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Dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 produced by UC 11065 were evaluated as anthelmintics. Assays used to examine these compounds included effects on the free-living nematode Caenorhabditis elegans, ability to clear target nematodes (Haemonchus contortus and Trichostrongylus colubriformis) from jirds, and clearance of Haemonchus contortus from lambs. A crude extract of UC 11065 containing dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 was active against C. elegans and against H. contortus in the jird. Purified and/or synthetic samples of dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 were tested in the jird model; only dioxapyrrolomycin exhibited appreciable activity against H. contortus (greater than or equal to 90.9% clearance at 0.33 mg/jird), while none of the compounds showed appreciable activity against T. colubriformis. Dioxapyrrolomycin cleared 99.9% of H. contortus from lambs at 12.5 mg/kg. An in vitro migration study using susceptible and closantel-resistant H. contortus showed there is cross-resistance between dioxapyrrolomycin and closantel. Dioxapyrrolomycin appears to be a narrow-spectrum anthelmintic which works through a closantel-like mode-of-action.

Virulent Treponema pallidum reacts poorly with the specific antibodies present in human and rabbit syphilitic sera, a phenomenon often attributed to an outer coat of host serum proteins. Here we present additional evidence that the limited antigenicity of virulent organisms actually is due to a paucity of proteins in the outer membrane. Initially, we used electron microscopy to demonstrate that the outer membrane is highly susceptible to damage from physical manipulation (i.e., centrifugation and resuspension) and nonionic detergents. Organisms with disrupted outer membranes were markedly more antigenic than intact treponemes as determined by immunoelectron microscopy (IEM) with rabbit syphilitic and antiendoflagellar antisera. Data obtained with a new radioimmunoassay, designated the T. pallidum surface-specific radioimmunoassay, corroborated these IEM findings by demonstrating that the major T. pallidum immunogens are not surface exposed; the assay also was unable to detect serum proteins, including fibronectin, on the surfaces of intact organisms. Furthermore, IEM of T. pallidum on ultrathin cryosections with monospecific anti-47-kDa-immunogen antiserum confirmed the intracellular location of the 47-kDa immunogen. On the basis of these and previous findings, we proposed a new model for T. pallidum ultrastructure in which the outer membrane contains a small number of transmembrane proteins and the major membrane immunogens are anchored by lipids to the periplasmic leaflet of the cytoplasmic membrane. This unique ultrastructure explains the remarkable ability of virulent organisms to evade the humoral immune response of the T. pallidum-infected host.

Haemonchus contortus- and Trichostrongylus colubriformis-infected jirds (Meriones unguiculatus) are useful for anthelmintic studies. With concurrent infections of these parasites established in the jird, questions of not only anthelmintic activity, but to some extent spectrum, could be assessed in a single model system. This report outlines a model using immunosuppressed (0.02% hydrocortisone in feed) jirds concurrently infected with H. contortus and T. colubriformis. Immunosuppressed jirds were inoculated with approximately 1,000 exsheathed infective larvae of each species, treated per os on day 10 postinoculation (PI), and killed on day 13 PI. Stomachs and small intestines were removed, opened longitudinally, incubated in distilled water at 37 C for 5 hr, fixed in formaldehyde solution, and stored for subsequent examination. Contents of both organs were examined using a stereomicroscope (15-45 x). Various standard anthelmintics were evaluated in the model; modern broad-spectrum ruminant anthelmintics (benzimidazoles, febantel, ivermectin, levamisole hydrochloride, and milbemycin D) are active uniformly and in most cases at doses comparable to those required for efficacy against these parasites in ruminants. This model, using worms of 2 genera living in distinct sites, allows preliminary evaluation of anthelmintic activity and spectrum for experimental compounds in a single cost- and resource-efficient experiment.

Protein profiles of in vivo propagated and in vitro cultivated Treponema pallidum subsp. pallidum (Nichols strain) were analyzed using two-dimensional gel electrophoresis (2D-PAGE). A comparative study between the two protein profiles (as detected by silver stain, Western blotting, and autoradiography) demonstrated two in vitro synthesized polypeptides with approximate molecular masses of 29- and 35 kDa (both with pI 5.62) which were absent in the protein profile of the in vivo propagated T. pallidum. It also was demonstrated that the in vitro cultivated organisms lacked an antigenic polypeptide (as judged by Western blots) of approximate molecular weight 35 kDa (pI 5.34) which was present in the in vivo propagated organisms. To determine the cellular location of these polypeptides, the outer membrane proteins of T. pallidum was extracted with Triton X-114. The extracted proteins were subjected to phase partitioning and the 2D-PAGE protein profiles of the detergent and aqueous phases were compared. The results indicated that in vitro cultivation caused some changes in the cytoplasmic protein profiles of T. pallidum, but no changes were detected in the profiles of the membrane proteins.

