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application/pdf, Previously, we have identified a gene encoding thrombospondin-related anonymous protein of Babesia gibsoni (BgTRAP), and have shown that the antisera raised against recombinant BgTRAP expressed in Escherichia coli inhibited the growth of parasites. In the present study, a recombinant vaccinia virus expressing the BgTRAP (VV/BgTRAP) was constructed. A specific band corresponding to a molecular mass of 80 kDa, which was similar to that of native BgTRAP on the merozoites of B. gibsoni, was detected in the supernatant of VV/BgTRAP-infected RK13 cells. Moreover, mice inoculated with VV/BgTRAP produced antibody that specifically reacted with the native BgTRAP on parasites. These results indicated that the recombinant vaccinia virus expressing BgTRAP might be a vaccine candidate against canine B. gibsoni infection.

Apoptosis has been found to help in the defence against pathogens. Infection with the obligate intracellular parasite Toxoplasma gondii is known to trigger host-cell apoptosis. When using a T. gondii-infected macrophage cell line, J774A.1, treatment with IFN-gamma significantly enhanced apoptosis in noninfected bystander cells while parasitized cells became relatively resistant. Infection and IFN-gamma treatment activated the expression of inducible nitric oxide synthase (iNOS), and the production of nitric oxide (NO) and treatment of cells with an iNOS inhibitor, N(G)-monomethlyl-L-arginine acetate (L-NMMA) reduced the apoptosis frequency. However, the reversal was only partial suggesting that not only NO, but also other, as of yet, unknown factors are induced. Finally, we studied the effect in vivo by infecting mice with either a virulent or an avirulent strain. Challenge with the virulent strain lead to a higher parasite burden, induced host-cell apoptosis in peritoneal cells, and produced higher levels of IFN-gamma and NO. Moreover, treatment of mice with a NO synthase inhibitor, aminoguanidine, partially inhibited the host-cell apoptosis induced by the parasite infection. Altogether, our findings indicate that apoptosis in bystander host cells is due to the secretion of NO and other soluble factors released by parasite-infected cells.

Polymerase chain reaction assays and culture were used to investigate 728 faecal samples from 404 calves (286 diarrhoeic, 118 healthy) and 324 lambs (230 diarrhoeic, 94 healthy) in Kashmir, India, for the presence of enterotoxigenic Escherichia coli (ETEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and salmonellae. Antimicrobial sensitivity patterns were also investigated. In total, 23 ETEC isolates were obtained from the diarrhoeic calves and 12 from diarrhoeic lambs. Most (74%) of the isolates from calves harboured the gene encoding heat-labile enterotoxin I, whereas 75% of the isolates from lambs possessed only the gene encoding for heat-stable enterotoxin a. The ETEC isolates belonged to 20 serogroups, among which serogroups O15 (five isolates) and O8 (four isolates) were the most frequent. Salmonella Typhimurium or S. Enteritidis was identified in three samples from diarrhoeic lambs. The ETEC isolates and the salmonellae showed multidrug resistance. No EAEC or DAEC was detected in any of the samples.

BACKGROUND After a brief episode of ischemia, myocardial function may be depressed for prolonged periods despite reperfusion. The mechanisms of postischemic dysfunction differ depending on the experimental model. Regional ischemia and reperfusion in the intact animal provide a clinically relevant model, but experimental variables are difficult to control. Experimental conditions can be well controlled in isolated cardiac muscle and myocyte preparations, but these models are limited by the assumptions used to mimic ischemia and reperfusion. This study combines the unique advantages of both preparations. We characterized in vivo alterations in regional two-dimensional finite strains with ischemia and reperfusion produced in the intact animal, then isolated cardiac myocytes from the region with postischemic dysfunction to characterize in vitro function of postischemic myocytes. METHODS AND RESULTS In seven anesthetized rabbits, three piezoelectric crystals were inserted in a triangular array to measure two-dimensional finite strains around the large coronary artery in the left ventricular anterior free wall. After 15 minutes of ischemia and reperfusion, strains were depressed at a stable level approximately 30% to 40% below control values between 1 and 6 hours after reperfusion. The direction of maximal shortening deformations was midway between circumferential and longitudinal directions during control and did not shift after reperfusion. In a second group of five rabbits, cardiac myocytes were isolated from the region with postischemic dysfunction after 15 minutes of ischemia and 45 minutes of reperfusion. We compared in vitro function in 45 postischemic myocytes with 48 cardiac myocytes isolated from five normal rabbits. Each rabbit (postischemic and control) contributed 9 +/- 1 (SD) myocytes to the study. All myocytes were studied within 1 hour after myocyte isolation (approximately 3 to 5 hours after reperfusion for postischemic myocytes). Myocytes were stimulated at 0.5 Hz and perfused with 2 mmol/L [Ca2+] Tyrode's solution to measure unloaded cell shortening. There was significantly less shortening in postischemic myocytes (12.4 +/- 2.1%) than control myocytes (16.2 +/- 1.2%). Maximal cell length (Lmax) was significantly longer in postischemic (134 +/- 7 microns) than control myocytes (122 +/- 7 microns), as was minimum cell length (Lmin) (118 +/- 8 versus 103 +/- 9 microns, respectively). The duration of shortening (time from stimulation to Lmin) was significantly shorter in postischemic (279 +/- 56 milliseconds) than control myocytes (405 +/- 44 milliseconds). Peak rates of cell shortening (-dL/dt) and lengthening (+dL/dt) did not differ. CONCLUSIONS In rabbits, 15 minutes of ischemia produced a stable depression in finite strains for 1 to 6 hours after reperfusion, with shortening deformations reduced by approximately 30% to 40% without a shift in direction. Cardiac myocytes isolated from postischemic myocardium display functional impairments in vitro similar to those measured in vivo, with an approximately 25% reduction in unloaded myocyte shortening and decreased contraction duration. This indicates that ischemia and reperfusion induce intrinsic impairments in contractility independently of external loading conditions. This model may be useful for examining cellular mechanisms of postischemic myocardial dysfunction.

