Determination and evolutionary significance of nucleotide sequences near to the 3''-end of 16S ribosomal RNA of mycobacteria

The nucleotide sequence at the 3'-end of 16Sr-RNA (nucleotides 1305-1508) was determined, by the primer extension method, for Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium vaccae, in addition to Mycobacterium leprae. No differences in nucleotide sequence were detected, indicating that this region of 16SrRNA is highly conserved among mycobacteria. The nucleotide sequence common to the four above-mentioned mycobacteria differs from that reported for species of other genera. For example, for helix 39 (nucleotides 1408-1491) the mycobacterial sequence has 58% similarity with the Escherichia coli sequence, 74% similarity with the Bacillus subtilis sequence and 93% similarity with the Streptomyces sequence. The observations support the assignment of M. leprae to the genus Mycobacterium.     

Symptoms of low blood pressure: a population study.

OBJECTIVE--To establish whether an association exists between blood pressures in the "low normal" range and common symptoms such as tiredness, dizziness, headache, and palpitation, as suggested by French and German medical practice but not English or American medical practice. DESIGN--Cross sectional population based survey (the health and lifestyle survey) of blood pressure measurements and self reported common symptoms. Results were analysed by combined stratification and logistic regression. SUBJECTS--7383 (82%) Adults aged 18 and over chosen from the electoral register in England, Wales, and Scotland for the health and lifestyle survey, in whom satisfactory physiological measurements were taken, from 9003 in the interviewed sample. MAIN OUTCOME MEASURES--Body mass index, smoking, social class, exercise, self declared physical illnesses, hours slept, use of drugs, and psychological illness as determined with the general health questionnaire assessed as potential confounders. RESULTS--True confounders were sex, age, taking of drugs, physical illness, exercise, and body mass index. A negative association was found between systolic blood pressure and self reported tiredness and feeling faint, which persisted after adjustment for the confounders. The association was strongest in women aged under 50. A negative association between systolic blood pressure and headache and a positive association between systolic blood pressure and palpitation were explained by confounding by age. CONCLUSIONS--Systemic hypotension is associated with persistent tiredness but treatment is not suggested as either possible or necessary. Instead, low blood pressures may be associated with opposite effects on mortality contrasted with morbidity.     

Selection of the T-cell repertoire in transgenic mice expressing a transplantation antigen in distinct thymus subsets

Transgenic mice that expressed a transplantation antigen, H-2Kb, in an unusual tissue distribution have been developed. Gene-regulatory elements from the immunoglobulin heavy-chain locus (Emu enhancer and heavy chain promoter) were linked to the class I Kb gene and the construct microinjected into fertilized mouse eggs of a different haplotype. It was expected that such gene-regulatory elements would direct expression of the foreign class I molecules only to B and T lymphocytes. However, expression was also detected in a subset of thymus medullary epithelium. The Kb molecules expressed on this thymic subset were unable to positively select T cells for passage to the periphery. The mice were, however, tolerant of the cell types expressing the foreign Kb molecules and were also tolerant of Kb presented as skin grafts. These results suggest that not all components of thymic epithelium are involved in positive selection of T cells and that transplantation antigens expressed on non-dendritic cells can induce tolerance.     

Amino acid sequence of crayfish troponin I

Troponin I is the actomyosin ATPase inhibitory subunit present in the thin filament regulatory complex. The complete amino acid sequence of crayfish tail muscle troponin I has been determined. The protein is composed of 201 amino acid residues and has a molecular weight of 23,547. The N terminus is blocked, likely by an acetyl group. Crayfish troponin I shows a rather low (20-25%) sequence identity with vertebrate troponin Is as compared to the 60-82% identity within the vertebrate phylum. Similar to vertebrate cardiac troponin I, crayfish troponin I contains a 30-residue-long N-terminal extension. In crayfish troponin I, this segment bears significant sequence homology with the heavy or light chains of particular myosins. The actin-binding domain of crayfish troponin I, which displays 57% sequence homology with vertebrate troponin Is, possesses 2 unusual trimethyllysine residues. The consensus sequence of this domain in five troponin Is is as follows: D-L-R-G-K-F-X-R*-P-X-L-R*-R*-V, where R+ stands for Arg/Lys, R* for Arg/trimethyllysine, and X for any amino acid residue. Troponin I possesses two Ca2+-dependent interactive sites for troponin C; one partly overlaps with the actin binding domain and is highly conserved, and the other, corresponding to the 30-residue-long segment following the N-terminal extension in vertebrate cardiac and crayfish troponin I, is poorly conserved in the different troponin Is. Troponin I also interacts with troponin T. The consensus sequence for the interacting site on troponin I is as follows: h-D- -X-D- -R+-Y-D-h-E-h, where h stands for a hydrophobic residue, D- for Asp/Glu, R+ for Arg/Lys, and X for any residue. The five troponin Is further possess one more 15-residue-long segment of high sequence identity near the C terminus. Its evolutionary conservation suggests that this domain is involved in protein-protein interaction.     

