An analysis of excessive running in the development of activity anorexia

Several plasmids that were isolated from complement-resistant Pasteurella multocida or Escherichia coli were evaluated for phenotypic markers. Plasmid p2267, isolated from a tetracycline-resistant, complement-resistant fowl cholera field isolate of P. multocida (PM2267), was used to transform a K-12 E. coli (C600); this resulted in increased complement resistance, which was eliminated by curing. Either of two plasmids (p1870 or p70-1, isolated from P. multocida and E. coli, respectively) conferred an increase in complement resistance and invasiveness to turkey epithelial cells when expressed in the Clemson University (CU) vaccine strain of P. multocida. Additionally, the CU strain containing p1870 was more virulent in turkey challenge, and the plasmid appeared amplified in vivo. No detectable differences in major outer-membrane proteins, capsule, or carbohydrate fermentation were found to be associated with the acquisition of these plasmids.     

Antigenic mapping of bacterial and animal cytochromes P-450

A peptide scanning (PEPSCAN) approach was used for antigenic mapping of two hepatic microsomal cytochromes P450 (rab1A2 and rab2B4) and the microbial cytochrome from Pseudomonas putida (P450 101 or P450cam). This approach includes simultaneous synthesis of pin-linked overlapping hexapeptides covering the whole sequences of three P450s and testing them by ELISA with corresponding polyclonal antisera. Microsomal cytochrome P450 maps were shown to vary depending on an antiserum used for testing the peptides, however, the most active linear B-epitopes were revealed with antisera from two animal species used. P450 linear B-epitopes were classified into individual and group-specific epitopes. While almost all P450 101 linear antigenic determinants are unique for this protein, rab1A2 and rab2B4 contain epitopes both individual for each protein, and subfamily- or even family-specific epitopes. These results point out the possibility of producing both monospecific and group-specific antipeptide antibodies against different P450s. The antigenic map of P450 101 was superimposed on the structural-functional map of this protein. Its linear B-epitopes were shown to coincide with boundaries of secondary structure elements, with surface-located, water accessible regions and with sites responsible for intermolecular interactions in the Pseudomonas putida monooxygenase system. Several known or predicted functionally active sites in microsomal cytochrome P450 rab1A2 and rab2B4 were also shown to coincide with linear B-epitopes. The peculiarities of epitope locations in the protein tertiary structure will allow to predict antigenic regions starting from protein structural information and vice versa, to structural protein models in accordance with antigenic mapping results. Antigenic regions which coincide with sites responsible for intermolecular interactions in monooxygenase systems may be synthesized as separate peptides and used as blockers of such interactions.     

In vitro characterization of a novel, tissue-targeted ultrasonic contrast system with acoustic microscopy

Targeted ultrasonic contrast systems are designed to enhance the reflectivity of selected tissues in vivo [Lanza et al., Circulation 94, 3334 (1996)]. In particular, these agents hold promise for the minimally invasive diagnosis and treatment of a wide array of pathologies, most notably tumors, thromboses, and inflamed tissues. In the present study, acoustic microscopy was used to assess the efficacy of a novel, perfluorocarbon based contrast agent to enhance the inherent acoustic reflectivity of biological and synthetic substrates. Data from these experiments were used to postulate a simple model describing the observed enhancements. Frequency averaged reflectivity (30-55 MHz) was shown to increase 7.0 +/- 1.1 dB for nitrocellulose membranes with targeted contrast. Enhancements of 36.0 +/- 2.3 dB and 8.5 +/- 0.9 dB for plasma and whole blood clots, respectively, were measured between 20 and 35 MHz. A proposed acoustic transmission line model predicted the targeted contrast system would increase the acoustic reflectivity of the nitrocellulose membrane, whole blood clot, and fibrin plasma clot by 2.6, 8.0, and 31.8 dB, respectively. These predictions were in reasonable agreement with the experimental results of this paper. In conclusion, acoustic microscopy provides a rapid and sensitive approach for in vitro chracterization, development, and testing of mathematical models of targeted contrast systems. Given the current demand for targeted contrast systems for medical diagnostic and therapeutic use, the use of acoustic microscopy may provide a useful tool in the development of these agents.     