The present study aimed to develop a colony hybridization method for the exhaustive detection and isolation of diarrhoeagenic Escherichia coli (DEC) from samples containing numerous coliform bacteria.Digoxigenin-labelled DNA probes were designed to detect seven pathotypes of DEC based on type-specific genes. A total of 615 meat, food and faeces samples identified as DEC-positive by multiple real-time PCR for the virulence genes (eae, stx, elt, est, virB, aggR, afaB and astA) were analysed by a colony hybridization method, which involved filtering enrichment cultures through hydrophobic grid-membrane filters. DEC were isolated from 72.5% (446/615) of samples by the colony hybridization method but were only detected in 26.3% (162/615) of samples by a conventional culture method. The hybridization method was particularly effective for isolating low-level contaminants, such as enterotoxigenic and Shiga toxin-producing E. coli, which were isolated from 51.8% (58/112) of samples identified as positive by PCR for the enterotoxin genes, in contrast to only 4.5% (5/112) of samples analysed by the conventional method.The developed colony hybridization system allows for the efficient and simultaneous isolation of all DEC pathotypes.The colony hybridization system described here permits the sensitive isolation of DEC and represents a suitable tool for ecological investigations of DEC.

A total of 480 samples, comprising 429 faecal samples from healthy adult birds and 51 tissue samples from dead birds, were collected from four government poultry farms in the Kashmir valley from September 2007 to April 2008. In all, 33 Salmonella isolates were obtained. Of these, 28 (84.85%) isolates were Salmonella Gallinarum, 3 (9.09%) were Salmonella Enteritidis and the remaining 2 (6.06%) were Salmonella Typhimurium. All the isolates harboured the invA, sefA, stn and spvC virulence-specific genes. However, the sopB gene was found in only 90.9% of the isolates. Pulsed-field gel electrophoresis analysis of representative isolates revealed that the majority were related but a few belonged to different clones. The majority of the isolates were resistant to cefpodoxime, nalidixic acid and sulphadiazine and sensitive to chloramphenicol, cefotaxime and tetracycline. Isolation of multidrug-resistant Salmonella, including the zoonotically important serovars, revealed a potential threat not only to poultry but also to human health in Kashmir.

Toxoplasma gondii is a zoonotic protozoan parasite of both medical and veterinary importance worldwide. The parasite can cause severe complications in immunocompromised individuals such as AIDS patients and transplant recipients, where up to 25% of patients will develop toxoplasmic encephalitis. Toxoplasmosis is a zoonosis that causes a public health concern in both developed and developing countries such as Thailand. Livestock development particularly in dairy cows of Thailand have been hampered by low production of milk and slow growth rate because of many pathogens including T. gondii. The objective of this study was to evaluate the serodiagnostic tool to be used for detection of T. gondii infection in dairy cows of Thailand. During 2006-2007, the sera of 700 cows from 55 small farm holders of the highest number of dairy cow population in the northern provinces were collected and analysed. Antibodies to T. gondii were assayed by latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT). The overall prevalence of T. gondii infection in dairy cows was 9.4% (66/700) by LAT and 17% (119/700) by ELISA. Sixty-three seropositive samples by LAT (95.5%) and 107 by ELISA (89.9%) were confirmed by IFAT. These results demonstrated that LAT had the highest specificity for detection of T. gondii infection.

SUMMARYPeptidases of parasitic protozoa are currently under intense investigation in order to identify novel virulence factors, drug targets, and vaccine candidates, except in Babesia. Leucine aminopeptidases in protozoa, such as Plasmodium and Leishmania, have been identified to be involved in free amino acid regulation. We report here the molecular and enzymatic characterization, as well as the localization of a leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from B. gibsoni (BgLAP). A functional recombinant BgLAP (rBgLAP) expressed in Escherichia coli efficiently hydrolysed synthetic substrates for aminopeptidase, a leucine substrate. Enzyme activity of the rBgLAP was found to be optimum at pH 8·0 and at 37°C. The substrate profile was slightly different from its homologue in P. falciprum. The activity was also strongly dependent on metal divalent cations, and was inhibited by bestatin, which is a specific inhibitor for metalloprotease. These results indicated that BgLAP played an important role in free amino acid regulation.

The surface antigen 1-related sequence 2 of Neospora caninum (NcSRS2) is considered as an immunodominant antigen. In this study, the gene encoding truncated NcSRS2 (NcSRS2t) lacking an N-terminal signal peptide and C-terminal hydrophobic regions was expressed in Escherichia coli, and its diagnostic potential in an enzyme-linked immunosorbent assay (ELISA) was evaluated. ELISA could discriminate clearly between known N. caninum-positive and -negative sera from cattle. Field serum samples collected from cattle in Brazil were examined for the diagnosis of N. caninum infection using ELISA. Of the 197 samples analyzed, 64 (32.5%) samples were positive for antibodies to N. caninum. Of the 64 ELISA-positive samples, 58 (90.6%) were confirmed as positive by Western blot analysis with whole-parasite antigens. These results suggest that ELISA with recombinant NcSRS2t is an effective method for diagnosis of N. caninum infection in cattle.

