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.     

Imaging findings of the developing temporal bone in fetal specimens.

PURPOSETo trace the development of the normal fetal temporal bone by means of plain radiography, MR, and CT.METHODSEighteen formalin-fixed fetal specimens, 13.5 to 24.4 weeks'' gestational age, were examined with a mammographic plain film technique, CT, and MR imaging at 1.5 T. Temporal bone development and ossification were assessed.RESULTSThe membranous labyrinth grows with amazing rapidity and attains adult size by the middle of the gestation period. The cochlea, vestibule, and semicircular canals are very prominent and easily recognized on MR images. The otic capsule develops from a cartilage model. Ossification of the otic capsule proceeds rapidly between 18 and 24 weeks from multiple ossification centers that replace the cartilaginous framework. The mastoid, internal auditory canal, vestibular aqueduct, and external auditory canal continue to grow after birth.CONCLUSIONThe study of fetal developmental anatomy may lead to a better understanding of congenital disorders of the ear. Faster MR scanning techniques may provide a method for in utero evaluation of the fetal temporal bone.     

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.     

Genetic and antigenic variation in the haemagglutinin of recently circulating human influenza A (H3N2) viruses in the United Kingdom

Summary Variation in the haemagglutinin (HA) gene of influenza A (H₃N₂) viruses isolated in the U. K. and abroad from 1992–1994, was determined by nucleotide sequencing of the HA1 domain of the HA gene. Viruses isolated in the U.K. early in the 1992–93 season were from the A/Beijing/353/89 lineage and were replaced later that season by viruses from the A/Beijing/32/92 lineage. Viruses from the new lineage continued to be isolated during the 1993–94 season, but were heterogeneous. Most of these isolates were more closely related to an A/Beijing/32/92 variant, A Hong Kong/23/92, but could be distinguished into three groups by serology (of which one group was circulating during the previous season) and four groups based on sequence variation in the HA gene. However, phylogenetic analysis of antigenically-distinct isolates showed that the HA gene is evolving along one lineage. Sequence analysis identified mainstream, subgroup and strain specific amino acid substitutions. There was a broad correlation between the observed amino acid changes and the antigenic sites of the HA. The results of this study highlight the value of regular molecular analysis of circulating viruses.     

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.     

Chronic anxiolytic treatment effects on conflict behavior in the rat

The present studies examined the effects of chronic posttest treatment with the antipanic agent alprazolam (ALP) or the traditional anxiolytic agents chlordiazepoxide (CDP) and phenobarbital (PhB) on conflict behavior. In daily ten-minute sessions, water-deprived rats were trained to drink from a tube which was occasionally electrified (0.25 or 0.5 mA). Electrification was signalled by a tone. Chronic ALP (10 mg/kg/day), CDP (40 mg/kg/day), PhB (80 mg/kg/day) or vehicle were injected IP after conflict testing (in some experiments again 12-16 hours later) for a minimum of 6 weeks. Chronic ALP (but not CDP or PhB) resulted in a time-dependent increase in punished responding, with a latency to onset of 3-4 weeks; this effect was not antagonized by the benzodiazepine antagonist Ro15-1788. These data support the hypothesis that conflict paradigms may serve as animal models for the study of antipanic agents. Moreover, these data suggest that not all anxiolytics will exhibit antipanic efficacy.