Homonucleotide tracts, short repeats and CpG/CpNpG motifs are frequent sites for heterogeneous mutations in the neurofibromatosis type 1 (NF1) tumour-suppressor gene

Glutamatergic synaptic potentials induced by micromolar concentrations of the potassium conductance blocker 4-aminopyridine (4-AP) were recorded intracellularly from rat neostriatal neurons in the presence of 10 microM bicuculline (BIC). These synaptic potentials originate from neostriatal cortical and thalamic afferents and were completely blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 100 microM D-2-amino-5-phosphonovaleric acid (2-APV). Their inter-event time intervals could be fitted to exponential distributions, suggesting that they are induced randomly. Their amplitude distributions had most counts around 1 mV and fewer counts with values up to 5 mV. Since input resistance of the recorded neurons is about 40 M omega, the amplitudes agree to quantal size measurements in mammalian central neurons. The action of a D2 agonist, quinpirole, was studied on the frequency of these events. Mean amplitude of synaptic potentials was preserved in the presence of 2-10 microM quinpirole, but the frequency of 4-AP-induced glutamatergic synaptic potentials was reduced in 35% of cases. The effect was blocked by the D2 antagonist sulpiride (10 microM). Input resistance, membrane potential, or firing threshold did not change during quinpirole effect, suggesting a presynaptic site of action for quinpirole in some but not all glutamatergic afferents that make contact on a single cell. The present experiments show that dopaminergic presynaptic modulation of glutamatergic transmission in the neostriatum does not affect all stimulated afferents, suggesting that it is selective towards some of them. This may control the quality and quantity of afferent flow upon neostriatal neurons.     

Assessment of renal osteodystrophy in dialysis patients: use of bone alkaline phosphatase, bone mineral density and parathyroid ultrasound in comparison with bone histology

Bone biopsies were studied in 73 patients to determine if a two-site radioimmunometric assay for serum bone alkaline phosphatase (BAP), total serum alkaline phosphatase (ALP), serum intact parathyroid hormone (iPTH), hand X-rays, regional bone mineral density (BMD) measurements and parathyroid enlargement detected by ultrasonography could accurately predict renal osteodystrophy. In the patients studied 57 had hyperparathyroid bone disease, 4 mixed renal osteodystrophy, 3 adynamic bone disease, 1 osteomalacia and 8 normal histology. Serum BAP, ALP and iPTH correlated positively with mineral apposition rate, osteoblastic, osteoid and eroded surface. In the diagnosis of hyperparathyroid bone disease serum iPTH was the most sensitive investigation, detecting 81% of patients at a level > 100 pg/ml but with a specificity of only 66%. Serum BAP was more sensitive, 70% at a level of > 10 ng/ml, than serum total ALP, 30% at a level of 300 IU/l, with similar specificities, 92 and 100%, respectively. Ultrasound detection of an enlarged parathyroid gland had a sensitivity of 64% and a specificity of 100% for the diagnosis of hyperparathyroid bone disease. Hand X-rays had a poor sensitivity, 47%, but a high specificity, 92%, for the detection of hyperparathyroid bone disease. The majority of patients had regional BMD values within the normal reference range and this test was of poor discriminatory value. The non-invasive markers were unable to distinguish between patients with low turnover, mild hyperparathyroidism and patients with normal histology. In conclusion the measurement of serum iPTH is a useful screening tool for the detection of hyperparathyroid bone disease which can be confirmed by the finding of a raised serum BAP or parathyroid enlargement. For definitive diagnosis, however, the gold standard remains bone biopsy and at present one cannot recommend any non-invasive method as an adequate substitute.     

Point prevalence of oropharyngeal carriage of fluconazole-resistant Candida in human immunodeficiency virus-infected patients

To estimate the prevalence of both clinically evident and asymptomatic carriage of fluconazole-resistant Candida, we prospectively surveyed 128 adults infected with human immunodeficiency virus (HIV). The patients had an average CD4 cell count of 206/mm3. Ninety-seven isolates of Candida were obtained from the oropharynx of 82 patients (64%). Of these 82 patients, 76% carried C. albicans alone; 18%, both albicans and non-albicans isolates; and 6%, non-albicans species alone. Oropharyngeal candidiasis was evident in only 38 (46%) of the 82 patients for whom a culture was positive and was never seen unless C. albicans was present. When MICs were measured by using the National Committee for Clinical Laboratory Standards M27-T methodology and grouped by using recently proposed breakpoints, we found that eight of the 38 patients with oropharyngeal candidiasis and six of the 44 patients who were asymptomatically colonized carried C. albicans isolates resistant to fluconazole (MIC, > or = 64 micrograms/mL); estimated rates of carriage were 21% (95% confidence interval, 10%-37%) and 14% (95% confidence interval, 5%-27%), respectively. Carriage of resistant isolates of C. albicans by HIV-infected adults is more common than previously suspected, and clinicians should be alert to the possible need for either higher doses of fluconazole or alternative treatment modalities.     

