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Post-translational maturation of cytochromes c involves the covalent attachment of heme to the Cys-Xxx-Xxx-Cys-His motif of the apo-cytochrome. For this process, the two cysteines of the motif must be in the reduced state. In bacteria, this is achieved by dedicated, membrane-bound thiol-disulfide oxidoreductases with a high reducing power, which are essential components of cytochrome c maturation systems and are also linked to cellular disulfide-bond formation machineries. Here we report high-resolution structures of oxidized and reduced states of a soluble, functional domain of one such oxidoreductase, ResA, from Bacillus subtilis. The structures elucidate the structural basis of the protein's high reducing power and reveal the largest redox-coupled conformational changes observed to date in any thioredoxin-like protein. These redox-coupled changes alter the protein surface and illustrate how the redox state of ResA predetermines to which substrate it binds. Furthermore, a polar cavity, present only in the reduced state, may confer specificity to recognize apo-cytochrome c. The described features of ResA are likely to be general for bacterial cytochrome c maturation systems.

Covalent attachment of heme to apocytochromes c in bacteria occurs on the outside of the cytoplasmic membrane and requires two reduced cysteinyls at the heme binding site. A constructed ResA-deficient Bacillus subtilis strain was found to lack c-type cytochromes. Cytochrome c synthesis was restored in the mutant by: (i) in trans expression of resA; (ii) deficiency in BdbD, a thiol-disulfide oxidoreductase that catalyzes formation of an intramolecular disulfide bond in apocytochrome c after transfer of the polypeptide across the cytoplasmic membrane; or (iii) by addition of the reductant dithiothreitol to the growth medium. In vivo studies of ResA showed that it is membrane-associated with its thioredoxin-like domain on the outside of the cytoplasmic membrane. Analysis of a soluble form of the protein revealed two redox reactive cysteine residues with a midpoint potential of about -340 mV at pH 7. We conclude that ResA, probably together with another thiol-disulfide oxidoreductase, CcdA, is required for the reduction of the cysteinyls in the heme binding site of apocytochrome c.

The effect of Mn2+/Mg2+ concentration on the activity of intact, homogeneous phosphoenolpyruvate carboxykinase (PEPCK) from leaves of the C4 grass, Guinea grass (Panicum maximum), have been investigated. Assay conditions were optimized so that PEPCK activity could be measured at concentrations of Mn2+/Mg2+ similar to those found in the cytosol (low micromolar Mn2+ and millimolar Mg2+). PEPCK activity was totally dependent on Mn2+ and was activated at low micromolar concentrations of Mn2+ by millimolar concentrations of Mg2+. Therefore, at physiological concentrations of Mn2+, PEPCK has a requirement for Mg2+. Assay at physiological concentrations of Mn2+/Mg2+ led to a marked decrease in its affinity for ATP and a 13-fold increase in its affinity for CO2. The Km (CO2) was further decreased by assay at physiological ATP to ADP ratios, reaching values as low as 20 microM CO2, comparable with the Km (CO2) of ribulose 1,5-bisphosphate carboxylase-oxygenase. This means that PEPCK will catalyze a reversible reaction and that it could operate as a carboxylase in vivo, a feature that could be particularly important in algal CO2-concentrating systems.

In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves. To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves. The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms. In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate. The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations. Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass. Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves. There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation.

The aim of this study was to investigate whether gluconeogenesis catalysed by phosphoenolpyruvate carboxykinase (PEPCK) occurs during leaf senescence. This was addressed by determining changes in the abundance and intercellular location of enzymes necessary for gluconeogenesis during the senescence of barley leaves and cucumber cotyledons. PEPCK was never present in barley leaves, despite the presence of large amounts of isocitrate lyase (ICL), a key enzyme of the glyoxylate cycle, and of its product, glyoxylate. Although PEPCK was present in non-senescent cucumber cotyledons, its abundance declined during senescence. Throughout senescence, PEPCK was only present in the trichomes and vasculature, whereas ICL was located in mesophyll cells. Pyruvate,Pi dikinase (PPDK) which, in concert with NAD(P)-malic enzyme, is also capable of catalysing gluconeogenesis, was present in non-senescent barley leaves and cucumber cotyledons, but in both plants its abundance decreased greatly during senescence. The abundance of ICL was greatly reduced in senescing detached barley leaves by either illumination or by co-incubation with sucrose, and greatly increased in darkened attached barley leaves. These results argue against the large-scale occurrence of gluconeogenesis during senescence catalysed either by PEPCK or PPDK. In cucumber cotyledons, PEPCK may play a role in metabolic processes linked to the export of amino acids, a role in which phosphoenolpyruvate carboxylase may also be involved. The amount of ICL was increased by starvation and during senescence may function in the conversion of lipids to organic acids, which are then utilised in the mobilisation of amino acids from leaf protein.

