A distinct cyclin A is expressed in germ cells in the mouse.

In this paper, the existence of two A-type cyclins in the mouse is demonstrated. In the adult mouse, the expression of cyclin A1, which has greatest sequence identity with Xenopus cyclin A1, is restricted to germ cells. In contrast cyclin A2, which has greatest sequence identity with human cyclin A and Xenopus cyclin A2, is expressed in all tissues analysed. In order to explore the function of cyclin A1 in germ cells, its expression during the meiotic cell cycle and its associated kinase subunits have been characterised in the testis. The levels of cyclin A1 mRNA rise dramatically in late pachytene spermatocytes and become undetectable soon after completion of the meiotic divisions; thus its expression is cell cycle regulated. In lysates of germ cells from adult testes, cyclin A1 is present in p13suc1 precipitates, and cyclin A1 immunoprecipitates possess histone H1 kinase activity. Three kinase partners of cyclin A1 were identified: p34cdc2, a polypeptide of 39 x 10(3) M(r) that is related to p33cdk2 and, in lesser quantities, p33cdk2. Cyclin A1 was also detected in oocytes; in metaphase I and metaphase II oocytes, a proportion of the cyclin A1 colocalises with the spindle, possibly suggestive of a functional interaction. These data indicate that mammalian germ cells contain cyclin A1-dependent kinases that either act as a substitute for, or in addition to, the cyclin A2-dependent kinases characterised in somatic tissues.

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5'end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191, 337-446], was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, Mr 46,265) and 469 amino acids (E3, Mr 49,193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-oxo-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.

Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.

Physicians' attitudes toward generic drug substitution by pharmacists.

Physicians in Southeast Texas were surveyed to obtain information on attitudes and practices concerning generic drug substitution. Questionnaires were sent to all practicing primary care physicians in the area, and responses were received from about 36% of them. The investigation revealed that a large percentage of physicians did not readily permit substitution. Two major reasons given for not permitting substitution were concerns about therapeutic inequivalency and doubts about pharmacists' ability to substitute safely.

A common structural motif in thiamin pyrophosphate-binding enzymes.

The amino acid sequences of a wide range of enzymes that utilize thiamin pyrophosphate (TPP) as cofactor have been compared. A common sequence motif approximately 30 residues in length was detected, beginning with the highly conserved sequence -GDG- and concluding with the highly conserved sequence -NN-. Secondary structure predictions suggest that the motif may adopt a beta alpha beta fold. The same motif was recognised in the primary structure of a protein deduced from the DNA sequence of a hitherto unassigned open reading frame of Rhodobacter capsulata. This putative protein exhibits additional homology with some but not all of the TPP-binding enzymes.

Adenosine kinase from human erythrocytes: kinetic studies and characterization of adenosine binding sites.

The reaction catalyzed by adenosine kinase purified from human erythrocytes proceeds via a classical ordered sequential mechanism in which adenosine is the first substrate to bind to and AMP is the last product to dissociate from the enzyme. However, the interpretation of the steady-state kinetic data is complicated by the finding that while AMP acts as a classical product inhibitor at concentrations greater than 5 mM, at lower concentrations AMP can act as an apparent activator of the enzyme under certain conditions. This apparent activation by AMP is proposed to be due to AMP allowing the enzyme mechanism to proceed via an alternative reaction pathway that avoids substrate inhibition by adenosine. Quantitative studies of the protection of the enzyme afforded by adenosine against both spontaneous and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated oxidation of thiol groups yielded "protection" constants (equivalent to enzyme-adenosine dissociation constant) of 12.8 microM and 12.6 microM, respectively, values that are more than an order of magnitude greater than the dissociation constant (Kia = 0.53 microM) for the "catalytic" enzyme-adenosine complex. These results suggest that adenosine kinase has at least two adenosine binding sites, one at the catalytic center and another quite distinct site at which binding of adenosine protects the reactive thiol group(s). This "protection" site appears to be separate from the nucleoside triphosphate binding site, and it also appears to be the site that is responsible for the substrate inhibition caused by adenosine.

Vaccination studies for the control of campylobacteriosis in Jamaican cattle.

