(E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2'-isobutyl-2'-(methanesulfonyl)-4-methylvalerohydrazide (Ro 32-7315), a selective and orally active inhibitor of tumor necrosis factor-alpha convertase.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by inflammatory cells, has been implicated in several inflammatory disease states. (E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2'-isobutyl-2'-(methanesulfonyl)-4-methylvalerohydrazide (Ro 32-7315), is a potent, orally active inhibitor of the TNF-alpha convertase (TACE), an enzyme responsible for proteolytic cleavage of the membrane bound precursor, pro-TNF-alpha. Ro 32-7315 inhibited a recombinant form of TACE (IC(50) = 5.2 nM) with selectivity over related matrix metalloproteinases. In a cellular assay system, THP-1 cell line, and in human and rat whole blood, Ro 32-7315 significantly reduced lipopolysaccharide (LPS)-induced TNF-alpha release with IC(50) values of 350 +/- 14 nM (n = 5), 2.4 +/- 0.5 microM (n = 5), and 110 +/- 18 nM (n = 5), respectively. Oral administration of Ro 32-7315 to Wistar rats caused a dose-dependent inhibition of LPS-induced release of systemic TNF-alpha with an ED(50) of 25 mg/kg. Treatment (days 0-14) of Allen and Hamburys hooded rats with Ro 32-7315 (2.5, 5, 10, and 20 mg/kg, i.p., twice daily) significantly reduced adjuvant-induced secondary paw swelling (42, 71, 83, and 93%, respectively) as compared with the vehicle group. In the Ro 32-7315-treated group, the reduced paw swelling was associated with improved lesion score and joint mobility. Furthermore, in a placebo-controlled, single-dose study, Ro 32-7315 given orally (450 mg) significantly suppressed ex vivo, LPS-induced TNF-alpha release in the whole-blood samples taken from healthy male and female volunteers (mean inhibition of 42% over a 4-h duration, n = 6). These data collectively support the potential use of such a compound for the oral treatment of inflammatory disorders.

Sex differences in investigation results and treatment in subjects referred for investigation of chest pain.

OBJECTIVE: To evaluate differences in investigation results and treatment between men and women referred for diagnostic treadmill exercise testing and coronary arteriography. DESIGN: Cohort study. SETTING: Tertiary cardiology centre. SUBJECTS: 1522 subjects referred by primary care physicians to an open access chest pain clinic for initial investigation of chest pain, of whom 485 were subsequently referred for coronary arteriography; and a similar cohort of 107 subjects referred directly by secondary care physicians for diagnostic coronary arteriography. MAIN OUTCOME MEASURES: Rates of positive exercise tests and rates for referral for arteriography and revascularisation according to sex. RESULTS: Overall, women were less likely to be referred for arteriography and revascularisation than men. However, men were more likely to have positive exercise tests, and for various exercise test diagnostic end points men were also more likely to have significant coronary artery disease. After taking this into account, there was no sex difference in referral rates for arteriography or revascularisation. CONCLUSIONS: There was no evidence of a sex bias resulting in inappropriate underinvestigation or undertreatment of women. However, the positive predictive value of treadmill exercise testing is low for women and further research is needed into how best to investigate women with chest pain.

Taxonomy of the Mycoplasma mycoides cluster.

Some of the mycoplasmas found in diseases of ruminants, Mycoplasma capricolum, M. mycoides subsp. capri (PG3), bovine group 7 (PG50), strain F38 and strain Y goat show various degrees of relationship to M. mycoides subsp. mycoides (PG1) and to one another, such that they are referred to as the M. mycoides cluster. Studies using serology, DNA homology, isoenzyme analysis and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of proteins have been carried out on the type and reference strains of these organisms and some representative strains in an effort to resolve this cluster into well-defined taxa. An ad hoc committee set up by the Subcommittee on the Taxonomy of the Mollicutes examined the known data, but found that it led to conflicting opinions on the classification of the unspeciated strains, partly because of the lack of guidelines on the relative weight to be given to each criterion, and partly because insufficient strains of each member of the cluster had been examined. Although classifications were proposed, total agreement was not achieved on any one proposal, and the committee believes that decisions on the taxonomy would have to await the results of repeat tests, in which several representative strains of each of the six groups would be included with the type strain.

Membrane proteins of Mycoplasma gallisepticum.

Mycoplasma gallisepticum membrane proteins were separated by two-dimensional gel electrophoresis. We found no evidence for the presence of a membrane glycoprotein.

Emmy Klieneberger-Nobel Award Lecture: The protein fingerprints of mycoplasmas.

The proteins of 91 strains of Mycoplasma from 19 established species and three groups of uncertain taxonomic position were separated by two-dimensional polyacrylamide gel electrophoresis. Considerable variations in protein patterns were found among strains from the same species or subspecies; the percentage of matching spots (% of congruence) ranged from 42%-100%, but many proteins were strongly conserved in all strains of a given species. Maps of these conserved proteins could be used to identify strains. It was concluded that (1) large colony-type strains of Mycoplasma mycoides subspecies mycoides are more closely related to M. mycoides subspecies capri than to small colony-type strains of M. mycoides subspecies mycoides; (2) Mycoplasma capricolum, Leach group 7, and the F38 group of mycoplasmas are all related to M. mycoides; (3) the 2D group of strains are not related to either M. mycoides or Mycoplasma bovis, (4) M. bovis and Mycoplasma agalactiae are closely related; (5) apart from a small overlap in pattern between Acholeplasma laidlawii and Acholeplasma granularum, the type strains of seven established species of Acholeplasma each had distinct protein patterns that justified their classification as separate species; and (6) analysis of two-dimensional protein patterns is a valuable method for resolving problems in mycoplasma taxonomy and for identifying strains.

