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.      

Removal of the circumsporozoite protein (CSP) glycosylphosphatidylinositol signal sequence from a CSP DNA vaccine enhances induction of CSP-specific Th2 type immune responses and improvesprotection against malaria infection

The C terminus of the circumsporozoite protein (CSP) is anchored to the parasite cell membrane by a glycosylphosphatidylinositol (GPI) glycolipid. This GPI signal sequence functions poorly in heterologous eukaryotic cells, causing CSP retention within internal cell organelles during genetic immunization. Cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization. Removal of the GPI signal sequence had a profound effect on induction and efficacy of CSP-specific immune response after genetic immunization of BALB/c mice with a gene gun. The CSP produced from the plasmid lacking the GPI anchor signal sequence (CSP-A) was secreted and soluble, but that produced by the CSP+A plasmid was not. The CSP-A plasmid induced a highly polarized Th2 type response, in which the CSP-specific IgG antibody titer was three- to fourfold higher, and the protective effect was significantly greater than that induced by the CSP+A plasmid. Thus, these two physical forms of CSP induced quantitatively and qualitatively different immune responses that also differed in protective efficacy. Engineering plasmid constructs for proper cellular localization of gene products is a primary consideration for the preparation of optimally efficacious DNA vaccines.     

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.      

Introduction of new clones of penicillin-nonsusceptible Streptococcus pneumoniae in Hong Kong

Analysis of penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) isolates in Hong Kong by use of a combination of antibiogram typing, serotyping, multilocus sequence typing, and pulsed-field gel electrophoresis indicated that the dissemination of PNSP was the result of the spread of international clones: variants of the Spain(23F)-1 or Spain(6B)-2 clones were the predominant PNSP isolates from 1994 to 1997 and remained so, but Taiwan(19F)-14 and Taiwan serotype 6B clones were disseminated in Hong Kong in 1999 and 2000. Concomitant changes in antibiotic susceptibility profiles, with the rate of susceptibility to chloramphenicol rising from 10% in the period from 1994 to 1997 to 31% (P < 0.001) in 1999 and 2000, were noted to accompany the shift of clones.     

Procarcinogen activation by rat and human mammary extracts

Sprague-Dawley rat mammary gland is extremely sensitive to tumorigenesisby single or multiple doses of several poly-cydk aromatic hydrocarbons.We obtained quantitative data on the in vitro mutagenic activationof several procarcinogens by 9000 g supernatant fraction (S9)from rat mammary gland using the Ames test. Mutagenic activationwas shown to be dependent on a nicotinamide adenine dinucleotidephosphate (NADPH) generating system. An S9 preparation frommammary tissue of lactating Sprague-Dawley rats was shown toactivate 2-aminoanthracene (2-AA). A polychlorinated biphenylmixture of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) givento rats greatly raised the specific activity (rever-tant TA98colonies/mg S9 protein) of the mammary tissue using 2-AA asa test carcinogen, and permitted detection of 2, 4-diaminoanisole(DAA) and 2, 7-diaminofluorene (DAF) activation. Procarcinogens2-aminofluorine (2-AF), benzo[a]-pyrene (BP) and aflatoxin (AFL)Bl were not detectably activated by mammary gland. Mutagenesisproduced in mammary S9 activation of 2-AA, DAA or DAF was significantlyinhibited by alpha-naphthoflavone (NF) but was inhibited minimallyby metyrapone (MP). Human mammary tumor cell lines (734B, SkBr3,MDA-MD-330) possessed inducible procarcinogen metabolizing activitiessimilar to those found in S9 of rat mammary tissue. We demonstrateda simple and convenient use of the Ames test to characterizeactivation of many potential mutagens and carcinogens for mammarygland. When a test compound such as 2-AA was used, selectiveenzyme induction and inhibition was demonstrated.     

Association of intraoperative hypothermia with oncologic outcomes following radical cystectomy

IntroductionIntraoperative hypothermia (IOH) has been suggested to adversely impact outcomes following surgery. The objective of this study was to evaluate the association between IOH and survival following radical cystectomy (RC).MethodsPatients who underwent RC for bladder cancer from 2003 to 2018 were identified in our cystectomy registry. Intraoperative temperatures were extracted from the anesthesia record. IOH was defined as a median intraoperative temperature <36°C, and severe IOH as ≤ 35°C. Time under 36°C was assessed as a continuous variable. Recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS) were estimated using the Kaplan-Meier method. Associations between IOH and outcomes were assessed with multivariable Cox proportional hazards models.ResultsA total of 852 patients were identified, among whom 274 (32%) had IOH. Median follow up among survivors was 4.9 years (IQR 2.4–8.7), during which time 483 patients died, including 343 from bladder cancer. Two-year survival was not significantly different between patients with and without IOH (CSS: 74% vs. 71%, P= 0.31; OS: 68% vs. 67%, P= 0.13). Following multivariable adjustment, neither IOH nor time under 36°C was significantly associated with survival. A total of 37 patients (4.3%) had severe IOH. These patients were observed to have significantly lower 2-year OS (56% vs. 68%, P= 0.005); however, this association did not remain statistically significant after multivariable adjustment (P= 0.92).ConclusionIOH was not independently associated with survival following RC. These data do not support IOH as a prognostic factor for cancer outcomes among patients undergoing RC.     

Development of a school-based nutrition intervention for high school students: Gimme 5

PURPOSE: To describe a 4-year intervention targeting fruit/vegetable consumption by high school students. DESIGN: This is a cohort study involving six pairs of schools (n = 12) matched on gender, race, enrollment, and location with schools randomly assigned within pairs to intervention or control conditions. SETTING: Twelve Archdiocese of New Orleans high schools. SUBJECTS: Cohort was defined as students (n = 2339) who were ninth-graders in the 1993-94 school year who provided baseline data. INTERVENTION: Four components of the intervention are: (1) school-wide media-marketing campaign, (2) school-wide meal and snack modification, (3) classroom workshops and supplementary subject matter activities, and (4) parental involvement. MEASURES: Focus groups were conducted for target population input and program development. Process evaluation included student feedback on media-marketing intervention materials and activities reported here. Process measures also included school meal participation, student characteristics, and verification of intervention activities. RESULTS: Focus groups identified barriers to increased consumption of fruit and vegetables as lack of availability, variety, and inconsistency in taste. Student attitudes were favorable regarding a school program to improve diet and parental involvement. Low consumption of fruits/vegetables was reported. After a 2-month school-wide program introduction utilizing various media-marketing materials and activities, 93% of students were aware of the program and 96% could identify the healthy eating message. CONCLUSIONS: Program development can be guided and enriched by student input via focus groups. Media-marketing activities effectively delivered health messages and attracted students' attention. Materials and activities used were acceptable channels for increasing awareness, positive attitudes, and knowledge about fruits/vegetables.