Following the first diagnosis of campylobacteriosis in Jamaican cattle a field study was undertaken to determine the pathogenicity of Campylobacter fetus subspecies venerealis Jam (Jamaican strain) and to evaluate the effectiveness of vaccination in controlling the disease. A total of 46 nonpregnant yearling heifers and four two-year-old bulls were used in two separate experiments. The results showed that C fetus subspecies venerealis Jam readily colonised the reproductive tract of susceptible heifers and persisted in some animals (68 per cent of unvaccinated and 33 per cent of vaccinated animals) for the duration of the experiment. Pregnancy was confirmed in 13 of 18 (72 per cent) culture-negative heifers but in only eight of 28 (29 per cent) of the heifers with two or more positive cultures. Vaccination appeared to be curative because 44 per cent of vaccinated heifers were cleared of infection whereas 85 per cent of unvaccinated, inoculated heifers remained infected for at least 17 weeks. Vaccination improved the fertility level of the infected heifers threefold. Infection was not established in vaccinated bulls used for breeding infected heifers.

Factors influencing mortality in rheumatoid arthritis.

The prognosis in rheumatoid arthritis respecting mortality was studied in a consecutive series of 489 hospital patients over a period of 18 years. The relative risk of mortality was raised in both men (2.6; p less than 0.001) and women (3.4; p less than 0.001). In the women the relative risk was also influenced by prior duration of RA and was characterised by a diminution in risk 5-9 years after first presentation. Relative risks for men were more uniformly distributed over time. Annual excess mortality rates were strongly associated with age at first presentation in women, the rate increasing with increasing age in both the group seen within 5 years of onset of disease (chi 2(1) for trend = 30.4; p less than 0.001) and in the later referral group (chi 2(1) = 34.0; p less than 0.001). A similar but much less marked effect was observed in men in the early referral group (chi 2(1) = 13.7; p less than 0.001) only. These results suggest that initially women may have a milder form of disease and that hormonal status may affect prognosis. Future long-term therapeutic studies in RA should take into account the prognostic factors of age, sex and duration of disease.

Cause of death in rheumatoid arthritis.

A series of 489 consecutive patients with definite or classical rheumatoid arthritis was followed for a mean of 11.2 years. Cohort-analysis of the series showed a three-fold increase in mortality overall in comparison with age- and sex-specific rates in the general population. In patients first seen in hospital early in the course of the disease (less than 5 years from onset), the excess of deaths from circulatory, respiratory and musculoskeletal disorders was highly significant. Malignant disease and digestive system disorders accounted for a small excess of borderline significance. Patients who were referred later (greater than 5 years from onset) experienced, in addition, a highly significant excess of deaths from infections and disorders of the digestive and genito-urinary system but not from malignant neoplasms.

Cancer morbidity in rheumatoid arthritis.

A consecutive series of 489 patients with rheumatoid arthritis seen at the centre was studied to determine their cancer morbidity. Overall the 36 cancers diagnosed in the series between 1964 and 1981 were not significantly in excess of the expected number, but there was a highly significant excess of tumours of the reticuloendothelial system. The excess was mainly due to 6 observed cases of lymphoma. We conclude that there is a highly significant association between rheumatoid arthritis and the subsequent development of lymphoproliferative malignancy in this series.

Lymphoproliferative malignancy in rheumatoid arthritis: a study of 20 cases.

A series of 20 patients with definite or classical rheumatoid arthritis who subsequently developed a lymphoproliferative malignancy are described. The mean time between the onset of the 2 diseases was 13.2 years. A wide range of types of non-Hodgkin's lymphoma and Hodgkin's disease were found; there were no unusual histological features in the lymphomas. Although many of the patients had had gold, penicillamine, and other second-line drugs, none of them had received cytotoxic drugs, and there was no evidence that therapy was a cause of their malignancies. The likely cause of the association is a predisposition to both diseases.

Thrombocytosis of active rheumatoid disease.

Two cross-sectional and one longitudinal study of patients with rheumatoid arthritis showed that platelet number correlated with both clinical and laboratory parameters of disease activity, including erythrocyte sedimentation rate, zeta sedimentation ratio, viscosity of plasma and blood, white cell count, liver enzymes, rheumatoid factor, and several acute-phase proteins. There was also an inverse relationship between platelet number and the haemoglobin and serum albumin levels. III Indium-labelled platelet survival was reduced in 4 patients with active rheumatoid arthritis despite a raised platelet count, with labelled platelets being localised to inflamed joints in the 2 patients studied. Platelet aggregation was normal. We suggest that the raised platelet count of active rheumatoid arthritis may be a useful index of disease activity and may represent a bone marrow stress (syndrome) response to shortened platelet survival, with platelet sequestration occurring in areas of synovial inflammation.