The modifying effects of administrating an ethyl acetate extract of "Kurosu" (EK), a vinegar made from unpolished rice, on development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of EK on proliferating cell nuclear antigen (PCNA) index in ACF, prostaglandin (PG) E2 expression in the colonic mucosa and activities of detoxifying enzymes of glutathione S-transferase (GST) and quinone reductase (QR) in the liver. To induce ACF, rats were given two weekly subcutaneous injections of AOM (20 mg/kg body wt). They also received drinking water containing 0, 0.05, 0.1 or 0.2% EK for 4 weeks, starting 1 week before the first dosing of AOM. AOM exposure produced 140 +/- 23 ACF/rat at the end of the study (week 4). EK administration dose-dependently inhibited ACF formation and inhibition by 0.2% EK was statistically significant (P0.002). Treatment with EK significantly lowered PCNA index in ACF and reduced PGE2 content in the colonic mucosa, while EK elevated liver GST and QR activities. These findings suggest that EK may be effective for inhibiting colonic ACF, through induction of liver GST and QR and possibly alteration of PGE2 production.

Neospora caninum is a veterinary medically important pathogen capable of causing abortion in cattle and neuromuscular paralysis in dogs. The surface antigen 1 of N. caninum (NcSAG1) is an important candidate for the development of a diagnostic reagent for neosporosis. In order to establish an effective diagnostic method, the gene encoding truncated NcSAG1 (NcSAG1t) lacking a signal peptide and C-terminal hydrophobic regions was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The purified GST-NcSAG1t was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of N. caninum antibodies in cattle. The ELISA with GST-NcSAG1t clearly differentiated between immunofluorescent antibody test (IFAT)-positive and -negative sera from cattle. In addition, the ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Toxoplasma gondii. Field serum samples collected from cattle in Brazil were examined for the diagnosis of neosporosis by using the ELISA. Of the 197 samples analyzed, 66 (33.5%) samples were positive for antibodies to N. caninum. Of the 66 ELISA-positive samples, 60 (90%) samples were confirmed as positive by Western blot analysis with whole parasite antigens. These results suggest that the recombinant NcSAG1t could be a reliable reagent for use as an antigen in ELISA for the serodiagnosis of N. caninum infection in cattle.

Interferon-gamma (IFN-γ) has a crucial role for host defence against parasite infection. It is not clear, however, how IFN-γ affects the parasite-infected host cells. The effect of IFN-γ on Neospora caninum-infected cells was investigated in murine fibroblasts and canine kidney cells in vitro. In the presence of IFN-γ, the viability of the infected host cell was decreased and apoptotic cell death occurred, as analysed by DNA stainings with propidium iodide and a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) and DNA fragmentation. The percentage of apoptotic cells depended on the dose of IFN-γ. Flow cytometric analysis indicated a significant increase of FasL expression on the IFN-γ-treated cells following N. caninum infection. Moreover, IFN-γ treatment down-regulated Bcl-2 expression in the cells cultured with N. caninum while parasite infection up-regulated Bcl-2 expression. The present study suggests that the IFN-γ induced increases of FasL expression and down-regulated Bcl-2 expression in N. caninum-infected cells are associated with apoptosis in vitro.

Nociceptive neurons receiving afferent input from the tooth pulp (TP) were recorded from the nucleus ventralis posteromedialis proper (VPM) and intralaminar nuclei of the thalamus in cats anesthetized with urethane and chloralose. The effects of stimulating the periaqueductal gray (PAG), or the nucleus raphe dorsalis (NRD) on responses of thalamic nociceptive neurons were investigated. Eight tooth pulp specific (TPS) and 7 wide dynamic range (WDR) neurons with TP input were observed around the periphery (shell region) of the posterior half of VPM. Following electrical stimulation of either the ventral PAG or the NRD, responses to TP stimulation were inhibited in all TPS and WDR neurons tested. Responses of these neurons to electrical stimulation of trigeminothalamic tract (TTT) fibers in the trigeminal medial lemniscus were also inhibited following PAG/NRD stimulation. These results suggest that PAG/NRD stimulation-produced inhibition of both TPS and WDR neurons may be partially mediated by an ascending antinociceptive mechanism. Intralaminar nociceptive neurons with TP input were observed in the nucleus centralis lateralis (CL), and parafascicularis (Pf). The effects of conditioning electrical stimulation of either the ventral PAG or the NRD on responses of intralaminar nociceptive nerons were studied. Of 15 intralaminar nociceptive neurons tested, 6 neurons were inhibited, 5 neurons were excited and 4 were unaffected following the conditioning stimulus. In neurons in which responses to TP stimulation were inhibited, responses elicited by electrical stimulation of the mesencephalic reticular formation (MRF) were also inhibited. These data suggest that although there is an ascending inhibitory pathway from PAG/NRD to intralaminar nuclei, this system is far less potent compared with the ascending inhibitory system acting upon the VPM.

The effects of intravenous morphine on responses of feline thalamic nociceptive neurons receiving afferent input from the tooth pulp (TP) were investigated. Morphine suppressed responses to TP stimulation in both tooth pulp specific (TPS) and wide dynamic range (WDR) neurons with TP input recorded from the nucleus ventralis posteromedialis (VPM). However, there was scant morphine effect on responses to stimulation of trigeminothalamic tract (TTT) fibers in the trigeminal medial lemniscus. Furthermore, in nociceptive neurons with TP input recorded from nucleus centralis lateralis (CL) and parafascicularis (Pf) of the intralaminar nuclei, intravenous morphine suppressed responses both to stimulation of the mesencephalic reticular formation (MRF) as well as TP stimulation. The suppressive action of morphine on responses elicited by TP stimulation of VPM, CL and Pf neurons was antagonized by intravenous naloxone (1 mg/kg). Results suggest that intravenous morphine suppresses synaptic transmission of nociceptive impulses in the intralaminar nuclei as well as in the lower brain stem but not in the VPM.

