, in persons exposed to asbestos. Perhaps this risk factor is due to lesions of the pericardium with consequences for heart function.

The risk of lung cancer and mesothelioma after cessation of asbestos exposure: a prospective cohort study of shipyard workers.

A prospective cohort study of 3,893 shipyard workers, mainly exposed to chrysotile, indicated no increased risk of lung cancer 7-15 yrs after exposure to asbestos had ceased. The shipyard workers, however, had an increased risk of pleural mesotheliomas with 11 observed cases versus 1.5 expected. An explanation for these observations may be that asbestos may have different carcinogenic mechanisms in causing lung cancer and mesothelioma. A non-increased risk of lung cancer some years after exposure to asbestos has stopped is in accordance with asbestos acting as a promotor. The high risk of mesothelioma, on the other hand, may indicate that asbestos acts as a complete carcinogen in developing this disease.

A study of possible predictors of mesothelioma in shipyard workers exposed to asbestos.

In a prospective cohort study of 3893 shipyard workers, we estimated the value of medical monitoring, including chest radiograph, spirometry, and questions about smoking habits, asbestos exposure, and respiratory symptoms, as predictors of the risk of developing mesothelioma. There was no strong association between different exposure parameters and risk of mesothelioma. Impaired lung function and smoking were not predictors of risk of mesothelioma. Pleural plaque was not found to be associated with an increased risk of mesothelioma. Respiratory symptoms were of low value as predictors of risk of mesothelioma. Thus, traditional methods in health monitoring seem to be of low value in identifying persons with a high risk of mesothelioma in populations exposed to asbestos.

Effects of post-menopausal oestrogen-progestogen replacement therapy on serum lipids and lipoproteins: a review.

There are strong indications that post-menopausal oestrogen replacement therapy has a protective effect on the cardiovascular system, due to changes in the lipid profile. There are also indications that the addition of progestogens to oestrogen therapy may negatively influence these effects on the lipid metabolism. The present survey was undertaken to provide a comprehensive summary of all the available data on the effect of combined oestrogen-progestogen treatment on the post-menopausal lipid profile published from 1984 onwards. It appears that many progestogens are capable of diminishing the beneficial changes induced by oestrogens. The ultimate effect depends on the type and daily dose of both the oestrogen and the progestogen component. Oestradiol or oestradiol valerate administered cyclically with progesterone or cyproterone acetate appear to have the most favourable impact on lipids and lipoproteins.

Estimating asbestos exposure: a comparison of methods.

The association between pleural plaques and asbestos exposure was investigated in a cross-sectional study of 951 male shipyard workers. Asbestos exposure was estimated by the men themselves and also by an expert panel whose judgment was based only on occupational title. The analysis was restricted to men who had had at least 20 years of exposure and who had not changed jobs during their period of employment in the shipyards. The results showed that there was a much closer correlation between the occurrence of pleural plaques and the men's own estimates of exposure than between the occurrence and the experts' estimates. Contrary to reports of other investigators, the authors found no association between smoking habits and the occurrence of pleural plaques (rate ratio = 1.0, chi 2 (1) = 0.3).

Cancer morbidity in Swedish shipyard workers 1978-1983.

The cancer morbidity in 3787 shipyard workers was studied between 1978 and 1983. In these shipyards the use of asbestos was abandoned in 1972. The overall cancer morbidity was found to be similar to that of the male population of the same city, but there were four cases of mesothelioma. There were 11 cases of lung cancer, as opposed to 9.8 expected cases. Men with both heavy and long exposure to asbestos had no increased risk of lung cancer. The occurrence of pleural plaques was not associated with the risk of developing cancer.

Pleural plaques and respiratory function.

This cross-sectional study was comprised of 202 nonsmoking shipyard workers with varying exposures to asbestos. Their chest X-rays were normal or contained no abnormality other than pleural plaques. They participated in a health examination of workers exposed to asbestos, which comprised a total of 3,904 persons. One hundred and fifteen of the 202 workers had no deviations from normal X-rays, and 87 had pleural plaques but no other finding on their X-rays. Three out of the 115 workers with normal X-rays and 13 of the 87 with pleural plaques had FVCs below the reference limits (p less than 0.005). The workers with plaques had an average of 6.9% lower FVC. Even after stratification for asbestos exposure, men with plaques were found to have lower FVCs than men without plaques. This difference was largest for those with heavy exposure to asbestos.