Relationships between strains of Mycoplasma mycoides subspp. mycoides and capri studied by two-dimensional gel electrophoresis of cell proteins.

Strains of Mycoplasma mycoides subsp. mycoides have been divided into small colony (SC) and large colony (LC) types (Cottew & Yeats, 1978). The protein patterns of representative strains of these two types and of M. mycoides subsp. capri were compared by a high resolution, two-dimensional gel electrophoretic method. The results suggest that the LC strains are more closely related to M. mycoides subsp. capri than to the SC strains of M. mycoides subsp. mycoides.

The nature and location of Acholeplasma laidlawii membrane proteins investigated by two-dimensional gel electrophoresis.

The high resolution, two-dimensional electrophoresis system for the separation of proteins described by O'Farrell, (O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007--4021) has been modified for the separation of Acholeplasma laidlawii proteins. Reproducible protein patterns have been obtained from A. laidlawii cell, membrane and soluble protein preparations. The isoelectric focusing of membrane proteins was greatly improved by removing the bulk of the membrane lipid before solubilizing the protein. A. laidlawii peripheral membrane proteins were removed from the membrane by low ionic strength washing and by treatment with EDTA. The effect of an exhaustive EDTA treatment and a rapid, warm EDTA treatment were compared. By comparing the protein patterns obtained in these ways it was possible to distinguish two separate groups of peripheral membrane proteins and one integral membrane protein group. The peripheral membrane proteins which were removed from the membrane at low ionic strength (group I) were also insoluble in Triton X-100, whereas additional peripheral membrane proteins extractable by subsequent EDTA treatment (group II) were soluble in Triton X-100. Exterior-facing membrane proteins were distinguished from the interior-facing ones by lactoperoxidase-catalyzed iodination of intact cells and membranes. Group I peripheral membrane proteins faced the cell interior whereas group II proteins faced the cell exterior. We counted approximately 320 individual whole cell proteins. Of these, about 140 were membrane associated and a maximum of 40 proteins were iodinated after iodinating intact cells. A. laidlawii was also grown in the presence of NaH232PO4 and whole cell proteins were separated by two-dimensional gel electrophoresis. One membrane protein and two soluble proteins were labelled.

Striated fibers of the rho form of Mycoplasma: in vitro reassembly, composition, and structure.

The rho-form of Mycoplasma contains a striated, axial fiber and associated terminal structure. The presence of this organelle was correlated with the synthesis of two proteins, A and B, of molecular weights of approximately 85,000 and 26,000, respectively, each accounting for about 10% of the total cell protein. Their amino acid compositions showed them to have distinct polypeptide chains. After osmotic lysis of rho-form cells the organelles disappeared; protein A accompanied the membrane fraction, whereas protein B was partly released in soluble form. After lysis by Nonidet P-40 in a medium composed of 4 M glycerol, 50 mM phosphate, and 10 mM MgSO4 at pH 6 (GPM-6), the organelles were preserved and released with ultrastructure unchanged. Protein A was recovered in the soluble fraction and protein B in the particulate (crude fiber) fraction. Treatment of the crude fiber fraction with 0.5 M NaCl in GPM-6 or with a solution containing 4 M glycerol, 10 mM morpholinoethanesulfonate, and 1 mM ethylenediaminetetraacetate at pH 7.0 caused the fibers to disassemble into subunits. By subsequent changes in the ionic conditions and temperature it was possible to cause the subunits to reassemble into ordered aggregates having the same ultrastructure as the native rho-fibers. The optimum temperature for reassembly in the presence of 4 M glycerol was 37 C, the optimum pH was 6.5 to 7.0, and the presence of Mg-2+, replaceable by Ca-2+, SR-2+, or Ba-2+, was essential. Protein B was the only protein detected in the purified, reconsituted fibers.

Occurrence and ultrastructure of a variant (rho) form of Mycoplasma.

The ultrastructure of a variant (rho) form of Mycoplasma is described. The rho-forms are characterized by dark-ground light microscopy as relatively rigid, unbranched, filamentous organisms with discoidal swellings, and by electron microscopy by the presence of an intracytoplasmic axial fiber extending throughout the length of the cell and associated with a terminal structure of characteristic appearance. In negatively stained preparations the fiber presents a pattern of transverse light and dark major bands, the dark band being divided by a central minor light band. The periodicity of the banding varies from 12.0 to 14.5 nm, and the width of the fiber varies from 40 to 120 nm. The fiber appears to be composed of fibrils aligned parallel to the long axis. The evidence indicates that the fiber contains protein and is devoid of nucleic acid. rho-Forms were commonly found in Mycoplasma strains derived from goats and occasionally in bovine strains. They may have a wider distribution, as the growth medium was shown to be important both for the expression of the rho-character and for the selection of the rho-variant. The functional significance, if any, of the fiber and the terminal structure is unknown.