The medulla oblongata caudal to the obex was explored for neurons responsive to tooth pulp (TP) stimulation in cats. Four different subclasses of TP neurons were found. The latter included TP specific (TPS) neurons, trigeminal wide dynamic range (trigeminal WDR) neurons with TP input, trigeminal subnucleus reticularis ventralis (trigeminal SRV) neurons with TP input and convergent reticular formation (convergent RF) neurons with TP input. TPS neurons were located in the dorsal marginal rim of the trigeminal subnucleus caudalis, i.e., in the marginal layer or the outer zone of substantia gelatinosa. WDR neurons with TP input were found in the neck region of medullary dorsal horn which corresponds to the lateral part of subnucleus reticularis dorsalis (SRD). Trigeminal SRV neurons with TP input were located in the lateral part of SRV. Convergent RF neurons with TP input were found in the middle third of the caudal bulbar RF consisting of SRD and SRV. Both TPS neurons and WDR neurons with TP input included trigeminothalamic neurons as evidenced by the antidromic activation from the nucleus ventralis posteromedialis of the contralateral thalamus. A significant proportion of both trigeminal SRV and convergent RF neurons with TP input were antidromically activated by stimulation of the nucleus centralis lateralis of the contralateral thalamus. The former two subclasses may subserve the sensory-discriminative aspect of toothache, while the latter two subclasses, the emotional-motivational aspect.

An improved method for constructing canine herpesvirus (CHV) recombinants expressing foreign genes by using the lacZ-TK gene cassette as a double selectional marker was developed. A recombinant CHV carrying the lacZ-TK gene at a targeted gene locus was constructed and used as a parental virus for generating new recombinants. The parental virus formed blue plaques and was sensitive to TK-specific drugs, while newly generated recombinants, in which the lacZ-TK gene was replaced with the desired foreign gene, become both resistant to the TK-specific drugs and formed white plaques. Recombinants were isolated by using the combination of drug selection and color selection. This improved method allows construction of recombinant CHV with great ease, because the drug selection can enrich the frequency of recombinant CHV from 0.01-0.1% to 10-80%. This method was employed to construct a recombinant CHV that expressed rabies virus (RV) glycoprotein (G protein).

To clarify the role of transforming growth factor-beta (TGF-beta) and its receptors in hepatocyte growth, we studied the expression of TGF-beta1 and its receptors and the sensitivity to growth inhibition by TGF-beta1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-beta type II receptor (TbetaR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-beta1 increased with time in primary culture. The cell surface TGF-beta receptor proteins (TbetaR-I, II, and III), examined by the receptor affinity-labeling assay using 125I-TGF-beta1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-beta1 protein was added at later times in culture, corresponding to the presence of increased TGF-beta receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TbetaR-I, TbetaR-II, TbetaR-III, IGF-II/M-6-PR, and TGF-beta1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-beta receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TbetaR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-beta1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-beta receptors and sensitivity to growth inhibition by TGF-beta1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture.

PC12 cells growth-arrested with bafilomycin A1 died showing apoptotic chromatin condensation in the nuclei. The bafilomycin A1-induced chromatin condensation was preceded by neurite outgrowth (NOG), required higher concentrations of bafilomycin A1 than NOG, and was suppressed by cycloheximide and aurintricarboxylic acid. NH4Cl (10 mM), another acidotropic pH perturbing agent, neither induced apoptotic chromatin condensation by itself nor suppressed that induced by bafilomycin A1, suggesting that bafilomycin A1-induced apoptosis occurs independently of intracellular pH in PC12 cells.

A simple and rapid method was developed for determination of oxytetracycline (OTC) in swine muscle and kidney by liquid chromatography (LC). The method involved homogenization of sample in acetonitrile-1M imidazole buffer containing 10 mM disodium ethylenediaminetetraacetic acid (Na2.EDTA) and 50 mM magnesium acetate (15 + 85) with added hexane, centrifugation, removal of the hexane phase, and ultrafiltration of the supernatant. L-column ODS (150 x 4.6 mm) with a mobile phase of acetonitrile-1M imidazole buffer containing 50 mM magnesium acetate and 10 mM Na2.EDTA (10 + 90) was used for the LC separation. A fluorescence detector was used at an excitation wavelength of 380 nm and an emission wavelength of 520 nm. The calibration graph was linear from 1.25 to 200 ng OTC. Recoveries of OTC from swine tissue fortified at levels of 0.05-1.0 microgram/g ranged from 58.0 to 67.3%. The quantitation and detection limits were 0.05 and 0.04 microgram/g, respectively.

We have reported previously that highly metastatic LuM1 cells derived from colon carcinoma colon 26 secrete larger amounts of gelatinase B than NM11 cells with poor metastatic potential, and that an increase in this gelatinase B secretion can be induced by autocrine factors (Hyup et A, Cancer Res., 54: 3611-3616, 1994). In the present study, a partial characterization was achieved by comparison of the autocrine factor preparation (fraction G) from serum-free medium conditioned with metastatic LuM1 cells with soluble factors known to stimulate gelatinase B secretion. Secretion of gelatinase B by LuM1 cells was augmented by tumor necrosis factor alpha, transforming growth factor beta1 (TGF-beta1), interleukin 1beta, or epidermal growth factor, and specific neutralizing antibodies abolished the induced increases. Platelet-derived growth factor and insulin-like growth factor 1 had no effect on gelatinase B secretion by LuM1 cells. The enhancement of gelatinase B secretion by fraction G was partially inhibited by the antibody to TGF-beta1. TGF-beta1 was detected in both active and latent forms in serum-free medium conditioned with LuM1 or NM11 cells, with the amount of TGF-beta1 higher in the former case. Gelatinase B secretion by LuM1 cells was enhanced by the addition of TGF-beta1 to the culture medium, but that by NM11 cells was not seriously affected, although the latter bound more of the factor. These results indicate the involvement of this growth factor in the autocrine stimulation of gelatinase B secretion by LuM1 cells. However, the autocrine factor effect was not fully explained by TGF-beta1 in the medium, and the involvement of some other unknown factor(s) was thus indicated.

