Detection of Bacteroides fragilis Enterotoxin Gene by PCR

Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.     

Nociceptin (Orphanin FQ) abolishes gestational and ovarian sex steroid-induced antinociception and induces hyperalgesia

Nociceptin (Orphanin FQ) is a newly discovered endogenous heptadecapeptide substrate for the opioid-receptor-like 1 receptor, a G protein coupled receptor that bears striking amino acid sequence homology to opiate receptors. In rats, intrathecal (i.t.) administration of nociceptin is without effect on basal thresholds for responsiveness to electric foot shock. However, during either late gestation or its hormonal simulation, when nociceptive thresholds are elevated by approximately 70%, i.t. nociceptin substantially attenuates jump thresholds in a dose-dependent fashion. This hypoalgesic effect of nociceptin is not limited to attenuating the gestational or sex steroid-induced increment in pain thresholds. Following the highest i.t. dose of nociceptin employed (20 nmol), the gestational or sex steroid-induced increment in jump thresholds is not only abolished but a significant hyperalgesia is observed. These results underscore the importance of the hormonal milieu to nociceptin hypoalgesic sensitivity. The potential contribution of spinal nociceptive pathways that utilize nociceptin to the etiology of extraordinary painful pregnancy and labor should not be ignored.     

Total cortisol,free cortisol,and growth hormone associated with brief social separation experiences in young macaques

Many behavioral, immunological, and physiological consequences of brief maternal separation in bonnet (Macaca radiata) and pigtail monkeys (Macaca nemistrina) have been documented. However, the impact of social separation on plasma cortisol and growth hormone is unknown for these particular species. In the present study, the behavioral and endocrinological consequences of a 2-week maternal separation in socially housed infant bonnet and pigtail monkeys were followed. In seven pairs (separated and matched control) of bonnet and six pairs of pigtail infants, plasma was obtained under baseline, separated, and reunion conditions twice weekly for the duration of the study. Blood samples were obtained from both infants of the pair in approximately 10 min. Plasma total cortisol, free cortisol, and growth hormone were measured in these samples. Focal animal behavioral observations were made on all subjects twice daily throughout the study period. In both species, total cortisol and free cortisol rose immediately following maternal separation in comparison to the matched nonseparated controls and returned to basal levels (e.g., that of matched non-separted controls) following reunion with the mother. In contrast, plasma growth hormone rose only in the pigtail infants over a time course that peaked around the time of reunion. Multiple regression techniques indicated for the first week of separation, in the separated but not control subjects, that mean plasma free and total cortisol was positively related to distress behaviors (vocalization and postural slouch) observed during this week and negatively related to social behaviors (play and proximity to others) noted during the same period. In contrast, plasma growth hormone was related to both species and sex of the subjects but unrelated to behavioral variables. © 1995 John Wiley & Sons, Inc.     

Congenital cardiac malformations in Adams-Oliver syndrome

Whole genomic hprt clones were used in Southern analysis to screen the integrity of the hprt gene in a family that includes a patient with HPRT enzyme deficiency causal to Lesch-Nyhan syndrome. A 5 kb DNA sequence deletion was found to have its endpoints in the first and third introns. The probes identified the carrier status of female family members, aided by an RFLP carried by the mother's normal X-chromosome.     

Immunization-induced inflammatory infiltration of the central nervous system in transgenic mice expressing a microbial antigen in astrocytes

Transgenic mice expressing a defined microbial antigen from central nervous system (CNS) cell type-specific promoters can be utilized to investigate the consequences of induction of peripheral immune responses to foreign antigens produced by different CNS cell types. Immunization of mice expressing β-galactosidase (β-gal) in astrocytes with this protein resulted in antigen-dependent infiltration of the CNS by mononuclear cells, principally CD4⁺ T lymphocytes and monocyte/macrophages. The perivascular and intraparenchymal infiltrates, which were located predominantly in the hippocampal formation and cerebellum, the areas of highest β-gal expression, were associated with astrocytosis, microgliosis, and a generalized increase in blood-brain barrier permeability. The resemblance of these pathological changes to aspects of human immune inflammatory CNS disorders e.g. multiple sclerosis, suggests that an initiating step in the process by which such complex diseases are produced could be the induction of peripheral immune responses to antigens expressed in astrocytes.     

Cannabinoid receptor down-regulation without alteration of the inhibitory effect of CP 55,940 on adenylyl cyclase in the cerebellum of CP 55,940-tolerant mice

The objective of this study was to determine whether the development of tolerance to CP 55,940, a potent cannabinoid agonist, was due to changes in the receptor or second messenger system. ICR mice treated with CP 55,940 (2 mg/kg) twice a day for 6 and one-half days developed a high degree of tolerance to the pharmacological effects of CP 55,940. The ability of CP 55,940 to produce motor hypoactivity, hypothermia and immobility was reduced 163-, 97- and 19-fold, respectively. Evaluation of 3H-CP 55,940 binding to rat brain membranes indicated no difference in receptor affinity between the vehicle- and CP 55,940-treated animals. However, these binding studies revealed a 50% decrease in receptor number in the cerebellum of the CP 55,940-tolerant mice. Although cAMP is generally considered to be the second messenger for cannabinoid receptors, little difference was observed in the inhibitory effects of CP 55,940 on adenylyl cyclase activity in cerebellum between vehicle and drug-treated mice. However, there was an increase in receptor mRNA which suggests a compensation for receptor loss. There are several possible explanations for these results. There may be sufficient spare receptors such that CP 55,940-tolerant mice are capable of producing a maximal effect on the second messenger system. On the other hand, one could conclude that cannabinoid receptor down-regulation does not account for the development of tolerance to all of the effects of CP 55,940 in mice.