Development of a monoclonal antibody to 6 beta-hydroxycortisol and its application in an enzyme-linked immunosorbent assay (ELISA) for 6 beta-hydroxycortisol in urine

A novel monoclonal antibody to 6 beta-hydroxycortisol (6 beta-OHC) was generated and incorporated into an antigen-coated indirect enzyme-linked immunosorbent assay (ELISA) using 6 beta-OHC-protein conjugate as the steroid-coating antigen. The monoclonal antibody is specific to 6 beta-OHC and 6 beta-OHC-3-carboxymethyloxime. Cross-reactivity with other structurally related steroids such as cortisol, cortisone, and 6 beta-hydroxycortisone was less than 10%. Two different clones (clone 5C1 and 19F) of the monoclonal anti-6 beta-OHC antibody have been developed, each with slightly different sensitivity and specificity. The sensitivity of the MAb clones was not significantly improved when compared to the rabbit polyclonal antibodies in this study, but still within the accepted detection limit for 6 beta-OHC in both human and laboratory animals. The assay had a detection limit of 200 ng/ml, an intraassay variation of 6.4% and an interassay variation of 7.3%. The application of the anti-6 beta-OHC-MAb-based-ELISA was tested by measuring the urinary output of 6 beta-OHC in human before and after enzyme induction by rifampicin treatment. The mean 24-h urine output of 6 beta-OHC in human subjects was 485 +/- 100 micrograms and 1478 +/- 281 micrograms before and after rifampicin administration, respectively. In conclusion, the monoclonal anti-6 beta-OHC antibody developed in this study has the required specificity and sensitivity as an alternative method for measuring urinary 6 beta-OHC in the detection of enzyme induction or enzyme inhibition of CYP3A in humans and laboratory animals.     

Training in firearm safety counseling in family practice residency programs

We have studied the function of partial orthotopic liver transplantation in the rat by evaluating prothrombin time (PT), liver blood flow, basal and glucose-stimulated insulin secretion and glucose tolerance, and the reticuloendothelial function (RES) in hepatectomized rats subjected to partial liver transplantation. A graft corresponding to 68% of a normal liver was transplanted to totally hepatectomized rats. Comparison was made between control rats and rats subjected to 32% liver resection. PT was not significantly different in the transplanted group compared with liver-resected and control rats. Laser Doppler flowmetry showed that at 28 days after surgery, blood flow had increased in the transplanted livers. Furthermore, on the third day after transplantation, basal plasma insulin was increased and the plasma insulin response to glucose was exaggerated, suggesting reduced insulin action and impaired insulin degradation. Finally, uptake of radioactive-labeled E. coli bacteria, as a measure of RES function, was not compromised in transplanted animals. Based on these results, we conclude that reduced-size liver transplant in out-bred rats results in fast normalization of liver function after surgery although, immediately after surgery, glucose intolerance is seen.     

Exclusion of BMP6 as a candidate gene for cleidocranial dysplasia

Cleidocranial dysplasia (CCD) is an autosomal dominant, generalized skeletal dysplasia in humans that has been mapped to the short arm of chromosome 6. We report linkage of a CCD mutation to 6p21 in a large family and exclude the bone morphogenetic protein 6 gene (BMP6) as a candidate for the disease by cytogenetic localization and genetic recombination. CCD was linked with a maximal two-point LOD score of 7.22 with marker D6S452 at theta = 0. One relative with a recombination between D6S451 and D6S459 and another individual with a recombination between D6S465 and CCD places the mutation within a 7 cM region between D6S451 and D6S465 at 6p21. A phage P1 genomic clone spanning most of the BMP6 gene hybridized to chromosome 6 in band region p23-p24 using FISH analysis, placing this gene cytogenetically more distal than the region of linkage for CCD. We derived a new polymorphic marker from this same P1 clone and found recombinations between the marker and CCD in this family. The results confirm the map position of CCD on 6p21, further refine the CCD genetic interval by identifying a recombination between D6S451 and D6S459, and exclude BMP6 as a candidate gene.     

Vasopressin receptor subtypes differentially modulate calcium-activated potassium currents in the horizontal limb of the diagonal band of Broca

Because many testicular toxicants cause damage to specific stages of spermatogenesis, the present study has investigated the utility of a model in which the testis is synchronized to contain only a few closely related spermatogenic stages. The susceptibility of different stages to 1,3-dinitrobenzene (1,3-DNB) toxicity was investigated in rats, the testes of which had been stage synchronized by a vitamin A depletion/repletion (VADR) procedure. 1,3-DNB (25 mg/kg, IP) or vehicle was injected 58, 61, or 78 d after vitamin A readministration, and testicular histopathology was evaluated 48 h later. At the time of sacrifice, testes in the three groups were synchronized to stages I-VI, VII-IX, or X-XIV+I. The data indicated that tubules in all stages of spermatogenesis, in both synchronized and unsynchronized animals, demonstrated histopathologic changes in response to 1,3-DNB. However, the lesion seen in synchronized animals was more severe and less stage specific than that seen in weight-matched, unsynchronized animals. This increase in degree of susceptibility could be partially explained by differences in toxicokinetics. Stage-synchronized testes could provide unique insights into stage-specific cellular and molecular events, especially for in vitro studies where the stage enrichment could be maximally exploited. However, results obtained from in vivo toxicity studies using animals subjected to VADR should be interpreted carefully in light of the confounding physiologic/metabolic perturbations potentially induced by the VADR procedure.