The effects of various concentrations of dithiothreitol, molecular oxygen, and several antioxidants upon the in vitro replication of Treponema pallidum were studied. The optimal dithiothreitol concentration was between 0.65 and 1.62 mM, and the optimum oxygen concentration was 3.0% +/- 0.5% in both the presence and absence of additional antioxidants. It was discovered that the reduced sulfhydryl concentration and the oxidation-reduction potential of the medium were stabilized after 5 days. The water-soluble antioxidants cobalt chloride, cocarboxylase, mannitol, and histidine were individually tested for their ability to increase treponemal growth in vitro. The optimum concentrations for these antioxidants were 21 nM, 4.3 nM, 0.55 mM, and 0.23 mM, respectively. When combined at these concentrations, the mixture of antioxidants stimulated the in vitro replication of T. pallidum. The number of treponemes in cultures with the antioxidants averaged a 59-fold increase, compared with a 43-fold increase in cultures lacking the antioxidants. It was further demonstrated that histidine and mannitol were the most critical components of this mixture. Catalase and superoxide dismutase were investigated for their ability to promote the growth and maintain viability of T. pallidum in tissue culture. The optimum concentrations for these enzymes were 10,000 U/liter and 25,000 U/liter, respectively. When these enzymes and the above antioxidants were combined and added to a chemically reduced modified Eagle medium, the treponemes increased an average of 70-fold, compared with an average of 35-fold in cultures lacking them. Furthermore, this medium, T. pallidum culture medium, supported the replication of T. pallidum at oxygen concentrations from 5 to 7% with little loss in yield or viability. The lipid-soluble antioxidants vitamin A and vitamin E acetate were also shown to enhance the in vitro growth of T. pallidum in this medium.

Sample solubilization techniques can influence the protein patterns obtained by SDS-PAGE. In this study we determined the effects of the amount of SDS present in the sample solubilization buffer on one and two dimensional (1D and 2D) electrophoretic protein profiles of radiolabeled T. pallidum as well as the antigenicity of treponemal proteins as judged by Western blots of 1D and 2D gels probed with pooled secondary human syphilitic sera. Although the total number of protein bands in 1D profiles obtained at SDS concentrations between 0-5% was not significantly different, there was an obvious shift in the location of the bands. However, a significant amount of variation (including isoelectric point and molecular weight shifts) was observed among the total number of protein species in 2D profiles. The greatest number of protein species (158 total) was obtained with 3% SDS in the solubilization buffer, followed by the 1% SDS giving raise to 143 protein species. The highest number of proteins (59 total) detected on Western blots of 2D profiles was obtained when no SDS was used. The number of antigenic proteins in 1D profiles followed a bell shaped curve with 2% SDS giving the highest number (32 total) of the antigenic proteins. With different concentrations of SDS, the number of antigenic proteins in 2D profiles decreased as the concentration of SDS increased. Thus, we conclude that, 2% SDS in the sample solubilization buffer is best for 1D profiles. However, SDS is not recommended to separate all of the antigenic proteins in 2D gels.

A number of parameters aimed at optimizing culture conditions for both Sf1Ep cells and Treponema pallidum have been investigated. Optimum temperature for replication of T. pallidum ranged between 33 and 35 degrees C. At 33 degrees C, replication occurred in the presence of atmospheric oxygen concentrations of less than 0.3 to 10%, the optimum range being 1.5 to 5%. No replication occurred in the presence of 12.5% oxygen. When both temperature and oxygen concentrations were varied between 33 and 35 degrees C and 1.5 to 5%, respectively, little differences in replication were noted. Although variation in the oxygen concentration within each temperature group had little or no effect on replication, it did have an effect on motility, which remained greater in the 5% oxygen concentration after 9 to 12 days of cultivation. Optimum concentration of fetal bovine serum in the culture medium was 20%, although replication occurred in concentrations ranging from 5 to 30%. If carefully screened, calf serum could be substituted for fetal bovine serum. Testis extract was an essential component of the culture medium. Although extract obtained from an adult rabbit--either normal or T. pallidum infected--was slightly superior, replication of T. pallidum occurred when rat or hamster testis extract was substituted.