Evidence for a role of collagen synthesis in arterial smooth muscle cell migration

Migration of smooth muscle cells (SMCs) and collagen synthesis by SMCs are central to the pathophysiology of vascular disease. Both processes can be induced shortly after vascular injury; however, a functional relationship between them has not been established. In this study, we determined if collagen synthesis was required for SMC migration, using ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl-4-hydroxylase, and 3,4-DL-dehydroproline (DHP), a proline analogue, which we demonstrate inhibit collagen elaboration by porcine arterial SMCs. SMCs exposed to EDHB or DHP attached normally to collagen- and vitronectin-coated substrates; however, spreading on collagen but not vitronectin was inhibited. SMC migration speed, quantified by digital time-lapse video microscopy, was significantly and reversibly reduced by EDHB and DHP. Flow cytometry revealed that expression of beta1 integrins, through which SMCs interact with collagen, was unaffected by EDHB or DHP. However, both inhibitors prevented normal clustering of beta1 integrins on the surface of SMCs, consistent with a lack of appropriate matrix ligands for integrin engagement. Moreover, there was impaired recruitment of vinculin into focal adhesion complexes of spreading SMCs and disassembly of the smooth muscle alpha-actin-containing cytoskeleton. These findings suggest that de novo collagen synthesis plays a role in SMC migration and implicates a mechanism whereby newly synthesized collagen may be necessary to maintain the transcellular traction system required for effective locomotion.     

Nephrectomy through lumbar endoscopy. Report of 3 cases

The major outer surface protein, OspA, of Borrelia burgdorferi is a lipoprotein which is a particular interest because of its potential as a vaccine candidate. However, serotypic and genetic analysis of OspA from both European and North American strains have demonstrated antigenic and structural heterogeneities. We purified OspA to homogeneity by exploiting its resistance to trypsin digestion. By treating spirochetes with trypsin and then using Triton X-114 extraction and ion-exchange chromatography, we obtained a yield of 2 mg of pure OspA protein per liter of culture. INtrinsic labeling with [14C]palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal end of the molecule. The reactivity of five anti-OspA murine monoclonal antibodies to nine different isolates of B. burgdorferi was ascertained by Western blot (immunoblot) analysis. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains. Further resolution of the epitope specificity to determine humoral and cellular immune responses to OspA has implications for vaccine development and for the utility of this protein as a reagent in diagnostic testing for Lyme borreliosis.     

Establishment and characterization of human gastric carcinoma cell lines

We report 8 newly established gastric-carcinoma cell lines (SNU-216, 484, 520, 601, 620, 638, 668, 719) from Korean patients. Morphologic study was carried out using light and electron microscopes. CEA, alpha FP, and CA 19-9 and TPA in supernatant and in cell lysate were measured by radioimmunoassay. p53 and c-Ki-ras gene mutations were screened and confirmed by sequencing. The cell lines, derived from tumors with moderate differentiation, grew as a diffuse monolayer, and those from tumors with poor differentiation and minimal desmoplasia grew exclusively as non-adherent. Out of the 8 gastric-cancer cell lines, 5 had detectable levels of CEA both in supernatant and in cell lysate; there was no expression or secretion of alpha FP in these cells; 4 cell lines showed high levels of CA 19-9 in cell pellets. All cell lines except SNU-484 had high concentrations of TPA both in cell lysate and in supernatants. p53 mutation was found in 6 cell lines (75%): 2 (SNU-216 and SNU-668) had mutations in exon 6, and other 3 in exon 8. The c-Ki-ras mutation was found in 2 cell lines (25%), SNU-601 and SNU-668. The former showed GGT-to-GAT transition mutation at codon 12, while the latter showed CAA-to-AAA transversion mutation at codon 61. DNA profiles using restriction endonuclease HinfI and polymorphic DNA probes ChdTC-15 and ChdTC-114 showed different unique patterns; which suggests that these cell lines are unique and not cross-contaminated. We believe that the newly characterized gastric-cancer cell lines presented in this paper will provide a useful in vitro model for studies related to human gastric cancer.     

Buccal capsule development as a consideration for phylogenetic analysis of Rhabditida (Nemata)

Bacterial feeding nematodes in the order Rhabditida including Zeldia punctata (Cephalobidae) and Caenorhabditis elegans (Rhabditidae) differ profoundly in the buccal capsule parts and associated cells. We carried out a range of tests to determine which buccal capsule parts and cells are evolutionarily homologous between the representative species of the two families. Tests included reconstruction of the buccal capsule and procorpus with transmission electron microscopy (TEM), nuclei position and morphology using 4, 6-diamidino-2-phenylindole (DAPI) staining, and cell lineage using four dimensional (4D) microscopy. The lining of the buccal capsule of Z. punctata and additional Cephalobidae includes four sets of muscular radial cells, ma, mb, mc and md, in contrast to C. elegans and additional Rhabditidae, which has two sets of epithelial cells (e1, e3) and two sets of muscle cells (m1, m2). Cell lineage of a nematode closely related to Z. punctata, Cephalobus cubaensis, supports the hypothesis that in cephalobids the e1 and e3 cells become hypodermal cells or are programmed to die. Our findings contradict all previous hypotheses of buccal capsule homology, and suggest instead that ma and mb in Z. punctata are homologous to m1 and m2 in C. elegans respectively. We also hypothesize that ma and mb could be homologous to primary and secondary sets of stylet-protractor muscle cells in the plant parasitic Tylenchida.