Multiplex PCR analysis of in vivo-arising deletion mutations in the hprt gene of human T-lymphocytes

A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of deletions of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes. The hprt clonal assay was used to isolate in vivo-arising hprt-deficient T-cells from six healthy males. Mutant frequencies ranged from 9-27 × 10^?6. Simple crude cellular extracts from 223 mutants were analyzed for hprt gene deletion. Sixteen (7.2%) were found to be due to total gene deletion and 22 (9.9%) were due to partial gene deletion. The relatively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand conformation polymorphism (SSCP) analysis of rearranged T-cell receptor (TCR)-γ genes. Eighteen of the 22 partial hprt gene deletion mutants were determined to be of independent origin based on a unique hprt mutation or SSCP-TCR-γ pattern. One-half (9/18) of the partial deletion mutants involved all or part of exon 4 alone, suggesting that this region of the hprt gene is prone to deletion. The small deletions effecting exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) would not have been detected by conventional Southern blot analysis and may represent a new, previously unrecognized class of mutations. The ready isolation of such intragenic deletions will allow the characterization of breakpoint junctions and may provide insights into the important processes of DNA breakage and rejoining. © 1994 Wiley-Liss, Inc.     

Cloning and expression analysis of the chick DAN gene, an antagonist of the BMP family of growth factors.

Differential screening-selected gene aberrative in neuroblastoma (DAN) is a member of a cystine knot protein family that includes Cerberus and Gremlin. First isolated in a screen to identify genes down-regulated in transformed rat fibroblasts, DAN has subsequently been cloned in Xenopus, mouse, and human. Overexpression of DAN suppresses the transformed phenotype and retards the cell's entry into S phase. Biochemical analyses have demonstrated DAN's ability to bind bone morphogenetic proteins and antagonize their signaling activity. In this study, chick DAN was cloned and sequenced, revealing a conserved cystine knot region as well as an N-glycosylation site. A riboprobe was designed from the 3' chick DAN coding sequence and used for analysis of DAN in the developing chick embryo by in situ hybridization. Chick DAN was expressed beginning at stage 10 in the developing somites and the medial otic epithelium. Expression in the neural layer of the eye became apparent at stage 14. By stage 17, expression had expanded to the base of the hindbrain. Limb bud labeling began at stage 20, whereas expression in the branchial arches appeared at stage 25. Chick DAN expression generally corresponded to that of mouse DAN expression as shown by comparative in situ hybridization. However, chick DAN was found in the otic epithelium and notochord, whereas mouse DAN was restricted to the overlying otic ectomesenchyme and was absent from the notochord. This observation suggests that DAN may play different roles in chick and mouse otic and notochord development.     

In vivo vs in vitro anticariogenic behavior of glass-ionomer and resin composite restorative materials.

OBJECTIVE: To evaluate the in vivo vs the in vitro anticariogenic potential of glass-ionomer and resin composite restoratives, utilizing a standardized interfacial gap model. METHODS: (a) In vitro study. Box shaped cavities were prepared at the buccal surfaces of extracted premolars limited to enamel. The incisal cavity walls received no treatment and were covered with 40 microm-thick metal spacers. The cavities were restored with a glass-ionomer (Ketac-Fil, n=8) and a fluoride-free resin composite (Scotchbond MP Plus/Z100, n=8). After 4 weeks immersion in an acidic gel (pH 4), thin sections were produced and examined under polarized-light microscopy.(b) In vivo study. Four low caries activity volunteers, with first four premolars, each planned to be extracted for orthodontic reasons, participated in the study. Cavities were prepared as before and filled contralaterally per patient with glass-ionomer (n=8) and resin composite (n=8). After 6 months in vivo, the teeth were extracted, sectioned and investigated by polarized-light microscopy, Raman microspectroscopy and SEM-EDS X-ray microanalysis. Unpaired t-test (lesion dimensions) and one-way ANOVA and Newman-Keuls tests (Ca, P wt%, Ca/P ratios) were used to identify statistically significant differences in lesion analysis (alpha=0.05). RESULTS: (a) In vitro study. All restorations developed lesions at incisal and cervical margins. At gap-free regions glass-ionomers showed reduced lesion dimensions compared to those of composites (p<0.05). At regions with gaps, no significant differences were found in lesion depth between the restorative groups tested. Lesion length was increased in composite, and decreased in glass-ionomer, whereas lesion depth in both restorative groups was increased in comparison to gap-free regions (p<0.05).(b) In vivo study. No lesions were observed at gap-free regions. At gap regions, 75.5% of glass-ionomer and 62.5% of composite restorations developed lesions. The lesion dimensions were significantly greater in glass-ionomer (p<0.05). A reduction in PO4(3-), CO3(2-), Ca and P was found in lesions compared to intact tissues. No F was detected and no CaF2 lattice vibrations were found at the enamel margins facing the gap adjacent to glass-ionomers. SIGNIFICANCE: In the presence of a standardized interfacial gap, no preventive effect was exerted in vivo from the glass-ionomer to protect the adjacent enamel wall from secondary caries attack. The lack of any correlation between the in vivo and in vitro models tested implies that artificial caries experiments have a negligible clinical relevance in predicting the in vivo effect.     