Ultrastructure of the synovial membrane in seronegative inflammatory arthropathies.

The ultrastructure of the synovial membrane has been studied in 6 patients with seronegative inflammatory arthropathies: Reiter's (2), Crohn's (2), Whipple's (1) and Behçet's disease (1). The most striking changes were found in the synovial B cells, many containing abnormally large mitochondria with altered cristae surrounded by fibrillar material. Similar material was present in dilated endoplasmic reticulum which was the probable source of groups of extracellular fibrillar spheroidal bodies. The B cells also contained electron dense granular lysosomes of very variable size which, in common with the abnormal mitochondria, were often associated with bundles of orientated microfilaments and large golgi complexes. Light microscopy of the synovial membrane was consistent with an inflammatory arthritis, as were the high white cell counts in the synovial fluid. Systemic activity in the patients was indicated by raised ESR and C-reactive protein (CRP).

Some observations and speculations on the factors influencing the concentration of phenylbutazone in synovial fluid.

Simultaneous plasma and synovial fluid samples have been obtained from patients, having reached a steady state on phenylbutazone, as a means of investigating factors influencing drug penetration into synovial fluid. Synovial fluid levels are lower than, but related to, plasma levels and are higher in rheumatoid disease (55-100% plasma levels) than in osteoarthritis (less than 50%). Patients with a more active form of the disease have higher synovial fluid drug concentrations. Results are discussed in the light of present knowledge and areas for further study are proposed.

Serum calcium levels in rheumatoid arthritis.

Total and corrected (for albumin) serum calcium levels were investigated in a cross-sectional study of 394 patients with rheumatoid arthritis, 4490 healthy subjects, and 2609 inpatients at a district general hospital. Patients with rheumatoid arthritis had lower mean calcium levels than the healthy subject (p less than 0.001), but had similar levels to inpatients at the district general hospital. Thirty-eight inpatients with rheumatoid arthritis at a hospital for rheumatic diseases had lower mean corrected and total calcium levels than all other groups (p less than 0.01). Corrected or total calcium levels higher than 2.60 mmol/l or corrected calcium levels lower than 2.20 mmol/l were uncommon in the patients with rheumatoid arthritis. A longitudinal study of serum calcium levels in 17 patients with rheumatoid arthritis over 6-48 months showed considerable temporal variation in total and corrected calcium levels. Transient hypercalcaemia and hypocalcaemia occurred occasionally, but for most of the time calcium levels were normal. Changes in calcium levels were not related to changes in clinical, haematological, or immunological parameters of disease activity. Mean serum calcium levels are lower in disease than health; this occurs in RA as well as other diseases.

Relationship of gold and penicillamine therapy to diffuse interstitial lung disease.

Seven cases of diffuse interstitial lung disease (DILD) are reported with an unequivocal temporal relationship between the development of the lung disease and treatment with gold (6 cases) and penicillamine (1 case). They were characterised clinically by the sudden onset of dyspnoea and crepitations and radiologically by diffuse bilateral pulmonary shadowing. Most showed evidence of hypersensitivity such as eosinophilia, a raised serum IgE level in response to gold, proteinuria, thrombocytopenia, or an immediate postinjection reaction. DILD is a serious complication of treatment with gold and penicillamine that is commoner than generally realised.

Campylobacteriosis in Jamaican cattle.

Infertility is a widespread problem among cattle herds in Jamaica and an intergovernmental project was initiated to investigate the problem. One aspect of the project included diagnosis of campylobacteriosis (vibriosis), which was suspected in some herds but had not been confirmed in the laboratory. Tests were undertaken in late 1978 using improved techniques for handling and transporting specimens to the laboratory. Campylobacter fetus subspecies fetus was isolated from five of six herds tested. The diagnosis of C. fetus infection has led to improvements in farm management and a better appreciation of more efficient artificial insemination and veterinary services.