Nursing plays an important contributory role in the multidisciplinary team approach to patient care. The nurse who is responsible for the care of liver transplant patients requires special preparation to meet the patient's needs during the critical phases of the liver transplant process. In the hospital setting, a primary nursing-care delivery system enhances the nurse's role in supporting the goal of patient recovery. Primary nursing places the accountability and responsibility for planning, administering, and evaluating the care of the liver transplant patient with the primary nurse. The transplant nurse acts as a key link in the communication network within the multidisciplinary team, and effective communication is essential in the care of the liver transplant patient. For achievement of a life-style of independence after transplantation, the patient is assisted by the nurse, who provides instruction in such areas as diet, medications, monitoring of vital signs, and record keeping. Although the nurse is only one member contributing to the care of liver transplant patients, the nurse's role in the success of treatment is vital.

Eggshells of the skink, Eumeces fasciatus, and of the rough green snake, Opheodrys vernalis, contain 28-40% of total shell mass as calcium at oviposition. After incubation, both calcium concentration and content were reduced 17-41%. Protein accounted for 33-65% of shell mass in Eumeces and Opheodrys eggshells at oviposition. Reductions in protein concentration and content from 22-86% following incubation were observed. Comparing amino acid compositions of eggshells collected following oviposition with those collected from the same clutch after hatching reveals similarities among all eggshells except those of Eumeces from Michigan which lack desmosine and isodesmosine. Concentrations of desmosines in eggshells from Missouri and Michigan Eumeces appeared to decrease following incubation by 49.7% and 12.2%, respectively. Although neither the mechanisms involved in apparent losses of calcium and protein, nor the fate of the mobilized materials are known, possible pathways for mobilization of eggshell constituents are outlined and their pertinence to the physiology of squamate eggs and the evolution of viviparity in reptiles is suggested.

Specific monoclonal antibody and Western blot analysis were used to examine the phenotypic expression of the major 47 kDa surface immunogen of Treponema pallidum among organisms cultivated in vitro. Tissue-cultured treponemes synthesized the 47 kDa immunogen as well as, or better than, organisms cultivated in vivo (rabbit testicles).

Recently, the successful in vitro cultivation of the Nichols strain of Treponema pallidum was achieved. Afterward, attempts were made to cultivate three other strains of T. pallidum and two strains of T. pertenue. The cultivation of the KKJ, Mexico A, and Bosnia A strains of T. pallidum was somewhat successful; the average increases were 10.8, 9.1, and 7.5-fold, respectively. The range of growth for each of these strains varied dramatically from experiment to experiment. The KKJ strain varied from 14.4 to 8.0-fold; the Mexico A strain from 12.8 to 5.4-fold; and the Bosnia A strain from 11.3 to 3.6-fold. However, the attempts to cultivate the Gauthier and the FB strains of T. pertenue were unsuccessful. The average increases were 1.7 and 1.9-fold, respectively. Although the maximum growth observed was about threefold with either of these strains of T. pertenue, over 50% of the treponemes remained motile for 10 d. These results suggest that although each of these strains of T. pallidum and T. pertenue has been shown to be genetically identical, they are very diverse biologically even among strains of the same species.

Polymorphonuclear leukocytes have been implicated in connective tissue injury in a variety of disease processes. To gain insight into mechanisms by which neutrophils might degrade connective tissue macromolecules in the presence of proteinase inhibitors, we have used a model system that allows neutrophils to be held in vitro under physiologic conditions in close proximity to a very proteinase-sensitive substrate, (125)I-labeled fibronectin. We have found: (a) neutrophils spread rapidly on the fibronectin substrate; (b) fibronectin proteolysis by neutrophils is largely attributable to released elastase, and is linearly related to cell number over the range of 2,000 to 30,000 cells per assay; (c) oxidants released from neutrophils stimulated by opsonized zymosan or phorbol myristate acetate do not protect released elastase from inhibition by alpha(1)-proteinase inhibitor or alpha(2)-macroglobulin; (d) neutrophil myeloperoxidase and enzymatically generated superoxide anion render alpha(1)-proteinase inhibitor ineffective against fibronectin proteolysis when neutrophils are added 30 min later; and (e) alpha(1)-proteinase inhibitor and alpha(2)-macroglobulin incompletely inhibit fibronectin proteolysis by neutrophils (79.8+/-6.3 and 73.5+/-12.0%, respectively.) The data suggested that proteolysis due to neutrophils that are in contact with susceptible macromolecules may occur due to partial exclusion of inhibitors from the cell-substrate interface. Although confirming that alpha(1)-proteinase inhibitor is ineffective against neutrophil-derived proteolysis after exposure to oxidants, these studies did not support the hypothesis that oxidants released from stimulated neutrophils enhance activity of proteinases they release in the presence of alpha(1)-proteinase inhibitor. We anticipate that further studies with this test system will be helpful in defining conditions that modulate inflammatory connective tissue injury in diseases such as pulmonary emphysema and rheumatoid arthritis.

