Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Heterogeneity of clara cell glutathione. A possible basis for differences in cellular responses to pulmonary cytotoxicants

Clara-cell populations show a high degree of variation in susceptibility to injury by bioactivated cytotoxicants. Because glutathione (GSH) is critical for detoxification of electrophilic metabolites, heterogeneity in Clara cell GSH levels may lead to a wide range of cytotoxic responses. This study was designed to define the distinct GSH pools within Clara cells, characterize heterogeneity within the population, and examine whether heterogeneity contributes to susceptibility. Using fluorescent imaging combined with high-performance liquid chromatography analysis, semiquantitative measurements were obtained by evaluation of GSH using monochlorobimane and monobromobimane. In steady-state conditions, the GSH measured in isolated cells was in the femtomole range, but varied 4-fold between individual cells. Clara cells analyzed in situ and in vitro confirmed this heterogeneity. The response of these cells to compounds that modulate GSH was also variable. Diethylmaleate depleted GSH, whereas GSH monoethylester augmented it. However, both acted nonuniformly in isolated Clara cells. The depletion of intracellular GSH caused a striking decrease in cell viability upon incubation with naphthalene (NA). The sulfhydryl-binding fluorochrome BODIPY, which colocalized with tetramethylrosamine, a mitochondrial dye, demonstrated by confocal microscopy that cellular sulfhydryls are highest in the mitochondria, next-highest in cytoplasm, and lowest in the nucleus. These pools responded differently to modulators of GSH. We concluded that the steady-state intracellular GSH of Clara cells exists in distinct pools and is highly heterogeneous within the population, and that the heterogeneity of GSH levels corresponds closely to the response of Clara cells to injury by NA.     

Self-selecting albino rats exhibit differential preferences for pure macronutrient diets: Characterization of three subpopulations

Analyses of natural feeding behavior in albino male Sprague-Dawley rats demonstrate that, when allowed to self-select from pure macronutrient diets (protein, carbohydrate and fat), these rats of the same genetic strain can be categorized into 3 subpopulations according to either their 24-h or their 12-h nocturnal patterns of nutrient intake. A majority of the animals (HC for high carbohydrate, 50% of the total population) consumed a diet rich in carbohydrate relative to protein or fat, while a smaller population of rats (HF, 30%) preferred the fat diet, and an even smaller population (HP, 20%) chose a high-protein diet. These 3 subpopulations, after a few weeks of maintenance on the diets, differed in their body weight, with the HF rats having a higher body weight than the HP animals, who tended to weigh more than the lightest HC rats. Whereas all subgroups exhibited a similar bimodal distribution of feeding during the nocturnal cycle, with peaks during the early and late dark periods, they were distinguishable on the basis of their nutrient consumption during specific phases of the dark cycle. This difference was most apparent in the early dark phase, when the 3 subgroups exhibited exaggerated preferences for the specific nutrient that was generally preferred over the 24-h cycle. This is in contrast to the middle dark phase, when diet preferences were attenuated or lost, and the late dark phase, when most rats were similar in showing an increased preference for protein and fat and a decreased preference for carbohydrate. The HF group was further distinguished by an unusually strong burst of feeding during the first 2 h of the dark period and an extra peak of feeding in the middle dark period (7th h), both of which were relatively high in fat content.     

Altered expression of the retinoblastoma gene product in human sarcomas

BACKGROUND. The retinoblastoma-susceptibility (Rb) gene is a prototype tumor-suppressor gene originally isolated from patients with heritable retinoblastoma. This gene encodes a nuclear phosphoprotein whose expression is altered in several types of human tumors. METHODS. We studied the expression of the Rb protein in 44 primary and 12 metastatic high-grade human sarcomas by means of immunohistochemical methods and Western blotting. Computerized image analysis was used to quantify the level of Rb gene product in individual tumor cells. The expression of the Rb gene was then correlated with clinical outcome in the patients with primary tumors. RESULTS. Of the 44 patients with primary sarcomas, 13 (30 percent) had tumors with normal, homogeneous expression of the Rb protein in essentially all tumor cells. Thirty-one patients with primary tumors (70 percent) had altered Rb expression; in 18 (40 percent) the Rb protein was heterogeneously expressed, and in 13 (30 percent) it was detected in fewer than 20 percent of the tumor cells. All 12 of the patients with metastatic sarcomas had altered expression of the Rb protein. When the findings in the patients with primary tumors were correlated with clinical outcome, survival was found to be significantly increased in the patients whose tumors had homogeneous Rb expression, as compared with those with either heterogeneous expression (P = 0.026) or no expression (P = 0.012). CONCLUSIONS. Tumors in which the expression of Rb gene product was decreased were more aggressive than tumors in which this protein was expressed by nearly all cells. The Rb gene product may be an important prognostic variable in patients with these tumors.     

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Polyvinylpyrrolidone modified bioactive glass fibers as tissue constructs: in vitro mesenchymal stem cell response

The sol-gel synthesis of a bioactive glass (BAG) sol with the incorporation of polyvinylpyrrolidone and the subsequent spraying of short, discontinuous fibers is reported. The incorporation of the polymer into the BAG sol allowed for increased control of the rheological properties and resulted in a more homogeneous fibrous material when sprayed through an air gun. Reaction kinetics and sol viscosity were monitored and analyzed during synthesis, and fibers were characterized using scanning electron microscopy and thermal analysis. Fibers were sintered at 900 degrees C and were examined for in vitro bioactivity in a simulated body fluid solution. The presence of hydroxyapatite crystals is confirmed by examination with scanning electron microscopy, energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). Both the proliferation rate and cell density of rat mesenchymal stem cells cultured on BAG fiber constructs of varying porosities were shown to be dependent upon fiber spacing.     

Total body water and the exchangeable hydrogen. II. A review of comparative data from animals based on isotope dilution and desiccation, with a report of new data from the rat.

Total body water (TBW) determination by tritium space could be factitiously elevated by exchangeable H+ contained within water-soluble chemical configurations. Should this nonaqueous (molecular) exchangeable H+ turn out to be a large fraction of total exchangeable H+, TBW measurement by tritiated water (THO) dilution would display a systematic upward and non-random error. TBW was measured by THO dilution and subsequently by total body desiccation in 21 rats (weight 227+/-83 g, mean+/-SD). TBW was 71.38+/-2.4% by THO dilution and 70.20+/-1.5% by body desiccation. Analysis of variance of TBW vs. body weight showed a highly significant correlation both with desiccation (P less than 0.0005, r=-0.78) and dilution (P less than 0.03, r= -0.50). Convariance analysis of both methods showed no difference in slope (P greater than 0.9). There was a difference in variance (P less than 0.001) and means (P less than 0.03). Tritium space is 1.2% of body weight larger than TBW measured by desiccation. TBW measured by THO dilution gives a 1.71% overestimation of TBW as measured by desiccation. TBW measurement by THO dilution is accurate within less than 2% error. These findings have particular significance in the light of our theoretical model of the total nonaqueous exchangeable H+ in fat, protein, and carbodhydrate in the living vertebrate.     

Maxillary artery embolisation in the management of epistaxis

Serious consideration needs to be given to the importance of early embolisation of the maxillary artery in severe and refractory epistaxis. This is particularly true in the young fit person with traumatic epistaxis and in the elderly person who is unfit for general anaesthesia. Embolisation is now a safe and reliable technique. Six patients were embolised. In the two institutions from 1999 - 2000. All responded well to embolisation without complication. This paper describes the technique used in our hospitals and suggests that with the advent of platinum coils and pre embolisation arteriography, the chances of cerebrovascular accident is very small.