NURSE-MIDWIFERY CARE TO VULNERABLE POPULATIONS Phase I: Demographic Characteristics of the National CNM Sample

The purpose of this article is to describe the extent to which certified nurse-midwives (CNMs) provide care to vulnerable populations in the United States and the source of reimbursement for this care. The data were obtained from the first phase of a national study to address the characteristics of women served and cost of care provided by CNMs. Results were analyzed nationally and by American College of Nurse-Midwives regions. Certified nurse-midwives in all types of practices are providing care to women from populations that are vulnerable to poorer than average outcomes of childbirth because of age, socioeconomic status, refugee status, and ethnicity. Ninety-nine percent of CNMs report serving at least one group of vulnerable women, and CNMs in the inner city and rural practices serve several groups. The vast majority of CNMs are salaried; only 11% receive their primary income from fee-for-service. Fifty percent of the payment for CNM services is from Medicaid and government-subsidized sources whereas less than 20% comes from private insurance. Source of income varies by type of setting in which the CNM attends births. The results suggest that CNMs, as a group, make a major contribution to the care of vulnerable populations.     

EXAMINATION OF DEVELOPMENTAL NEUROTOXICITY BY THE USE OF TISSUE CULTURE MODEL SYSTEMS

1. The rotation-mediated three-dimensional reaggregate culture system is uniquely suited for studies on developmental neurotoxicity. In this system, it is possible to reconstruct central neuronal pathways and follow their development. 2. Exposure to drugs of abuse including methamphetamine and methylenedioxyamphetamine or the appetite suppressant, fenfluramine, reduces monoamines in the cultures in a dose-dependent manner and interrupts normal monoaminergic development. 3. While the monoaminergic neurones may attain normal rates of development following drug removal, the affected neurones are not capable of overcoming the drug-induced insults and a deficiency in monoamines persists throughout development. 4. In addition, the production of immortalized monoclonal hybrid cells obtained by fusion of fetal mesencephalic neurones with a neuroblastoma has yielded cell lines expressing a dopaminergic phenotype. 5. Such cells have been useful in establishing the relationship of neurotoxicity to cell lineage and can serve as models for the study of the cellular and molecular mechanisms of neurotoxicity.     

Portion Sizes in Dietary Assessment: Issues and Policy Implications

Accurate estimates of energy and nutrient intake of individuals and populations are contingent on the reliability of information obtained about food intake, food composition, and portion size. This article reviews issues related to the definition, determination, and use of portion sizes in dietary assessment and public education.     

Geriatric care on a ward without nurses

The Multiphasic Environmental Assessment Procedure (MEAP; Moos and Lemke, 1984) was used to assess three long-stay settings within a geriatric hospital, one of which is a non-nursing unit committed to the philosophy of residents viewing the setting as their own home. Findings suggest positive outcomes for residents on the nonnursing unit, and support the view that types of care fostering independence and personal responsibility of elderly residents in their setting may be associated with increased mental functioning and activity. The lack of trained nursing staff had no detrimental effect on any measure of resident life, and some specific caring practices on the unit may be interpreted as having a positive outcome for residents.     

Flexible Sigmoidoscopy Screening in an Industrial Setting

Little is known about the yield of colorectal cancer screening programs in an industrial setting. We therefore established a flexible sigmoidoscopy screening program at a chemical manufacturing plant and offered testing to all employees over the age of 40. After a Fleet enema preparation had been administered, a digital rectal examination and sigmoidoscopy were performed on each volunteer worker in the medical office of the plant. The plant had an average census of about 650 workers; 202 were screened during a 2-yr period. The mean (+/- SEM) age of participants was 52 +/- 0.4. Sixty-four employees had polyps (31.7%); data on follow-up colonoscopy were available in 69%. Colonoscopy revealed adenomatous polyps in 23 workers (53.5%), hyperplastic polyps in 10 (23%), and no evidence of neoplasia in 10 (23%). Seven workers did not arrange for follow-up colonoscopy and 12 individuals could not be contacted. No cancers were detected. In the 40- to 50-yr age group, polyps were detected in 19.5% of employees (25% adenomatous). Incidental findings were common, and included prostatic nodules, hemorrhoids, diverticulosis, and proctitis, among others. We conclude that screening sigmoidoscopy can be conveniently and economically performed at the workplace, with a high yield and good worker acceptance. The high yield suggests a possible association between polyp formation and work in a chemical plant. The finding of adenomatous polyps in the younger patients suggests that the threshold for flexible sigmoidoscopy at age 50 needs to be reassessed.