A deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene causes the clinical phenotype of the fragile X syndrome

The vast majority of individuals with the fragile X syndrome show expanded stretches of CGG repeats in the 5' non-coding region of FMR1. This expansion coincides with abnormal methylation patterns in that area resulting in the silencing of the FMR1 gene. Evidence is accumulating that this directly causes the fragile X phenotype. Very few other mutations in FMR1, causing the fragile X phenotype have been reported thus far and all concerned isolated cases. We, however, report a family, in which 11 individuals have a deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene. Although fragile X chromosomes were not detected, all 4 affected males and 2 of the carrier females show characteristics of the fragile X phenotype. Using RT-PCR we could demonstrate that FMR1 is not expressed in the affected males, strongly suggesting that the FMR1 promoter sequences 5' to the CGG repeat are missing. The deletion patients have approximately 45 CGG repeats in their FMR1 gene, though not interspersed by AGG triplets that are usually present in both normal and expanded repeats. It is hypothesized that prior to the occurrence of the deletion, an expansion of the repeat occurred, and that the deletion removed the 5' part of the CGG repeat containing the AGG triplets. Transmission of the deletion through the family could be traced back to the deceased grandfather of the affected males, which supports the hypothesis that the FMR1 gene product is not required for spermatogenesis. Finally, the data provide additional evidence that the fragile X syndrome is a single gene disorder.     

Tachykinins influence interdigestive rhythm and contractile strength of human small intestine

The effect of the putative enteric neurotransmitters neurokinin A and substance P were investigated on human small intestinal motility. Either neurokinin A, at doses of 6-25 pmol/kg/min, or substance P at doses of 1-6 pmol/kg/min were administered intravenously to healthy volunteers over 4 hr. Neurokinin A dose-dependently increased the fraction of phase II of the migrating motor complex, contraction frequency, motility index, and amplitude of contractions. At the highest dose, neurokinin A induced a phase II-like pattern, disrupting the migrating myoelectric complex. Substance P dose-dependently increased phase II of the migrating motor complex. The contraction frequency increased slightly at the highest dose, but neither motility index nor contraction amplitude changed. It is concluded that neurokinin A and substance P stimulate small intestinal motility in man, and it can be speculated that they play a role in the control of human small intestinal motility.     

Cytotoxic and antiparasitic activity from Annona senegalensis seeds

Several extracts of Annona senegalensis (Annonaceae) seeds were tested for antiparasitic activity against Leishmania major, Leishmania donovani, Trypanosoma brucei brucei, and for cytotoxic activity against KB and VERO cell lines. Fractionation of seeds extracts was mainly guided by means of a biocidal assay against Artemia salina nauplii. The biological activities resulted from extract-isolated acetogenins.     

Evaluation of agonist-antagonist properties of nitrogen mustard and cyano derivatives of delta 8-tetrahydrocannabinol

delta 8-Tetrahydrocannabinol (delta 8-THC) is a naturally occurring cannabinoid with a characteristic pharmacological profile of in vivo effects. Previous studies have shown that modification of the structure of delta 8-THC by inclusion of a nitrogen-containing functional group alters this profile and may alkylate the cannabinoid receptor, similar to the manner in which beta-funaltrexamine (beta-FNA) alkylates the micro-opioid receptor. Two novel analogs of delta 8-THC were synthesized: a nitrogen mustard analog with a dimethylheptyl side chain (NM-delta 8-THC) and a cyano analog with a dimethylpentyl side chain (CY-delta 8-THC). Both analogs showed high affinity for brain cannabinoid receptors and when administered acutely, produced characteristic delta 9-THC-like effects in mice, including locomotor suppression, hypothermia, antinociception and catalepsy. CY-delta 8-THC shared discriminative stimulus effects with CP 55,940; for NM-delta 8-THC, these effects also occurred, but were delayed. Although both compounds attenuated the effects of delta 9-THC in the mouse behavioral tests, evaluation of potential antagonist effects of these compounds was complicated by the fact that two injections of delta 9-THC produced similar results, suggesting that acute tolerance or desensitization might account for the observations. NM-delta 8-THC, but not CY-delta 8-THC, attenuated the discriminative stimulus effects of CP 55,940 in rats several days following injection. Hence, addition of a nitrogen-containing functional group to a traditional cannabinoid structure does not eliminate agonist effects and may produce delayed attenuation of cannabinoid-induced pharmacological effects.     