Molecular phenotypes in cultured maple syrup urine disease cells. Complete E1 alpha cDNA sequence and mRNA and subunit contents of the human branched chain alpha-keto acid dehydrogenase complex

The activity of the branched-chain alpha-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 alpha subunit of the human enzyme complex. The composite human E1 alpha cDNA consists of 1783 base pairs encoding the entire human E1 alpha subunit of 400 amino acids with calculated Mr = 45,552. The human E1 alpha and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 alpha mRNA and E1 alpha and E1 beta subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 alpha mRNA is present in normal quantity, whereas the contents of E1 alpha and E1 beta subunits are reduced; Type III, where the level of E1 alpha mRNA is markedly reduced with a concomitant loss of E1 alpha and E1 beta subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.     

Comparison of the immunosuppressive properties of milk growth factor and transforming growth factors beta 1 and beta 2

The effects of the newly isolated bovine milk growth factor (MGF) which shows N-terminal homology to transforming growth factor beta 2 were compared with the effects of porcine transforming growth factor beta 1 and beta 2 (pTGF-beta 1 and -beta 2) on human T lymphocyte activation. Freshly isolated human PBMC were stimulated with either PHA, anti-CD3 + phorbol-12,13-dibutyrate (PDBu), or with a combination of ionomycin + PDBu. MGF, pTGF-beta 1, and pTGF-beta 2 decreased mitogen-induced [3H]thymidine incorporation by 30 to 75% in a dose-dependent manner. The maximum degree of inhibition was obtained at 1 ng/ml (40 pM) and could not be increased by increasing the concentration of teh transforming growth factor 10-fold. Stimulation of fresh T cells with the recall Ag tetanus toxoid was also inhibited (85%) by MGF at pM concentrations as was the proliferation of a human T cell clone specific for purified protein derivative. The effects of MGF and pTGF-beta 1 on anti-CD3-mediated increase of intracellular Ca2+ (Cai2+) was investigated by using the Fura-2 method. Neither MGF nor pTGF-beta 1 inhibited this increase in Cai2+ induced by a mitogenic concentration of anti-CD3 antibody. In order to determine whether TGF-beta preferentially inhibited the CD4+ or CD8+ subpopulation of human T cells, a limiting dilution analysis system, which allows every T cell to proliferate, was used. pTGF-beta 1 at a concentration of 5 ng/ml decreased the frequency of proliferating T cell precursors of both the CD4+ and CD8+ subsets to a similar extent. Furthermore, MGF, pTGF-beta 1, and pTGF-beta 2 also decreased IL-2 mediated [3H]thymidine incorporation into human PBL Con A blasts and the IL-4-mediated [3H]thymidine incorporation of purified T lymphocytes costimulated with PDBu by 70%. In conclusion, bovine MGF exerts suppressive effects on human T cells stimulated with Ag, mitogens, or interleukins, and the degree of T cell suppression is similar (or identical) to those of pTGF-beta 1 or -beta 2.     

Recognition of specific Physarum alpha-tubulin isotypes by a monoclonal antibody. Sequence heterogeneity around the acetylation site at lysine 40

The monoclonal antibody 6-11B-1 recognises specifically the acetylated form of alpha-tubulin. The acetylation event occurs on a unique lysine residue, lysine 40. Using 6-11B-1, acetylated alpha-tubulin was detected in myxamoebae but not plasmodia of Physarum polycephalum. Following chemical acetylation plasmodial alpha-tubulin was detected by 6-11B-1. The monoclonal antibody KMP-1 recognises certain Physarum alpha-tubulin isotypes but only in non-acetylated form. Whilst recognising all the non-acetylated fraction of myxamoebal alpha-tubulin only a proportion of plasmodial alpha-tubulin was recognised by KMP-1. Peptides were synthesised corresponding to the acetylation domains (containing lysine 40) of myxamoebal alpha-tubulin and the inferred acetylation domains of two plasmodial-specific alpha-tubulin isotypes. The only difference between the two peptides was at a single residue corresponding to amino acid 44 in the polypeptide. Tyrosine was present in myxamoebal alpha-tubulin and glycine was present in the plasmodial specific peptides; the peptides are referred to as the Tyr44 and Gly44 peptides respectively. Both peptides in acetylated form blocked 6-11B-1 reactivity towards acetylated myxamoebal alpha-tubulin. The Tyr44 but not the Gly44 peptide blocked KMP-1 reactivity towards non-acetylated myxamoebal alpha-tubulin. Tyrosine at position 44 is not found in any other known alpha-tubulin. Thus a unique antigenic determinant exists in certain Physarum alpha-tubulin isotypes, close to the acetylation site at lysine 40. This antigenic determinant forms part of the KMP-1 recognition epitope and explains the unique isotype selectivity of this monoclonal antibody.     

ATP-dependent peptide release from mitochondria of reticulocytes

ATP-dependent release of TCA-precipitable peptides from mitochondria-containing stroma (MCS) is described. The process is independent of ubiquitin, but is sensitive to hemin and to heat treatment. Neither chloramphenicol nor EGTA inhibit. 50% of the activity is dependent on charged tRNA. The peptides released from MCS possess a molecular mass of about 1–5 kDa and are degraded to TCA-soluble compounds by a cytosolic protease system (fraction II) without ubiquitin.