Research fellowship in behavioral AIDS research

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Research fellowship in behavioral AIDS research     

Effect of dexamethasone and combinations of hormones on 7-day chick embryo lung cells

Seven-day chick embryo lung was dissociated into single cells and the capacity of these cells to incorporate 1-3H-glucosamine and 1-14C-glycine in a rotational-mediated aggregation system upon the addition of hormones was determined. Incorporation of 1-3H-glucosamine was stimulated 50% by 10(-8) M dexamethasone while 1-14C glycine uptake was not markedly influenced. Dexamethasone, 10(-8) M and 10(-6) M insulin gave rise to a 90% enhancement in 1-3H-glucosamine incorporation and a 20% increase in 14C-glycine uptake. When the aggregating cells were incubated with 10(-8) M dexamethasone and 10(-8) M 3,3',5-tri-iodo-thyronine, glucosamine incorporation was augmented by 40% and glycine by 25%. The combination, 10(-8) M 3,3',5-tri-iodo-thyronine, 10(-8) M dexamethasone and 10(-6) M insulin led to a 70% stimulation in glucosamine labelling and a 50% increase in 1-14C-glycine uptake. Very little change in the incorporation of the hexosamine and glycine into the soluble culture media was observed by adding either of the hormones; however, there appeared to be a slight increase in the incorporation of both tracers when the cells were incubated with the combination of dexamethasone and insulin.     

Clonal Diversity and Turnover of Streptococcus mitis bv. 1 on Shedding and Nonshedding Oral Surfaces of Human Infants during the First Year of Life

Streptococcus mitis bv. 1 is a pioneer colonizer of the human oral cavity. Studies of its population dynamics within parents and their infants and within neonates have shown extensive diversity within and between subjects. We examined the genetic diversity and clonal turnover of S. mitis bv. 1 isolated from the cheeks, tongue, and primary incisors of four infants from birth to 1 year of age. In addition, we compared the clonotypes of S. mitis bv. 1 isolated from their mothers' saliva collected in parallel to determine whether the mother was the origin of the clones colonizing her infant. Of 859 isolates obtained from the infants, 568 were unique clones. Each of the surfaces examined, whether shedding or nonshedding, displayed the same degree of diversity. Among the four infants it was rare to detect the same clone colonizing more than one surface at a given visit. There was little evidence for persistence of clones, but when clones were isolated on multiple visits they were not always found on the same surface. A similar degree of clonal diversity of S. mitis bv. 1 was observed in the mothers' saliva as in their infants' mouths. Clones common to both infant and mothers' saliva were found infrequently suggesting that this is not the origin of the infants' clones. It is unclear whether mucosal immunity exerts the environmental pressure driving the genetic diversity and clonal turnover of S. mitis bv. 1, which may be mechanisms employed by this bacterium to evade immune elimination.     

Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease

AIMS: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). METHODS: The coding sequence and intron-exon boundaries of the cardiac beta myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. RESULTS: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. CONCLUSIONS: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.     

Marked non-uniformity of fiber-type composition in the primate suboccipital muscle obliquus capitis inferior

 Obliquus capitis inferior (OCI) is a monoarticular suboccipital muscle linking the transverse process of the atlas (C1) to the spinous process of the axis (C2). Histochemical analysis of fiber-type composition showed that the muscle has a marked gradient of fiber-type distribution in which type I fibers comprise 95–100% of fibers in the deepest region but less than 10% of fibers in the superficial layer. Step-like changes in fiber-type proportions occurred between groups of fascicles. In most instances the boundaries between these fascicles did not exhibit different perimysial features from those fascicles with similar fiber-type proportions. OCI contained large numbers of muscle spindles, which were concentrated in deep regions rich in type I fibers. The degree of nonuniformity in fiber-type distribution seen in OCI is unusually large when compared with patterns described in other primate muscles, and has implications for the way that the muscle is studied anatomically and physiologically. Received: 11 August 1998 / Accepted: 23 September 1998     