Effects of okadaic acid (OA), a protein phosphatase inhibitor, on chromatin structure and phosphorylation of histones were examined using HeLa and N18 cells. The chromatin condensation in HeLa cells was mild and resemble prometaphase nuclei, while the condensation in N18 cells was extensive and chromatin became a compact body. H2A in HeLa cells was extensively and consistently phosphorylated at the same site throughout the cell cycle, and H3 was demonstrated to be phosphorylated at the mitosis-specific site Ser10. In contrast, H1 phosphorylation was rapidly decreased in most sites within 3 h. The reduction of H1 phosphorylation was accompanied by a quantitative change in the set of H1 phosphopeptides. During the early phase of the OA treatment, H1 phosphorylation was transiently elevated in tandem, whereas H3 phosphorylation reached a maximum somewhat later. The results suggest that mitosis-specific events (cdc2/H1 kinase activation, H1 superphosphorylation, mitosis-specific H3 phosphorylation and chromatin condensation) induced by OA are sequentially associated. The changes appear to reflect a molecular mechanism similar to that operating in normal mitosis.

Tumor necrosis factor-alpha (TNF-alpha) is an endogenous mediator for several effects of endotoxin. To evaluate whether TNF-alpha mediates endotoxin-induced left ventricular (LV) dysfunction, we measured LV function (sonomicrometers) and serum TNF-alpha (cytolytic assay) in anesthetized rabbits given endotoxin (100 micrograms/kg iv). In the control group (n = 8), systolic depression (defined by a > 10% increase in end-systolic volume at a matched end-systolic pressure) developed in four rabbits and diastolic dilation (> 10% increase in end-diastolic volume at a matched end-diastolic pressure) developed in three rabbits. Neither the increase in end-systolic volume nor the increase in end-diastolic volume correlated with the increase in TNF-alpha, which reached a peak of 2,875 +/- 762 U/ml. In a second group of rabbits (n = 7), a goat polyclonal anti-rabbit antibody to TNF-alpha was given 30-60 min before endotoxin. Anti-TNF-alpha antibody alone did not alter LV function. Although the TNF-alpha response to endotoxin was effectively blunted (peak TNF-alpha remained < 100 U/ml), all seven rabbits developed systolic depression (P = 0.08 compared with control group) and diastolic dilation (P = 0.03). We conclude that serum TNF-alpha does not mediate endotoxin-induced LV systolic depression or diastolic dilation in this model.

Organ specificity in the lacI mutant frequency (MF) induced by dimethylnitrosamine (DMN) was analyzed in lung, liver, kidney, bone marrow, urinary bladder, and testis of Big Blue mice. Cell proliferative activity was also analyzed in some of these tissues by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Clastogenicity of DMN was concomitantly analyzed by the peripheral blood micronucleus assay with the same animals used for the lacI mutation assay. Five daily intraperitoneal (i.p.) treatments with DMN (1 mg/kg) increased MF in liver (6.2 x control), kidney (2.4 x control), and lung (2.1 x control). These are known target organs for DMN carcinogenesis. No MF increase was observed in nontarget organs studied, i.e., bone marrow, bladder, and testis. Single ip treatment with DMN also increased lacI MF in liver but the increases were smaller than in a 5-daily-treatment regimen. This result suggests that multiple dosing is more effective in the transgenic mutation assay. The enhancement of cell proliferation observed was in bronchial epithelia 7 days after treatment. No micronucleus induction in peripheral blood was observed 24 hours after 2 and 3 daily ip treatments with 1 mg/kg DMN. An increase in the incidence of micronucleated reticulocytes in peripheral blood was observed 48 hours after single ip treatment with 5 or 10 mg/kg DMN. The present study demonstrated organ-specific induction of gene mutations by DMN which suggests a relevance of this assay for the prediction of organ-specific carcinogenesis.

We examined endotoxin-induced myocardial depression in 31 anesthetized rabbits using left ventricular end-systolic and end-diastolic pressure-volume relationships (sonomicrometers). In the control group, endotoxin (100 micrograms/kg iv) induced systolic depression (> 10% increase in end-systolic volume at matched end-systolic pressure) in 9 of 16 and diastolic dilation (> 10% increase in end-diastolic volume at matched end-diastolic pressure) in 8 of 16 rabbits within 7 h, unrelated to hypotension, acidosis, or hypoxia. Seven rabbits were pretreated with nitrogen mustard (1-2 mg/kg iv 4 and 2 days before) to decrease circulating neutrophils and monocytes by 98%. Endotoxin did not induce systolic depression in any rabbit (P = 0.01 compared with control), and diastolic dilation developed in one rabbit (P = 0.12). In eight rabbits pretreated with dimethylthiourea (DMTU; 500 mg/kg iv 30 min before), an intracellular free radical scavenger, systolic depression developed in one (P = 0.05) and diastolic dilation in five (P = 0.44). We conclude that cells inhibited by nitrogen mustard (e.g., neutrophils, monocytes, or macrophages) mediate endotoxin-induced left ventricular systolic depression. DMTU inhibited endotoxin-induced systolic but not diastolic dysfunction.

The virulence traits that mediate Vero cytotoxin-producing E. coli (VTEC) adherence are unclear. Many VTEC strains possess the eaeA gene which is involved in the attaching and effacing effects of enteropathogenic E. coli (EPEC). Most eae-positive VTEC adhered to HEp-2 cells in a localized manner; however some strains did not adhere. Thus we investigated the adhesion of poorly adherent strains, especially those of serogroups O111 and O157. To establish a model, the adherence to HEp-2, INT407 and Caco-2 cells of 12 O157 VTEC and six O111 VTEC isolated from cases of human infection were studied after growth of the bacteria under different conditions. For adhesion tests mannose is usually added during prior broth culture of the bacteria, and during the period of attachment, so that any adhesion due to mannose-sensitive type 1 pili is inhibited. Bacteria cultured in peptone water in the absence of mannose adhered to all three lines; there were localized clusters of bacteria on 1%-82% cells, whether mannose was present during the attachment period or not. Bacteria grown in the presence of D-mannose, or any other sugar that was metabolized, showed little adherence (range 0-9%). alpha-Methyl-glucoside also caused marked inhibition of adhesion. It was concluded that inhibition of adhesion was due to catabolite repression.

