Expression and distribution of leptospiral outer membrane components during renal infection of hamsters.

The outer membrane of pathogenic Leptospira species grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41. The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during renal infection. Hamsters were challenged with host-derived Leptospira kirschneri to generate sera which contained antibodies to antigens expressed in vivo. Immunoblotting performed with sera from animals challenged with these host-derived organisms demonstrated reactivity with OmpL1, LipL41, and several other proteins but not with LipL36. Although LipL36 is a prominent outer membrane antigen of cultivated L. kirschneri, its expression also could not be detected in infected hamster kidney tissue by immunohistochemistry, indicating that expression of this protein is down-regulated in vivo. In contrast, LPS, OmpL1, and LipL41 were demonstrated on organisms colonizing the lumen of proximal convoluted renal tubules at both 10 and 28 days postinfection. Tubular epithelial cells around the luminal colonies had fine granular cytoplasmic LPS. When the cellular inflammatory response was present in the renal interstitium at 28 days postinfection, LPS and OmpL1 were also detectable within interstitial phagocytes. These data establish that outer membrane components expressed during infection have roles in the induction and persistence of leptospiral interstitial nephritis.

Characterization of leptospiral outer membrane lipoprotein LipL36: downregulation associated with late-log-phase growth and mammalian infection.

We report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL36 gene was identified. Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral outer membrane lipoprotein we described in a previous study (E. S. Shang, T. A. Summers, and D. A. Haake, Infect. Immun. 64:2322-2330, 1996). The deduced amino acid sequence of LipL36 would constitute a 364-amino-acid polypeptide with a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. LipL36 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL36 exclusively into the hydrophobic, detergent phase. LipL36 is intrinsically labeled during incubation of L. kirschneri in media containing [3H]palmitate. Processing of LipL36 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. After processing, LipL36 is exported to the outer membrane along with LipL41 and lipopolysaccharide. Unlike LipL41, there appears to be differential expression of LipL36. In early-log-phase cultures, LipL36 is one of the most abundant L. kirschneri proteins. However, LipL36 levels drop considerably beginning in mid-log phase. LipL36 expression in vivo was evaluated by examining the humoral immune response to leptospiral antigens in the hamster model of leptospirosis. Hamsters surviving challenge with culture-adapted virulent L. kirschneri generate a strong antibody response to LipL36. In contrast, sera from hamsters surviving challenge with host-adapted L. kirschneri do not recognize LipL36. These findings suggest that LipL36 expression is downregulated during mammalian infection, providing a marker for studying the mechanisms by which pathogenic Leptospira species adapt to the host environment.

Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species.

We report the cloning of the gene encoding a surface-exposed leptospiral lipoprotein, designated LipL41. In a previous study, a 41-kDa protein antigen was identified on the surface of Leptospira kirschneri (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe.A Lambda ZAP II library containing EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL41 gene was identified. The deduced amino acid sequence of LipL41 would encode a 355-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinant His6-LipL41 fusion protein was expressed in Escherichia coli in order to generate specific rabbit antiserum. LipL41 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL41 exclusively into the detergent phase. At least eight proteins, including LipL41 and the other major Triton X-114 detergent phase proteins, are intrinsically labeled during incubation of L. kirschneri in media containing [3H] palmitate. Processing of LipL41 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. Triton X-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin), LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36, only LipL41 and OmpL1 were exposed on the surface of intact organisms. Immunoblot analysis of a panel of Leptospira species reveals that LipL41 expression is highly conserved among leptospiral pathogens.

The rare outer membrane protein, OmpL1, of pathogenic Leptospira species is a heat-modifiable porin.

The outer membranes of invasive spirochetes contain unusually small amounts of transmembrane proteins. Pathogenic Leptospira species produce a rare 31-kDa surface protein, OmpL1, which has a deduced amino acid sequence predictive of multiple transmembrane beta-strands. Studies were conducted to characterize the structure and function of this protein. Alkali, high-salt, and urea fractionation of leptospiral membranes demonstrated that OmpL1 is an integral membrane protein. The electrophoretic mobility of monomeric OmpL1 was modifiable by heat and reduction; complete denaturation of OmpL1 required prolonged boiling in sodium dodecyl sulfate (SDS), 8 M urea, and 2-mercaptoethanol. When solubilized in SDS at low temperature, a small proportion of OmpL1 exhibited an apparent molecular mass of approximately 90 kDa, indicating the existence of an SDS-unstable oligomer. OmpL1 dimers and trimers were demonstrated by nearest neighbor chemical cross-linking. In order to generate purified protein for functional studies, the ompL1 gene was ligated into the pMMB66 expression plasmid under control of the tac promoter. Although expression in Escherichia coli was toxic, most of the OmpL1 produced was found in the outer membrane, as determined by subcellular fractionation. Purified recombinant OmpL1 was reconstituted into planar lipid bilayers, demonstrating an average single channel conductance of 1.1 nS, similar to the major porin activity of native leptospiral membranes. These findings indicate that OmpL1 spans the leptospiral outer membrane and functions as a porin.