A number of N-hydroxylated indole derivatives have been fed to rats and the urinary metabolites compared with those obtained on feeding the corresponding N-H indoles. 1-Methoxy- and 1-acetoxy-indole were metabolized to compounds normally observed on feeding indole. 1-Hydroxy- and 1-methoxy-indole-3-carboxylic acids were both partially excreted unchanged and reduced to indole-3-carboxylic acid which was excreted along with its glucuronide. Indole-3-glyoxylic acid and its 1-methoxy derivative were excreted almost entirely unchanged. 1-Hydroxyindole-3-glyoxylic acid was mostly excreted unchanged, but some reduction to indole-3-glyoxylic acid took place. The stability of these acids is associated with their low pKa values. The significance of the removal in vivo of groups attached through oxygen to the 1 position of indoles in relation to the synthesis of new potential medicinal agents is noted.

(1) Over 15 months, 532 consecutive admissions to the CCU at the Radcliffe Oxford were studied; of these 333 were cases of myocardial infarction, and 319 were first admissions for this condition. Information about survival and return to work was collected for 300. A further 30 had artificial pacemakers inserted; there were 141 (26%) of the 532 cases which did not require the special care offered by the CCU. (2) Of 300 patients for whom data were available, 27 were recorded as having received DC shock. In hospital, case fatality was significantly higher among those requiring DC shock than among the remainder. Overall the 3-year survival rates were 47 per cent among those receiving shock, and 62 per cent among the remainder, compared with an expected 91 per cent for a population of the same age and sex. (3) Among men aged under 65 years, 6 of 11 who received shock, compared with 117 (77%) who did not receive shock, returned to work after leaving hospital. (4) Rates of admission to the CCU of cases of myocardial infarction per 1000 standardised population among people living in the areas around Oxford City were estimated as being 58 per cent of admission rates of cases among residents of the city. (5) The case incidence of ventricular fibrillation and the case fatality rate were both higher among those living in the environs than among those living in the city, but these differences were not statistically significant. (6) It is also concluded that insufficient is known about the factors underlying the general practitioner's decision to commit a case of myocardial infarction to other than short ambulance journeys or about the effects of such journeys on prognosis.

Some definitions of need for health care as a basis of planning health services are discussed. A model proposed by Donabedian (1974) relating need to resources is used to consider the problems faced by the Resource Allocation Working Party (RAWP) (Department of Health and Social Security, 1976). The paper concludes that need should be defined in relation to the procedures available to meet it and the resources that permit those procedures to be used. The procedures include the whole gamut of prevention and screening, cure and care, research and development. This necessarily raises moral and ethical issues which stretch beyond the limits of medicine and concern patients, their relatives, and the public in general. Neither the medical profession nor the lay public can be expected to help define need for health care unless they understand the underlying issues. This is a challenge to those responsible for professional and public education.

A study carried out in East Anglia identified a number of aspects of the lives of elderly people living alone and some of these are reported here. A sample of 2000 elderly people from 20 general practitioner practices was selected for interview. Overall, there was a response rate of 88 per cent. Of those age 75 and over, 43 per cent lived alone, this proportion being higher in urban areas, in council, privately rented and sheltered housing and amongst women. There is a high level of informal support for elderly people living alone, although there is also a large number who do not have regular frequent visitors. Of the statutory services, home helps visit nearly a quarter of those aged 75 and over living alone, on a weekly basis. In terms of at least one activity of daily living, shopping, those living alone exhibit a higher level of independence than those living with others, but nearly a quarter of those aged 75 and over living alone do rely on someone else entirely to do their shopping for them. The study identified a number of aspects of ill health in elderly people. There was a substantial proportion with urinary incontinence and, amongst those aged 75 and over living with others, a significant proportion with a degree of faecal incontinence. There was also a high proportion of people unable to cut their own toenails.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of N,N'-dibenzyl-1,2-diaminobenzene (2) successively with thionyl chloride and then m-chloroperbenzoic acid gave N,N'-dibenzyl-1H,3H-2,1,3-benzothiadiazole 2,2-dioxide (4), which gave (via routes analogous to standard epinephrine syntheses) four bicyclic catecholamine analogues 7a-d. Hydrogenolysis of 4 yielded the parent heterocycle 5 in the first practicable synthesis avoiding expensive sulfamide (Scheme I). The trifluoromethanesulfonamidoacetophenones 8m and 8p on similar elaboration gave triflanilide catecholamine analogues 14m, 14p,17m, and 17p (Scheme II). 4,4'Dimethoxybenzhydrylamine (15) is recommended for the regiospecific synthesis of primary amines from epoxides (Scheme II). Series 7,14, and 17 were inactive in animal cardiovascular screens. Selected compounds were also screened in bronchodilator and in in vitro dopamine-, clonidine-, and prazosin-receptor binding assays as appropriate; again no activity was observed. Steric lipophilicity, and acidity factors are discussed, and the inactivity is ascribed to the high acidity of both systems (pKa approximately equal to 4).