1. Dynamic changes in left atrial (LA) function during treadmill exercise were studied in ten conscious dogs instrumented to measure left ventricular (LV) pressure and diameter, LA pressure and diameter, and pulmonary venous blood flow (PVF, transit time flowmeter). 2. Systolic PVF volume (reservoir volume; a measure of LA reservoir function) increased from 38 +/- 4% of total PVF volume at baseline to 52 +/- 8% of total PVF volume during exercise, and diastolic PVF volume (conduit volume; a measure of LA conduit function) decreased from 62 +/- 5% at baseline to 48 +/- 8% during exercise (P < 0.005). 3. The increases in reservoir volume and the decrease in conduit volume were due not only to a greater decrease in diastolic interval than systolic interval but were also caused by a significantly greater increase (P < 0.05) in the mean systolic filling rate (93%) than in the mean diastolic filling rate (51%). 4. During exercise the pattern of LV filling derived from changes in LV diameter showed that a greater percentage of LV filling occurred during the second half of diastole at the time of atrial contraction (P < 0.05), suggesting that LA booster function was enhanced. 5. Changes in LA dimension revealed that during exercise more blood volume was reserved in the LA during systole and that this change was associated with an increase in the LA dimension at the beginning of LA contraction (r = 0.61, P < 0.05). 6. We conclude that LA reservoir and booster functions were augmented during exercise, whereas conduit function was not. Increased reservoir function may play an important role in accelerating LV filling by helping to maintain an enhanced atrioventricular pressure gradient during diastole and also by increasing LA booster function through an increase in LA preload.

We previously reported that murine tumor cells with a high spontaneous metastatic potential to the lung secrete higher amounts of M(r) 95,000 gelatinase (matrix metalloproteinase 9, MMP9) than do poorly metastatic cells. The present study, conducted to clarify the mechanisms underlying the increase in MMP9, revealed an autocrine factor that enhances the secretion of M(r) 95,000 gelatinase (MMP9). The secretion of MMP9 by highly metastatic colon carcinoma LuM1 cells, detected by zymography, was augmented 10-fold when cultured in medium supplemented with serum-free medium conditioned with LuM1 cells. Because the secretion of M(r) 60,000 gelatinase (MMP2), as well as total protein, by the same cells was not affected under these conditions, the augmentation appears specific for MMP9. The steady-state level of MMP9 mRNA was elevated in LuM1 cells cultured in the presence of the supernatant. The amount of the factor in the culture medium increased with time in culture, indicating that it was produced by the LuM1 cells. It was found to be heat stable but sensitive to trypsin digestion. Conditioned medium from poorly metastatic NM11 cells did not stimulate the secretion of gelatinases by NM11 cells, suggesting that autocrine stimulation of MMP9 secretion is a characteristic of metastatic cells. This factor could account for the augmented secretion of MMP9 by murine tumor cells with spontaneous metastatic potential to the lung.

The phenotype of isolated rat hepatocytes changes during in vitro cultivation, but it is not clear whether this process turns back when the cells are returned to an in vivo milieu. Glutathione S-transferase placental form (GST-P), which is not expressed in hepatocytes in vivo, is induced within a few days of cultivation, whereas cytochrome P-450 constitutionally expressed in hepatocytes in vivo rapidly diminishes. We explored the dynamic nature of hepatocytic phenotypes using these two markers and intrahepatic and intrasplenic transplantation of cultured hepatocytes.Hepatocytes of F344 rats were isolated and cultured for 5 days to form spheroidal aggregates and then implanted into the livers and spleens of congenic analbuminemic rats. Expression of GST-P and P-450 was then examined immunocytochemically. In the intrahepatic case, the implanted hepatocytes could be distinguished from surrounding host hepatocytes by albumin immunostaining.The intrahepatically implanted hepatocytes turned GST-P-negative within 5 to 10 days, and re-expressed P-450 to a comparable level to that in surrounding host hepatocytes after 2 days. On the other hand, the hepatocytes implanted within spleens continued to express GST-P for 10 days, and started to express P-450 at extraordinarily high levels after 5 to 10 days.The phenotype of hepatocytes under in vivo and in vitro conditions has proven to be changeable from one to another.

Eight chronically instrumented conscious dogs were used to test the hypothesis that left ventricular (LV) relaxation is accelerated during cardiac tamponade. The time constant of LV transmural pressure fall was measured before and during intrapericardial (IP) saline infusion (baseline) with and without beta-adrenergic blockade (propranolol 1 mg/kg iv). Heart rate was controlled by atrial pacing. Increasing IP pressure caused a progressive linear decrease in stroke volume before and during beta-blockade in each animal. The time constant of LV transmural pressure fall also decreased continuously with an increase in IP pressure from 26 +/- 7 ms during baseline to 18 +/- 5 ms during severe cardiac tamponade (P < 0.01) before beta-blockade. However, after beta-blockade, the time constant of LV transmural pressure fall was constant over a wide range of IP pressures despite a continuous decrease in LV end-diastolic volume. The time constant of LV transmural pressure fall was not altered by vena caval occlusions that caused the same decrease in LV preload observed during cardiac tamponade. We concluded that despite decreased pump function, LV relaxation was accelerated progressively during graded cardiac tamponade, and this change was dependent not on changes in loading conditions but on an intact beta-adrenergic influence.