Worsening right flank pain over a 24-hr period.

AML is a benign renal tumor composed of variable quantities of mature vascular, smooth muscle and fatty elements. They occur as an isolated finding, classically in middle-aged females, or in association with tuberous sclerosis. When symptomatic, they typically present with flank pain secondary to hemorrhage. CT is the diagnostic imaging modality of choice. The diagnosis can usually be made based on the recognition of fat within the lesion. When discovered, asymptomatic lesions are generally monitored by follow-up imaging studies, and if they remain stable, no intervention is required. Arterial embolization has become the recommended treatment of choice in some instances, particularly in cases with associated hemorrhage.

The value of duplex sonography after peripheral artery angioplasty in predicting subacute restenosis.

OBJECTIVE: The purpose of this study was to determine if abnormal findings on duplex sonographic examination after peripheral artery angioplasty correlate with the subsequent recurrence of a stenosis. SUBJECTS AND METHODS: We used duplex sonography to examine 35 stenoses in 23 patients within 48 hr after the patients had angioplasty to treat these stenoses. Patients were followed up for 3 years by using one or more of the following: assessment of signs and symptoms, monitoring of peripheral pulses, pulse volume recordings, and angiography. Life tables were constructed to compare long-term patency with the presence of abnormal findings seen on duplex sonograms. Abnormal findings at the dilated segment included a blood-flow velocity greater than 120 cm/sec or a residual elevated velocity ratio greater than 1.4 or 2.0 immediately after angioplasty. RESULTS: Twelve (34%) of 35 angioplasty sites showed recurrent stenosis before 36 months. Patency at 24 months was calculated for velocities less than 120 cm/sec vs velocities of 120 cm/sec or greater (41% vs 68%), for velocity ratios less than 1.4 vs ratios of 1.4 or greater (63% vs 57%), and for velocity ratios less than 2.0 vs ratios of 2.0 or greater (54% vs 74%). We found no significant difference in patency between those patients with normal findings and those with abnormal findings on duplex sonographic examination after angioplasty. CONCLUSION: Abnormal findings on duplex sonograms obtained immediately after peripheral angioplasty cannot be used to predict subacute restenosis.

Matrix mineralization in hypertrophic chondrocyte cultures. Beta glycerophosphate increases type X collagen messenger RNA and the specific activity of pp60c-src kinase.

The phenomenon of chondrocyte hypertrophy is accompanied by the expression of type X collagen and the appearance of matrix mineralization. These events are also associated with changes in the phosphorylation of intracellular proteins. In this study the addition of 10 mM beta-glycerophosphate to hypertrophic chondrocytes resulted in stimulation of type X collagen synthesis up to 10 days in culture and an increase in the expression of type X collagen mRNA. This was followed by the onset of mineralization and the appearance of calcium hydroxyapatite. In contrast, the addition of beta-glycerophosphate to non-hypertrophic chondrocytes failed to induce expression of type X collagen or to produce changes in calcium and phosphate. The increased formation of type X collagen and of mineral in hypertrophic chondrocytes was accompanied by changes in the tyrosine kinase pp60c-src. While the level of c-src protein decreased approximately 2.5-fold in hypertrophic chondrocytes after 17 days of beta-glycerophosphate treatment, the specific activity of pp60c-src kinase increased approximately 3-fold in the cells that could be induced to mineralize but remained unchanged in cells that did not exhibit this property. Regulation of kinase activity may be an important event in endochondral ossification.

Antegrade selective catheterization of femoral vessels with a 4- or 5-F catheter and safety wire.

A method of antegrade catheterization of the superficial femoral artery or femoral bypass grafts with a 4- or 5-F catheter and safety wire is described. This method has advantages of smaller size and the use of torque wires when compared with previously described methods.

Dynamics of a human seminal vesicle specific protein.

The present paper is concerned with the temporal alterations and tissue localization of a seminal antigen secreted by the human seminal vesicle. This antigen is recognized by antibody MHS-5, which is one of a set produced in mice by immunization with human sperm. The respective clone produced an antibody of the IgG1 subtype, which reacted with seminal fluid from over 400 normal donors and 21 semen samples from vasectomized men. Incubation of seminal vesicle secretion with either prostatic fluid or prostate specific antigen (PSA) resulted in degradation on the antigen. The experiments showed that MHS-5 antigen is a substrate for the serine protease PSA: Immunohistochemical studies suggested that MHS-5 is a "sperm-coating" antigen and is exclusively synthesized and secreted by the seminal vesicle.

Monoclonal antibodies to type X collagen. Biosynthetic studies using an antibody to the amino-terminal domain.

