Evaluation of novel agars for the enumeration of Campylobacter spp. in poultry retail samples

The purpose of this study was to examine the performance of novel agars for the identification and enumeration of Campylobacter species. The analytical sensitivity and specificity of Campylobacter Selective agar (CASA), Brilliance CampyCount agar (BCCA) and CampyFoodIDagar (CFA) for 84 Campylobacter spp. isolates and 50 non-Campylobacter spp. isolates from 37 distinct genera were of 100% sensitivity, with a 98% specificity for BCCA and CFA, and a 100% specificity for CASA. The application of these selective agars for Campylobacter spp. enumeration in comparison to the conventional agars, modified charcoal cefoperazonedeoxycholate agar (mCCDA) and Campy-Cefex (CCA) was examined using Campylobacter jejuni and Campylobacter coli inoculated samples. From C. jejuni inoculated samples, recovery on BCCA was significantly greater than other media (p < 0.05). Recovery on CASA was not significantly different from mCCDA and CCA (p > 0.05). With C. coli inoculated samples, recovery was significantly greater on BCCA and CASA than with other media (p < 0.05). The recovery of both C. jejuni and C. coli from inoculated samples with CFA was significantly less than with other media (P < 0.05). CASA was able to effectively inhibit and differentiate Campylobacter spp. from background microflora while false positive organisms occurred with BCCA and CFA. An examination of 483 randomly selected suspect Campylobacter colonies from naturally contaminated samples demonstrated a colony confirmation rate for CCA, CFA, BCCA, mCCDA, and CASA, of 84%, 87%, 88%, 90%, and 100%, respectively. The media evaluated present an alternative to conventional selective agars for the identification and enumeration of thermotolerant Campylobacter spp. from samples of poultry origin through the farm to fork continuum.     

Development of in vivo somatic mutation system using antibody against hemoglobin Preparation and use of an anti-hemoglobin antibody for identifying C57BL/6 red cells in artificial mixture of DBA/2 and C57BL/6 red cells

A cellular specific-locus mutation test is described for detecting mutant cells in mammals. The test is based upon the use of specific anti-C57BL/6J mouse hemoglobin antibody that binds hemoglobin “single” (hemoglobin s, present in C57BL/6J mouse) and not hemoglobin “diffuse” (hemoglobin d, present in DBA/2J mouse). Attempts to purify such antibody from pony and rabbit antisera through cross-absorption were unsuccessful. Immunization of LP/J mouse with C57BL/6J hemoglobin produced antiserum that reacted with s hemoglobin but not with d hemoglobin. In a fluorescent antibody technique, this antibody was found to label fixed red blood cells from C57BL/6J mice but not from DBA/2J mice. In a mixture of C57BL/6J and DBA/2J red cells, the C57BL/6J cells could be differentiated by their bright fluorescence from the non-fluorescent DBA/2J cells. Reconstruction experiment with artificial mixtures of DBA/2J and C57BL/6J cells showed that s hemoglobin bearing cells could be detected in DBA/2J red cells at frequencies as small as 0.4×10^?6. Thus, the system is sensitive enough to detect d → s mutation in DBA/2J mice. Amino acid comparison of the globin chains of s and d hemoglobins shows that our antibody can probably detect mutations leading to a substitution of serine or proline by alanine at β²⁰ position and/or a substitution of threonine by alanine at β¹³⁹ position.     

Simple recessive mutation in ENAM is associated with amelogenesis imperfecta in Italian Greyhounds

We report a familial enamel hypoplasia in Italian Greyhounds resembling non‐syndromic autosomal recessive amelogenesis imperfecta (AI) of humans. The condition uniformly affects deciduous and permanent teeth and is manifested by enamel roughening/thinning and brownish mottling. Affected teeth are often small and pointed with increased gaps. However, basic tooth structure is usually maintained throughout life, and fractures and dental cavities are not a serious problem as in humans. No tissues or organs other than teeth were affected by this mutation, and there was no relationship between enamel hypoplasia and either autoimmunity or periodontal disease, which also are prevalent in the breed. The enamel hypoplasia was associated with a 5‐bp deletion in exon 10 of the enamelin (ENAM) gene. The prevalence of the enamel defect in Italian Greyhounds was 14%, and 30% of dogs with normal teeth were carriers. Genome analyses suggest that the trait is under inadvertent positive selection. Based on the deletion detected in the ENAM gene, a genetic test was developed for identifying mutation carriers, which would enable breeders to manage the trait.     

Studies on the cytotoxic,apoptotic and antitumoral effects of Au(III) and Pt(II) complexes of 1, 10-phenanthroline on V79 379A and A549 cell lines

In the present study, Au(III) and Pt(II) complexes of 1, 10-phenanthroline (phen) were synthesized and used as the test compounds. The structure elucidation of the synthesized compounds was performed by IR, ¹H-NMR and MASS spectroscopic data and the results of elemental analyses. The cytotoxic and apoptotic effects of test compounds were elucidated on V79 379A (Chinese hamster lung fibroblast like) and A549 (human lung carcinoma epithelial like) cell lines. Cytotoxicity was measured with MTT assay and antitumoral effect was determined by colony forming ability methods. In addition, nuclear fragmentation and activation of apoptotic enzyme (caspase-3) and DAPI staining were used to detect the apoptotic effect of the compounds. All the test compounds induced time and concentration-dependent cytotoxic and antitumoral effects. Significant increases in the levels of apoptosis were observed with increasing exposure concentration.     