Antiallergic effects of AL-3264 (N-[4-[4-(diphenylmethyl)-1-piperazinyl]butyl]-3-(6-methyl-3- pyridyl)acrylamide, CAS 118420-47-6) were compared with those of ketotifen, oxatomide, azelastine and tranilast in experimental animals. AL-3264 inhibited passive cutaneous anaphylaxis (PCA) in rats with an ED50 value of 6.1 mg/kg p.o. In inhibiting PCA, AL-3264 was the most potent among the antiallergic drugs examined. The anti-PCA effect of AL-3264 was long-lasting. Tolerance was not produced by repeated administration of AL-3264. AL-3264 inhibited antigen-induced bronchoconstriction in actively sensitized rats and in passively sensitized guinea pigs, with ED50 values of 14.5 and 0.44 mg/kg p.o., respectively. In the in vitro experiments, AL-3264 inhibited 5-lipoxygenase activity of guinea pig leukocytes with an IC50 value of 4.9 mumol/l, being the most potent among antiallergic drugs examined, and suppressed the antigen-induced histamine release from rat peritoneal mast cells with an IC50 value of 12.2 mumol/l. AL-3264 antagonized histamine-induced contractions in isolated guinea pig trachea with an IC50 value of 0.16 mumol/l. These results suggest that AL-3264 is an orally active, potent and long-lasting antiallergic compound which inhibits 5-lipoxygenase activity, histamine release and histamine H1 receptors at the similar concentrations.

A series of 3-nitro-1,2,4-triazole (NTA) derivatives with -(CH2)mC(= Y)NH(CH2)nZCH3(Y, Z = O or S; m = 1 or 2; n = 2 or 3) group in the side chain at the N-1 position of NTA and their fluorinated analogs were synthesized. Their physicochemical properties and radiosensitizing activities in vitro and in vivo were investigated with respect, particularly, to the effects of sulfur substitution on the side chain of triazoles. The sulfur substitution of the oxygen atom in the side chain of NTA derivatives increased the partition coefficient (P value), but had little effect on the one-electron reduction potential. The derivatives bearing a thioether group in the side chain were more effective in vitro on hypoxic EMT6/KU cells, but were less effective in vivo on SCCVII tumor than their oxygen analogs. The thioamide compounds showed almost the same or slightly higher sensitizing activities in vitro compared with their oxygen analogs.

Left atrial booster pump function produces variable effects on cardiac output. Generally, cardiac output decreases by only 15-20% when atrial fibrillation occurs, however, in some cases, hemodynamic collapse occurs through loss of left atrial contraction. We evaluated the relative significance of left atrial booster pump function in acute or chronic load and in myocardial ischemia using the left ventricular volume curve. Blood entering into the left ventricle during the left atrial contraction phase (FVLA) represents the left atrial volume work, and the ratio of FVLA to the left ventricular filling volume during one cardiac cycle (%FVLA) represents the relative significance of left atrial booster pump function in cardiac output. In dog experiments, we calculated the change in FVLA and %FVLA by measuring the the left ventricular internal minor axis diameter and using Pombo's method. We also measured the change of the left atrial segment length as a direct indicator of left atrial contraction. In the acute change in preload, FVLA changed with stroke volume, but %FVLA remained unchanged. The change in FVLA correlated with the direct indicator of the left atrial excursion; the extent of the left atrial segment length (LASL). During acute change of left ventricular afterload, both FVLA and %FVLA were unchanged. In regional myocardial ischemia, both FVLA and %FVLA were increased, suggesting an increase in the left atrial booster pump function. In clinical study, we calculated FVLA and %FVLA from the left ventricular diameter using M-mode echocardiography. In chronic volume overloading (aortic regurgitation), FVLA increased while %FVLA was maintained unchanged. The same FVLA-%FVLA relationship was observed in acute volume loading. In cases of left ventricular hypertrophy (LVH) and old myocardial infarction (MI), both FVLA and %FVLA were increased, suggesting the increased left atrial booster pump function. In these cases, the left ventricular rapid filling velocity decreased, suggesting that impairment of rapid filling caused the increase of left atrial preload and hence increased left atrial volume work. The results of this study show that in old MI and in LVH, both left atrial volume work and the relative significance of left atrial booster pump function increase. We concluded that prevention of atrial fibrillation may be very important in these diseases.

UDP-GlcNAc:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) catalyzes an essential first step in the conversion of high-mannose N-glycans to hybrid and complex N-glycans. Cloning of the gene encoding this enzyme was carried out by mixed oligonucleotide-primed polymerase chain reaction amplification of rabbit liver single-stranded cDNA using sense and antisense 20- to 24-base-pair (bp) primers. A rabbit liver library in phage lambda gt10 yielded a 2.5-kilobase (kb) cDNA with a 447-amino acid coding sequence. None of the nine asparagine residues were in an Asn-Xaa-(Ser or Thr) sequence, indicating that the protein is not N-glycosylated. There is no sequence homology to other previously cloned glycosyltransferases, but GnT I appears to have a domain structure typical of these enzymes--i.e., a short amino-terminal domain, a transmembrane domain, a "neck" region, and a large carboxyl-terminal catalytic domain. RNA was transcribed off the 2.5-kb cDNA, and in vitro translation with rabbit reticulocyte lysate yielded a 52-kDa protein with GnT I activity.

Escherichia coli was inoculated into the uterine lumen of ovariectomized rats, and the endometrial surfaces were examined by scanning electron microscopy. Adherence of E. coli to the epithelium and destruction of the surface leading to purulent endometritis were noticed. When rats were treated previously with estradiol, adherence of E. coli was not detected.