Monoclonal antibodies to chick type X collagen have been used to study the structure, biosynthesis, and location of type X in cartilage. The antibodies were produced by injecting purified type X collagen into female SJL/J mice and then fusing their spleen cells with Sp2/0 myeloma cells. Hybridoma culture supernatants were screened for antibodies to type X collagen by enzyme-linked immunosorbent assay and Western blots. Positive supernatants did not cross-react with other collagen types (I, II, IX, XI) or with fibronectin. Three monoclonal antibodies were chosen for further characterization. Two of them (1A6 and 6F6) recognize a pepsin-sensitive domain of type X collagen. Rotary shadowing showed that 1A6 and 6F6 both recognize the same end of type X, probably the aminoterminal non-triple helical domain. Amino acid sequencing of the intact protein and of the epitope-containing peptide confirmed that the antibody recognition sites for 1A6 and 6F6 are within the amino-terminal domain. Monoclonal antibody 2B3 reacts with the pepsinized (45 kDa) and weakly with the nonpepsinized (59 kDa) forms of type X collagen. The monoclonal antibodies were used for immunolocalization of type X in hypertrophic chondrocytes and reacted only with tissue samples from areas undergoing endochondral ossification, e.g. growth plate and fracture callus. Antibody 6F6, when coupled to Sepharose, selectively binds to type X collagen from cell and organ cultures. In a pulse-chase experiment, no processing of the 59-kDa form of type X could be detected. Two components with molecular masses of approximately 70 and 85 kDa, arising from a disulfide-bonded aggregate, were synthesized by both the permanent and calcifying cartilage organ cultures but did not react with the antibody, suggesting that these proteins are not related to type X. In summary, the pulse-chase results and the immune precipitation with monoclonal antibody 6F6 did not detect biosynthetic precursors larger than 59 kDa or proteolytically processed forms of type X.

Characterization of a monoclonal antibody to a conserved epitope on human seminal vesicle-specific peptides: a novel probe/marker system for semen identification.

A novel sperm-coating antigen from the human seminal vesicles was discovered. We identified a monoclonal antibody MHS-5, recognizing an epitope with characteristics of a forensic semen marker: conservation in all vasectomized or normal semen samples tested (421); absence in all human tissues or biological fluids other than semen; and immunolocalization on the surface of ejaculated sperm. Western blots of ejaculates allowed to liquefy for 5 min demonstrated the MHS-5 epitope to be located on peptides of a wide range of molecular masses from 69 to 8 kDa. After 15 h of semen liquefaction, immunoreaction peptides of higher molecular mass were undetectable in semen, while peptides of lower molecular mass from 8 to 21 kDa retained antigenicity. Three peptides of 10, 11.9, and 13.7 kDa were the most immunoreactive species in semen liquified for 15 h. Using the MHS-5 monoclonal, an enzyme-linked immunosorbent assay (ELISA) was developed sensitive to 1 ng of seminal protein. This assay showed that the MHS-5 antigen was undetectable in semen of common domestic animals and monkeys but was present in chimpanzee, gorilla, and orangutan semen. ELISA of homogenates from human organs and reproductive tissues demonstrated the antigen only in samples of seminal vesicles. Epididymal sperm obtained at vasovasostomy lacked the MHS-5 epitope, a fact that, together with immunolocalization on ejaculated sperm, demonstrated that the MHS-5 antigen functions as a "sperm-coating antigen." The MHS-5 monoclonal detected semen in sexual-assault evidence obtained six months previously and in mixtures of semen with vaginal or cervical fluid. Assay systems employing the MHS-5 monoclonal may be useful for identification of semen in sexual-assault casework. The MHS-5 epitope resides on novel seminal vesicle-specific peptides whose functions, aside from sperm coating, are uncharacterized.

Phosphorylation of a chromaffin granule-binding protein by protein kinase C.

Protein kinase C was detected in a group of Ca2+-dependent chromaffin granule membrane-binding proteins (chromobindins) on the basis of Ca2+-, phosphatidylserine-, 1,2-diolein-, and phorbol myristate acetate-stimulated histone kinase activity. When the chromobindins were incubated with [gamma-32P]ATP, Ca2+, and phosphatidylserine, 32P was incorporated predominantly into a protein of mass 37 +/- 1 kilodaltons (chromobindin 9, or CB9). Phosphorylation of this protein was also stimulated by diolein and phorbol myristate acetate, indicating that it is a substrate for the protein kinase C activity present in the chromobindins. Maximum phosphate incorporation into CB9 in the presence of 1 mM Ca2+, 75 micrograms/ml of phosphatidylserine, 2.5 micrograms/ml of diolein, and 12.5 micrograms/ml of dithiothreitol was 0.53 mol/mol of CB9 in 5 min. Eight 32P-labeled phosphopeptides were resolved in two-dimensional electrophoretic maps of trypsin digests of CB9. Phosphoamino acid analysis revealed that phosphorylation was exclusively on serine (94%) and threonine (6%) residues. Incubation of the chromobindins with chromaffin granule membranes in the presence of [gamma-32P]ATP resulted in the incorporation of 32P into eight additional proteins besides CB9 that could be separated from the membranes by centrifugation in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We suggest that phosphorylation of CB9 or these additional eight proteins may regulate events underlying exocytosis in the chromaffin cell.