The in vitro interaction between Treponema pallidum and Sf1Ep cells during treponemal replication was investigated by using transmission electron microscopy. The Sf1Ep cells grown on Teflon-treated cover slips after 12 days of cocultivation were fixed in situ, overlaid with agar, embedded, and vertically sectioned. Large numbers of treponemes were found extracellularly not only at the upper cell surfaces but also in the narrow spaces between the cells and between the cells and the cover slips. These narrow spaces supported treponemal growth and survival, as did those at the upper cell surfaces. Although few in number, organisms were also seen in cell vacuoles either surrounded by a membrane or free in the cytoplasm. Some extracellular treponemes attached to host cells by body spirals or the terminal end and formed electron-dense layers at attachment sites. Some treponemes were often surrounded with amorphous, extracellular material which appeared to "connect" them to host cell surface. After 12 days of cocultivation, host cells showed excessive vacuolation and appeared to be damaged. This did not seem to be due to treponemal infection alone, because cells from uninfected cultures also showed similar vacuolation.

An enzyme-linked immunosorbent assay was developed to enumerate Treponema pallidum cells. The assay could detect from 2 X 10(7) to 4 X 10(8) treponemes per ml. Reactive rabbit serum and goat anti-rabbit immunoglobulin G (peroxidase conjugate) were used in the assay. Optimum results were obtained when 2,2'-azino-di(ethylbenzthiazolinesulfonic acid) was used as the dye for the enzyme reaction and the reactions were allowed to run for 45 min. Interestingly, assays in which in vivo-cultivated T. pallidum was used produced lower absorbance values than those in which T. pallidum was cultivated in vitro.

In vitro propagation of Treponema pallidum can be achieved by cocultivation with Sf1Ep cells. This study had two objectives: (i) to achieve suspension cultivation of Sf1Ep cells and (ii) to develop procedures for achieving the replication of T. pallidum in those cell cultures. Seven suspension cultures of Sf1Ep cells yielded an average of 7.2 x 10(8) T. pallidum (36-fold increase) after 12 days.

The enzymatic carboxylation of phosphoenolpyruvate by cell-free extracts of Neisseria gonorrhoeae was examined and determined to be similar to the reaction catalyzed by phosphoenolpyruvate carboxylase (PEPC). This was shown by the irreversibility of the reaction and nucleotide independency. The enzyme was found to have some characteristics different from the other bacterial PEPCs reported. The enzyme showed catalytic activity in the presence of cobalt ions as well as magnesium and manganese ions, was not inhibited by succinate in fresh extracts, and displayed a low Michaelis constant for bicarbonate (0.27 mM), as compared with other PEPCs. The significance of this low Michaelis constant is discussed with respect to the growth of the organism and the importance of this enzyme to protein and nucleic acid synthesis.

In a series of seven experiments, the virulent Nichols strain of Treponema pallidum was shown to attach and replicate on the surface of tissue culture cells of cottontail rabbit epithelium (Sf1Ep) growing in conventional monolayer cultures under an atmosphere of 1.5% oxygen. Five days after inoculation of 10(6)T. pallidum, the number of treponemes had increased to between 8 x 10(6) and 2.59 x 10(7). The viability of harvested organisms ranged from 86 to 97%. The number of T. pallidum continued to increase, generally reaching a plateau between days 9 and 12 of incubation, with increases ranging up to 100-fold and averaging 49-fold. There appeared to be a ceiling of multiplication of about 2 x 10(8) irrespective of the inoculum, which ranged from 10(6) to 10(8)T. pallidum. Concurrent deoxyribonucleic acid assays were performed on the cultures containing T. pallidum to obtain further evidence of replication. Significant increases in treponemal deoxyribonucleic acid were observed when the inocula ranged from 10(6) to 10(7), with the greatest increases, as might be expected, being in the former group. There was also excellent correlation in the amount of deoxyribonucleic acid per treponeme; the averages for the 10(6), 2.5 x 10(6), and 10(7) groups were 3.46 x 10(-14), 3.28 x 10(-14), and 2.79 x 10(-14) g per treponeme, respectively (3.14 +/- 0.72 x 10(-14) g per treponeme). In each experiment, organisms were harvested from the group inoculated with 10(6)T. pallidum after 7 days of incubation to test for virulence. In all instances, the organisms were virulent; erythematous, indurated, treponeme-containing lesions were produced from an average of six to seven organisms. Scanning electron microscopy revealed that during the course of replication many microcolonies of treponemes formed on the surface of the cells.