To assess the influence of hormones on uterine infections, Escherichia coli was infused into uterine lumens of ovariectomized or adrenoovariectomized rats receiving exogenous administration of various doses of ovarian hormones. Large numbers of E. coli were recovered from the rat uterine lumens, irrespective of hormonal influences. The number of leukocytes in the uterine flushings, representing the magnitude of purulent inflammation, differed significantly depending upon the hormonal regimen given to each host. Purulent endometritis was induced by E. coli in ovariectomized rats receiving progesterone or corn oil (hormone vehicle). Infections were asymptomatic in rats receiving estradiol, but promethazine-treated uterine horns were susceptible to infection. When progesterone was administered along with estradiol, purulent inflammation was caused by E. coli, but the number of leukocytes in the uterine lumens was significantly less than that obtained from the rats treated with progesterone or corn oil. These effects of ovarian hormones on uterine infections were observed in adrenoovariectomized rats as well as in ovariectomized rats. It is suggested that estradiol alters the nature of endometrial epithelium and prevent manifestation of purulent endometritis; progesterone antagonizes estradiol. Adrenal hormones appear not to participate in the pathogenesis of endometritis induced by E. coli.

The influence of ovarian hormones on the adhesion of Escherichia coli to endometrial epithelial cells was investigated in an in vitro system. Endometrial cells liberated by collagenase from rat uteri were used. Optimal test conditions were obtained when 5 X 10(8) E. coli bacteria were added to 10(5) epithelial cells and incubated for 60 min. The adhesion of the organisms was inhibited by the addition of either mannose or alpha-methyl-D-mannopyranoside. When epithelial cells collected from uteri of estradiol-treated rats were used, the number of E. coli adhering to the cells was markedly lower than that adhering to epithelial cells collected from control rats. These results suggest that E. coli adheres to endometrial epithelial cells with so-called type 1 pili and that estradiol alters the nature of the endometrial epithelium and prevents the adherence of the organisms to the cells.

Escherichia coli was inoculated into the uterine lumen of rats and rabbits at different estrous stages; one uterine horn of each animal was ligated at the cervical end. In rats, a large number of E. coli were retained in the ligated horns regardless of the estrous stage. E. coli inoculated at diestrus or pseudopregnancy induced purulent endometritis, but when inoculated at proestrus-estrus the organism caused asymptomatic infection. In nonligated horns, few E. coli were recovered, and marked histopathological changes were not observed. Large numbers of E. coli were retained in the nonligated horn at proestrus as a result of physiological constriction of the cervix. E. coli inoculated at proestrus never caused purulent endometritis in either the ligated horn or the nonligated horn. In rabbits, E. coli infused into ligated horns brought about purulent inflammation irrespective of ovarian states. The number of recoverable E. coli was reduced rapidly at the follicular phase as compared with the luteal phase. These results suggest that the stage of the estrous cycle when animals are inoculated with E. coli influences the course of the uterine infection.

This paper examines similarities between rhesus macaques and humans in growth and remodeling of the long bones of the extremities. Cross-sectional moments of inertia were measured in three locations of the femur, humerus and tibia of M. mulatta using photon absorptiometry. Differences between males and females were evaluated with respect to age and weight. Remodeling was assessed from histologic sections at the same locations. Sex differences in structural properties of the long bones occur only at mid-diaphysis. Differences reflect the greater need for powerful hindlimb propulsion in males. The growth rate in males is twice as fast as that in females. Several periods of growth acceleration were detected in both males and females. Intracortical remodeling in growing macaques is relatively slow and may be more like that in elderly humans than that in growing children.

SGP140 glycoprotein, a major cell surface sialoglycoprotein with an apparent m.w. of 140,000, was detected on the human T lymphoblastoid cell line P12/Ichikawa by labeling with periodate-tritiated sodium borohydride, followed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Then SGP140 was purified from P12/Ichikawa cells for study of its biochemical character and its distribution in various cell lines. The purification was performed by 0.2% Triton X-100 solubilization from crude membranes, DEAE-Sephacel column chromatography, WGA-agarose column chromatography, Blue-Sepharose 6MB column chromatography, and Sephadex G-150 gel filtration. Antiserum raised against SGP140 was then prepared, and immunoprecipitation and membrane immunofluorescence assay were performed on various cell lines. SGP140 was detected on P12/Ichikawa, Raji, P3HR-1, Daudi, Namalva, BALL-1, MOLT-4B, TALL-1, NALL-1, and K562 cells, but was not detected on HL-60 cells. When HL-60 cells were treated with dimethyl sulfoxide, retinoic acid, or 12-O-tetradecanoylphorbol-13-acetate, SGP140 was detected on cell surfaces. We discuss the possibility that SGP140 may be a differentiation antigen.

14C-N-Ethylmaleimide-S-cysteinylglycine was used to investigate the role of dehydropeptidase-I in the metabolism of glutathione conjugates. The dipeptide was rapidly hydrolyzed to 14C-N-ethylmaleimide-S-cysteine in isolated rat renal cells, and subsequently acetylated to 14C-N-ethylmaleimide-S-N-acetylcysteine. Cilastatin, a specific inhibitor of dehydropeptidase-I, strongly inhibited the hydrolysis of the dipeptide by the isolated cells. In rat kidney homogenates, the marked inhibitory effect of cilastatin was also observed on the hydrolysis of cystinyl-bis-glycine and leukotriene D4, which are dipeptide intermediates in the biotransformation of oxidized glutathione and endogenous glutathione conjugate, respectively. In contrast, the inhibitory effect of bestatin, a potent inhibitor of aminopeptidase-M, was much smaller than that of cilastatin on the hydrolysis of these dipeptides by the renal cells and homogenates. These results suggest that dehydropeptidase-I plays a more important role in the metabolism of glutathione and its conjugates than aminopeptidase-M does.