Improved oocyte quality is obtained with follicle stimulating hormone alone than with follicle stimulating hormone/human menopausal gonadotrophin combination

The aim of this study was to compare the efficacy of pure follicle stimulating hormone (FSH) with that of FSH/human menopausal gonadotrophin (HMG) combination in downregulated cycles. A total of 357 patients was evaluated retrospectively. Sixty percent of patients in the FSH group and 55% in the FSH/HMG group were new; the others were repeat patients. Ovulation was suppressed with leuprolide acetate in all patients, followed by either FSH (n = 218) or FSH/HMG (n = 119). There was no difference in patients' age, infertility factors, number of ampoules used, length of stimulation, oestradiol levels on day of human chorionic gonadotrophin (HCG) administration, number of oocytes recovered or the number of embryos transferred. Also, nuclear maturity at aspiration and fertilization rates were not different between the two groups. FSH stimulation resulted in a significantly higher percentage of mature oocytes that showed the typical 'mature' morphological characteristics (P < 0.0001). The clinical pregnancy rates per transfer were 40 and 28% in patients stimulated with pure FSH and FSH/HMG respectively (P < 0.05). The significantly higher number of immature oocytes matured in vitro in the FSH/HMG group (P = 0.001) suggests a possible effect on in-vitro maturation, due to luteinizing hormone present in HMG. The difference in mature oocyte quality may be an important determinant in the higher pregnancy rates for the FSH- stimulated patients.      

Contributions of the ubiquitin–proteasome pathway and apoptosis to human skeletal muscle wasting with age

The primary mechanism that contributes to decreasing skeletal muscle strength and size with healthy aging is not presently known. This study examined the contribution of the ubiquitin–proteasome pathway and apoptosis to skeletal muscle wasting in older adults (n = 21; mean age = 72.76 ± 8.31 years) and young controls (n = 21; mean age = 21.48 ± 2.93 years). Subjects underwent a percutaneous muscle biopsy of the vastus lateralis to determine: (1) ubiquitin ligase gene expression (MAFbx and MuRF1); (2) frequency of apoptosis; and (3) individual fiber type and cross-sectional area. In addition, a whole muscle strength test was also performed. A one-way ANOVA revealed significant increases in the number of positive TUNEL cells in older adults (87%; p < 0.05), although no significant increase in caspase-3/7 activity was detected. Additionally, ubiquitin ligase gene expression, individual muscle fiber type and CSA were not different between old and young subjects. Muscle strength was also significantly lower in old compared to young subjects (p < 0.05). In conclusion, this study indicates a preferential role for apoptosis contributing to decreases in muscle function with age.     

Cross-correlation of EMG reveals widespread synchronization of motor units during some slow movements in intact cats

It is commonly assumed that the motor units comprising a single mammalian muscle will be recruited asynchronously at subtetanic firing rates to produce smoothly modulated force output. However, electromyograms from certain neck muscles, recorded by implanted bipolar "patch" electrodes having large contacts, often exhibited a rhythmic clustering of spike activity whose patterns suggested that motor-unit firing was synchronized both within and across muscles. We have developed a computerized processing system that digitizes EMG activity and calculates auto- and cross-correlation products of selected segments. The presence or absence of synchronization caused by neural mechanisms can be identified and differentiated from that due to the rhythmicity of the behavior itself (e.g. shaking) or due to cross-talk, according to the shapes of the resultant correlograms. These methods have so far been applied to the study of hindlimb and neck muscle EMG during various natural motor behaviors, but they provide a general, quantitative tool for the study of an important aspect of motor control that may be overlooked by conventional sampling and smoothing techniques.     

New Data on the Methohexital-Thiopental-Arrhythmia Issue

Previous reports of a much greater incidence of arrhythmias with thiopental anesthesia for electroconvulsive therapy (ECT) compared to methohexital were reexamined and the possible role of hypercapnia reconsidered. Review of 50 treatments with each agent in 13 patients who had received each at our facility revealed only one brief episode of arrhythmia. The importance of postictal hyperventilation in preventing hypercapnia and associated arrhythmias was suggested.