Biofeedback training in treatment of childhood constipation: a randomised controlled study

BACKGROUND: Because abnormal defaecation dynamics, which can be modified by biofeedback, are considered to be the underlying problem in constipation, biofeedback training may be a useful treatment for constipation. This treatment has mainly been studied in uncontrolled trials. We evaluated defaecation dynamics and clinical outcome in chronically constipated children in a randomised study comparing conventional treatment and conventional treatment with biofeedback training. METHODS: Patients, 5 to 16 years old, were referred to the Academic Medical Center in Amsterdam by general practitioners, school doctors, paediatricians, and psychiatrists. They had to fulfil at least two of four criteria for paediatric constipation and were included if they had been treated medically for at least one month before randomisation. Patients had a medical history, abdominal and rectal examination, and anorectal manometry at the start and end of the 6-week intervention period. The conventional group received laxative treatment with additional dietary advice, toilet training, and maintenance of a diary of bowel habits. The biofeedback group received the same conventional treatment and additionally five biofeedback training sessions. During the first 3 weeks, patients visited the outpatient clinic weekly; two subsequent visits were twice monthly. FINDINGS: 94 patients were randomised to conventional treatment (CT) and 98 to conventional treatment with additional biofeedback training (CT+BF). Normal defaecation dynamics increased in the CT group from 41% to 52% (not significant) and in the CT+BF group from 38% to 86% (p = 0.001). At 6 weeks, more patients in the CT+BF group showed normal defaecation dynamics, compared to the CT group (p < 0.001). This result was unaltered by controlling for baseline status in a logistic regression model. At 1 year, successful treatment (defaecation frequency > or = 3/week, soiling and/or encopresis < 2/month, and no laxatives) was accomplished in 59% of the CT and 50% of the CT+BF group (p = 0.24). The results were maintained after 1 1/2 years follow-up. No association was found between achievement of normal defaecation dynamics and clinical outcome. INTERPRETATION: Additional biofeedback training compared to conventional therapy did not result in higher success rates in chronically constipated children. Furthermore, achievement of normal defaecation dynamics was not associated with success: abnormal defaecation dynamics seem not to play a crucial role in the pathogenesis of childhood constipation. Intensive conventional laxative treatment should remain the first choice in chronically constipated children.     

Glucocorticoid-induced formation of tight junctions in mouse mammary epithelial cells in vitro

Phenotypically stable cultures of untransformed mouse mammary epithelial cells (denoted 31EG4) were established and utilized to investigate the lactogenic hormone (glucocorticoids, insulin, and prolactin) regulation of tight junction formation. When 31EG4 cells were grown on permeable supports for 4 days in medium containing the synthetic glucocorticoid dexamethasone and insulin, confluent cell monolayers obtained a transepithelial electrical resistance (TER) of 1000-3000 omega.cm2. In contrast, over the same time period, confluent monolayers treated with insulin or insulin and prolactin maintained a low TER (35-150 omega.cm2). Consistent with the formation of tight junctions, apical to basolateral paracellular permeability was decreased from 12% to 1% for [14C]mannitol and 3.3% to 0.3% for [3H]inulin when cells were cultured in dexamethasone. This effect of dexamethasone on TER required extracellular calcium, de novo protein synthesis, dose-dependently correlated with glucocorticoid receptor occupancy, and was not due to an increase in cell density. As shown by direct and indirect immunofluorescence microscopy, dexamethasone treatment did not modulate the production or location of filamentous actin, the tight junction protein ZO-1, or the cell adhesion protein E-cadherin. Our results suggest that glucocorticoids play a fundamental role in the function and maintenance of cell-cell contact in the mammary epithelia by inducing the formation of tight junctions.     

Human cytomegalovirus infection in a retinoblastoma cell line in vitro

BACKGROUND: The focus of these studies was to determine whether the Y79 human retinoblastoma cell line could function as a good in vitro model system for studying human cytomegalovirus (HCMV) infection. METHODS: Y79 cells were exposed to an HCMV mutant carrying a LacZ gene, and the resulting beta-galactosidase expression in infected cells was assessed by flow cytometry. The extent to which the three classes of viral gene products immediate early, early, and late proteins - were expressed was analyzed by immunohistochemical staining and Western blotting. Infected Y79 cells were also co-cultivated on human foreskin fibroblast (SF cell) cultures to recover virus. RESULTS: Infection of Y79 cells with the virus resulted in beta-galactosidase expression as detected by flow-cytometric analysis. Immunohistochemical staining revealed that a portion of Y79 cells expressed antigens reactive to monoclonal antibodies against immediate early, early, and late HCMV proteins. The 43-kDa early gene product was also detected by Western blotting. Infected Y79 cells co-cultivated on SF cell cultures yielded infectious foci, which turned blue following X-gal staining, demonstrating productive HCMV infection in the Y79 cells. CONCLUSION: These results demonstrate that while HCMV can productively infect Y79 cultures, it does so in a highly inefficient manner, leading these authors to conclude that this cell line does not provide a particularly good model system to study HCMV infection.