Partial amino acid sequence and molecular cloning of the encoding gene for the major outer membrane protein of Chlamydia trachomatis.

The first 25 N-terminal amino acids of the major outer membrane protein of Chlamydia trachomatis serovar L2 were determined. The amino acid sequence was used to construct an oligonucleotide probe specific for the major outer membrane protein gene. Using this oligonucleotide as a hybridization major outer membrane protein gene. Using this oligonucleotide as a hybridization probe, we discovered one recombinant clone that produced a 15-kilodalton polypeptide which reacted with a monoclonal antibody directed against the major outer membrane protein type-specific epitope. In a separate set of experiments, we uncovered another recombinant clone that produced a 51-kilodalton polypeptide which was reactive with an anti-major outer membrane protein subspecies-specific monoclonal antibody. The expression of these recombinant DNA plasmids in Escherichia coli is discussed.     

Retinoic acid reduces induction of monocyte tissue factor and tissue factor/factor VIIa-dependent arterial thrombus formation

Agents that downregulate the induction of monocyte/macrophage tissue factor (TF) activity may attenuate the thrombotic risk associated with mechanical restoration of vessel patency or artificial arterial grafting. In such events, procoagulant macrophages in the atherosclerotic plaque and procoagulant monocytes adherent to artificial materials may be exposed to the blood stream. Ishii et al (Blood 80:2556, 1992) reported that induction of endothelial TF is downregulated by all-trans retinoic acid (ATRA), and Conese et al (Thromb Haemost 66:662, 1991) reported that retinoids downregulate monocyte procoagulant activity (PCA). These findings led us to investigate the effect of ATRA on monocyte TF expression, and to study the effect of ATRA on monocyte-induced thrombus formation in a model system of human arterial thrombogenesis. Induction of PCA in human peripheral blood monocytes by 0.5 microgram/mL lipopolysaccharide (LPS) was dose dependently reduced by ATRA, reaching a reduction of 56% at 10(-5) mol/L ATRA (P < .0001). A 38% reduction (P < .0007) in LPS- induced TF antigen expression was observed at an ATRA concentration of 10(-6) mol/L. Adherence of monocytes to plastic cover slips (Thermanox, Miles Laboratories, Naperville, IL) also triggered induction of cellular PCA, which was inhibited by more than 80% by an anti-TF monoclonal antibody (MoAb) (P < .002). Inclusion of ATRA (10(-6) mol/L) reduced this PCA by 40% (P < .03), and the TF antigen expression by 30% (P < .0001). Exposure of Thermanox adherent monocytes to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber device at an arterial wall shear rate of 650 s-1 elicited significant fibrin deposition and platelet thrombus formation. Partial interruption of this thrombus formation was achieved by 10(-6) mol/L ATRA, which reduced the fibrin deposition by 80% (P < .02) and platelet thrombus formation by 50% (P < .05). In comparison, incubation of adherent monocytes with the anti-TF MoAb before the blood exposure, reduced the fibrin deposition by 83% (P < .02) and platelet thrombus volume by 75% (P < .0008). Thus, ATRA is an effective down-regulator of monocyte TF- PCA, and may reduce thrombotic complications at sites of plaque rupture, at plaque disruption after percutaneous transluminal angioplasty procedures, or on surfaces introduced by artificial arterial grafting.     

Variable major proteins of Borrelia hermsii. Epitope mapping and partial sequence analysis of CNBr peptides

The variable major proteins (VMP) of serotypes 7 and 21 of the relapsing fever agent Borrelia hermsii were isolated by detergent extraction and high performance liquid chromatography. Cyanogen bromide (CNBr) digestion of the isolated VMP yielded two peptides of apparent molecular weights 20,000 (20 K) and 16 K from VMP7, and three peptides of 14.5, 14, and 7 K mol wt from VMP21. Serotype-specific monoclonal antibodies bound in Western blots to one of each of the two or three CNBr fragments from the homologous VMP. A single monoclonal antibody bound to the whole cells, the isolated VMP, and a CNBr fragment of both serotype 7 and serotype 21. (This crossreactive antibody did not, however, bind to any of four other serotypes examined.) Regional conservation of structure between VMP7 and VMP21 was also shown by amino acid sequence analysis of the N-termini of the five CNBr fragments. One pair of aligned fragments from VMP7 and VMP21 had 80% amino acid homology in sequence; a second pair had 40% homology. The partial amino acid homologies between two VMP suggest that these proteins are products of members of a polygene family.     

Immunologic quantification of fibrin deposition in thrombi formed in flowing native human blood

We describe a new method for quantification of fibrin in thrombi formed in native human blood at venous and arterial shear conditions in a parallel-plate perfusion chamber device. Thrombi consisting of various proportions of fibrin and platelets were digested by plasmin. Fibrin deposition (μg/cm²) was calculated from the measured D-dimer levels.
Fibrin deposition in thrombi formed on a tissue factor (TF)-rich surface increased with increasing shear rate from 37 μg/cm² at 100/s to 77 μg/cm² at 2600/s (significant at 95%, ANOVA). The plasma levels of thrombin–antithrombin III complexes (TAT) increased in concert. In contrast, fibrin deposition in thrombi formed on collagen fibrils and the corresponding TAT plasma levels were independent of the shear rate and much lower than those elicited by the TF-rich surface (significant at 95%, ANOVA). The intra-individual variation in fibrin deposition was on average 10%, whereas the inter-individual differences were >500%. Such a large inter-individual difference has not been detected by morphometry which usually is employed in similar studies.
The present method is more accurate and less time-consuming than the morphometric approach. The novel method measures fibrin on the surface and in and around the thrombi, thus total deposited fibrin. In contrast, the morphometry approach quantifies surface coverage with fibrin only, thus being semiquantitative at best.     

Mouse Immunoglobulin Heavy Chains Are Coded by Multiple Germ Line Variable Region Genes

The N-terminal 20 residues of 13 heavy immunoglobulin chains from myeloma protein of the BALB/c mouse are compared with the same residues of 15 other heavy chains described in the literature. Sixteen of 28 sequences are different from one another. These proteins fall into four major sets, with 18 of the proteins in the largest set being further divisible into at least five subsets. This pattern of diversity suggests there are at least eight germ line genes coding for the variable regions of mouse heavy chain. Many of the immunoglobulins from which these heavy chains are derived exhibit binding activity for various haptens. The differing hapten specificities are closely correlated with distinct primary amino-acid sequences.