The incidence of myocardial infarction and the return to work for survivors were studied among the employees of an English car assembly plant by analysing 12 811 medical records of persons employed during the seven years between January 1966 and December 1972. The standardized morbidity ratio of myocardial infarction found in this study calculated on the basis of incidence rates reported by Kinlen )1973) for the Oxford community in which the factory was situated was 90. The standardized morbidity ratio from production line workers only was 66 and that for the monthly paid staff 272. Of the production line workers who survived the attack 22 (90%) returned to their previous jobs without undue difficulty and with two exceptions within four months of the onset of their illness; there was no relation between length of absence and age at the time of attack. These findings suggest that workers in mass production jobs such as car assembly are not special risk from myocardial infarction, and most of those who survive a heart attack are able to return to their former work. Taken with Kinlen's (1973) study and that of Armstrong et al. (1972) in Edinburgh, they also bear out mortality data by indicating that in Oxfordshire the incidence of coronary heart disease is lower than the British average.

The DNA-binding properties of the anti-cancer drug amsacrine and a 9-aminoacridine analogue substituted at the 4 position with a 4-methanesulphonanilido-group, have been examined by means of unwinding, melting and equilibrium binding experiments. These find that the latter compound is at least as effective as a DNA-binder and intercalator as amsacrine itself. Molecular modelling and energetic calculations have confirmed this, and have produced plausible intercalation geometries. These show that there are subtle differences in the low-energy minor groove arrangements adopted by the substituents of the two drugs. Speculation is advanced that these differences may be relevant to the marked differences in cytotoxicity shown by the two compounds.

A case-control study of the relationship between use of oral contraceptives and cancer of the breast, cancer of the corpus uteri, cancer of the ovary, and benign breast diseases was undertaken among women in the age group 20--44 years who had been admitted to several large hospitals in Connecticut. No significant association was seen between use of oral contraceptives and breast cancer. It was found that the longer a woman had used sequential oral contraceptives, the lower her relative risk of benign breast disease. Use of combination oral contraceptives, however, did not appear to influence the risk of benign breast diseases. The numbers of cases with cancer of the ovary and of the corpus uteri in this age group were too small for any conclusions to be reached.

In a survey of the general population the presence or absence of osteoarthrosis of the hand was determined radiologically in 685 adults (300 males and 385 females). Of these, 261 (124 males and 137 females), chosen randomly, were given a complete clinical examination of the musculoskeletal system which included x-ray of joints elsewhere in the body. Osteoarthrosis (OA) scores for the hand and for all body sites were computed for each subject by summing the number of affected joints. For all subjects soical class, height, weight, total serum protein, serum uric acid, haemoglobin, antistreptolysin O, (ASO), C-reactive protein (CRP), and rheumatoid factor were also measured. Analyses were carried out by simple comparison of means and by calculating multiple regressions and correlations.

The multifactorial control of serum uric acid levels is discussed from an epidemiological point of view and the principles at issue related to the epidemiology of osteoarthrosis. It is shown that in osteoarthrosis the clinical characteristics vary from one joint group to another. Association of x-ray changes with pain varies between joints and between sexes for the various joints. The disease tends to be commoner in females but not in all age groups. There is an association between obesity and osteoarthrosis in the hands and knees but not the hips or ankles. These differences raise questions about the homogeneity of generalised osteoarthrosis. Such questions might be answered by epidemiological studies which consider individual joint groups and are carefully directed towards specific hypotheses, the development of which could be facilitated by the use of iconic models.

A study of the workload of trainers and their trainees in general practice, was undertaken throughout East Anglia. Thirty trainer/trainee pairs returned data over a two-week period and follow-up information was obtained from five pairs, eight months later. A simple encounter form was used.Trainee workloads were found to be similar to those of earlier studies, though there was considerable variation between the workloads. Trainees had little experience with elderly patients, and there was a lack of teamwork between members of the practices in providing care for the elderly.

Thirty-nine randomly selected general practitioners in the Cambridgeshire Health Area were surveyed in an attempt to determine whether there were prior differences between those practitioners who choose to become involved with undergraduate medical students and those who choose not to. Teachers were found to have graduated more recently, subscribe to more journals and were more likely to use medical libraries than non-teachers. All the solo practitioners in the study and two of the four practitioners in two-man groups belonged to a subgroup of non-teachers who had never had students and did not want students in the future. This subfroup also subscribed to fewer journals than the other groups, bought fewer textbooks and used medical libraries less. The only prior differences between teachers and non-teacher that have been demonstrated are year of graduation and type of practice. Other differences could well be attributed to the influence of the students themselves. Studies are needed to clarify this issue, especially as it realates to continuing medical education.