Changes in the metabolism of glial fibrillary acid protein (GFAP) during chronic relapsing experimental allergic encephalomyelitis in the strain 13 guinea-pig

Spinal cords were removed from strain 13 guinea pigs in various stages of chronic relapsing experimental allergic encephalomyelitis (CREAE). Levels of glial fibrillary acidic protein (GFAP) in cord extracts were determined by radioimmunoassay and protein synthesis was monitored by incubating tissue prisms with [³⁵S]methionine. Analysis of cytoskeletal enriched preparations from these incubations by SDS-polyacrylamide electrophoresis and fluorography permitted evaluation of the metabolism of discrete polypeptides; the identity of a labelled band at 51,000 daltons as GFAP was confirmed by immunoprecipitation with a specific antibody. Total protein synthesis increased 4-fold during the acute phase of CREAE but fell to control values with clinical recovery, while over the same period GFAP synthesis increased by 6–7-fold and remained elevated in the post-acute phase at about 200% of control values. During this time there was no increase in the GFAP content of the cord indicating an increased turnover of this protein rather than net synthesis. In later stages of CREAE however, GFAP levels were raised, correlating with a further increase in the incorporation of precursor into GFAP, this being most pronounced in animals in clinical relapse.     

Ultraviolet light-induced mutation of diploid human lymphoblasts

Ultraviolet irradiation (254 nm) of immortal diploid human lymphoblasts killed cells, caused mutation at three genetic loci studied, and transiently inhibited ³H-TdR uptake into DNA. A shoulder of about 6 J/m² and a D₀ of 6 J/m² was observed for survival. Mutation rose in a monotonic non-linear fashion through 6 J/m²; above 6 J/m², complex behavior approximating a plateau in induced mutation was observed. Irradiation at 4.4 J/m² caused a transient increase in the number of cells synthesizing DNA and a decrease in the rate of DNA synthesis relative to mock-irradiated controls. The parameter of rate of DNA synthesis per cell in DNA synthetic phase showed a rapid recovery toward control values between 2 and 4 h after irradiation and a slower recovery to control values by 22 h post-irradiation.Fractionated dose schedules were used to measure the effects of allowing a time interval between doses at nontoxic fluences (2.2 j/m²), moderately toxic fluences (8.8 J/m²) and toxic fluences (17.6 J/m²). These measurements indicate that in the non-toxic range of fluences common to human exposure, mutational response is mediated by a post-irradiation process which seems to show to shkow enchanced ability to protect against mutation induced by subsequent irradiation. However, at moderately toxic fluences there was little effect of dose fractionation, and at toxic fluences, a time-dependent increase in mutation fraction was observed at separation times greater than 7 h. We suggest that these latter observations arise primarily from cell cycle heterogeneity with regard to sensitivity to UV killing and mutation.     

TIME COURSE OF BIOCHEMICAL AND IMMUNOHISTOLOGICAL ALTERATIONS DURING EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS

A comprehensive biochemical, immunological and histological study was undertaken during different stages of experimental allergic encephalomyelitis (EAE). Wistar rats with EAE induced by sensitization with bovine myelin showed a maximum decrease of body weight 14–16 days post-inoculation (dpi), coincident with the appearance of the paralysis symptom (acute period). Quantitation of some brain components indicated a temporal dissociation among the alterations observed. The higher diminution of myelin basic protein (MBP) occurred at 6 dpi and then increased to reach 21 dpi, a normal value. Also, the activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase was reduced by 40% with respect to control animals only at 6 dpi. The total lipid content was normal; however, among the individual lipids, sulfatides were principally degraded during the acute stage but the amount of cerebrosides was decreased during the recovery period (29–40 dpi). Free cholesterol was similar in both groups of animals, whereas cholesterol esters were detected in EAE animals from 14 to 40 dpi. Central nervous system meningeal and parenchymal infiltration with mononuclear cells was recognized principally at 14 dpi, but some of cells were still present at 40 dpi. Deposits of immunoglobulins in the infiltrated regions as well as in spinal cord motor neurons were observed among 14–29 dpi. Total circulating antibodies to MBP began to increase at 14 dpi, reaching a plateau at 21 dpi and then maintaining this value until 40 dpi. However, the population of anti-MBP antibodies that also recognizes the neuronal protein synapsin was only present at 14 dpi. The present results suggest that the neurological symptoms can be related to some early changes in the myelin membrane followed by alterations involving neuronal structures. The existence of immunological factors against some epitopes in MBP that also recognize a synaptosomal protein might account, at least in part, for the axonal damage and disruption of the normal interneuronal activity in EAE and lead together with the alterations in some specific myelin constituents and the concomitant CNS inflammatory process to the observed hindlimb paralysis. Copyright © 1996 Elsevier Science Ltd     

Decreased tubulin synthesis in synoviocytes from adjuvant-induced arthritic rats

The first microscopical alterations along adjuvant arthritis induction in rats seem to appear in the synovium. We have studied the protein synthesis pattern of the cells constitutively present in synovial membrane (synoviocytes) and have found an impairment of synthesis of some protein when synoviocytes are derived from adjuvant arthritic rats. One of these polypeptides was identified β tubulin by two-dimensional gel electrophoresis, a membrane transfer assay using a specific monoclonal antibody and peptide mapping. We postulate that a repressed synthesis of tubulin may be an initial step in the triggering of the disease, since the effect was evident at pre-arthritis stages, when infiltration by inflammatory cells had not yet occurred.     

Discovery and SAR of a novel series of 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles as N-type calcium channel inhibitors

A novel series of substituted 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles were investigated as N-type calcium channel blockers (Ca_v2.2 channels), a chronic pain target. One compound was active in vivo in the rat CFA pain model.