Molecular analysis of the distal enhancer of the mouse alpha-fetoprotein gene.

The mouse alpha-fetoprotein (AFP) gene is transcribed at high levels in the visceral endoderm of the yolk sac and fetal liver and at much lower rates in the endoderm of the fetal gut. Expression of the gene in vivo requires the presence of at least one of three enhancers which lie in its 5' flanking region. In this report, we establish that the most distal AFP enhancer directed consistent expression of a linked AFP minigene in all three endodermal tissues in transgenic mice. The enhancer is composed of three domains, each of which is essential for full enhancer function by transient transfection assays. DNase I footprinting identified three regions of the enhancer which are protected by human hepatoma nuclear extracts, one of which corresponded to a consensus site for HNF-3 binding. Site-directed mutations in this site caused a 10-fold reduction in enhancer function by transient transfection. In transgenic mice, however, the mutation resulted in sporadic expression of the transgene, dependent on the site of integration. A similar acquisition of position-dependent sporadic expression of the transgene was observed with a mutation in a second protein binding site, despite the fact that this mutation had very little effect on enhancer function as assessed by transient transfection. These studies underscore the value of examining the functions of specific protein binding sites in vivo.

The accuracy of environmental tobacco smoke exposure measures among asthmatic children.

This study determined the reliability and validity of parent-reported measures of environmental tobacco smoke (ETS) exposure among 91 asthmatic children. Test-retest reliability assessments were conducted for environmental, biological and parent-reported measures of ETS exposure. All measures except a urine cotinine assay resulted in satisfactory levels of reliability. The parent-reported measures of ETS exposure were compared to the environmental filter measure of nicotine as well as submitted to a construct validity test. Parent-reported home exposure to ETS proved moderately and significantly correlated to the filter measure. Approximately 80% of all hypothetical constructs agreed with the observed relationships for convergent, divergent and discriminant validity. It was concluded that middle class Caucasian parents' reports of their asthmatic child's residential ETS exposure are reliable and valid. These parent-reported measures should be valuable tools for epidemiological investigations and for clinical programs designed to reduce asthmatic children's residential exposure to ETS.

Reduction of environmental tobacco smoke exposure among asthmatic children: a controlled trial.

STUDY OBJECTIVE: This randomized clinical trial tested a behavioral medicine program designed to reduce asthmatic children's exposure to environmental tobacco smoke (ETS) in the home. DESIGN: Families were randomly assigned to an experimental preventive medicine counseling group, a monitoring control group, or a usual treatment control group. Families were measured six times over 1 year. PARTICIPANTS: Ninety-one families were recruited from four allergy clinics. INTERVENTION: The experimental group received a 6-month series of counseling sessions designed to decrease ETS exposure. This group also monitored smoking, exposure, and children's asthma symptoms. The monitoring group did not receive counseling and the usual treatment control group received outcome measures only. MEASUREMENTS AND RESULTS: Parents reported the daily number of cigarettes children were exposed to during the week preceding interviews. A nicotine air monitor and construct validity analysis confirmed the validity of exposure reports. Exposure to the parent's cigarettes in the home decreased for all groups. The experimental group attained a 79 percent decrease in children's ETS exposure, compared with 42 percent for the monitoring control and 34 percent for the usual treatment control group. Repeated-measures analysis of variance resulted in a significant (F([10,350] = 1.92, p < 0.05) group by time effect. At the final 12-month visit, the experimental/counseling group sustained a 51% decrease in children's exposure to cigarettes in the home from all smokers, while the monitoring control group showed an 18% decrease and the usual treatment control group a 15% decrease from pre-intervention [corrected]. CONCLUSION: A behavioral medicine program was successful in reducing exposure to ETS in the home for these asthmatic children.

Parent smoking and asthmatic children's exposure patterns: a behavioral epidemiology study.

Exposure of nonsmokers to environmental tobacco smoke (ETS) has become an important public health issue; it is generally agreed that increased exposure is related to morbidity and mortality. Precise prevalence estimates of exposure are not yet available, and measurement methodology for ETS exposure rates is still in its formative stage. Recent interventions have attempted to reduce ETS exposure, particularly in children of smoking parents. Studies have relied primarily upon reduction of parents' smoking rates to indirectly reduce children's ETS exposure. In order to effectively design interventions to achieve reductions in ETS exposure, more attention must be given to smoking behaviors which lead to passive exposure. Ninety-one families with at least one smoking parent and an asthmatic child were recruited from four allergy clinics, and interviewed regarding their smoking history, current residential smoking patterns, and the children's exposure patterns. Descriptive data are presented. It is striking that the most smoking and most exposure did not occur in the same locations, times, or during the same activities. It is recommended that interventions focus closely on these patterns rather than on reduction of smoking rates alone, in order to effect reduction in ETS exposure.

Disruption of the cytokeratin filament network in the preimplantation mouse embryo.

The distribution of the cytokeratin network in the intact preimplantation mouse embryo and the role of cytokeratin filaments in trophectoderm differentiation were investigated by means of whole-mount indirect immunofluorescence microscopy and microinjection of anti-cytokeratin antibody. Assembled cytokeratin filaments were detected in some blastomeres as early as the compacted 8-cell stage. The incidence and organization of cytokeratin filaments increased during the morula stage, although individual blastomeres varied in their content of assembled filaments. At the blastocyst stage, each trophectoderm cell contained an intricate network of cytokeratin filaments, and examination of sectioned blastocysts confirmed that extensive arrays of cytokeratin filaments were restricted to cells of the trophectoderm. Microinjection of anticytokeratin antibody into individual mural trophectoderm cells of expanded blastocysts resulted in a dramatic rearrangement of the cytokeratin network in these cells. Moreover, antibody injection into 2-cell embryos inhibited assembly of the cytokeratin network during the next two days of development. Despite this disruption of cytokeratin assembly, the injected embryos compacted and developed into blastocysts with normal morphology and nuclear numbers. These results suggest that formation of an elaborate cytokeratin network in preimplantation mouse embryos is unnecessary for the initial stages of trophectoderm differentiation resulting in blastocyst formation.

Effect of gamma irradiation on the microflora of freshwater fish. 3. Spoilage patterns and extension of refrigerated storage life of yellow perch fillets irradiated to 0.1 and 0.2 megarad.

Maximal shelf life was determined and microbial flora were compared for irradiated (0.1 and 0.2 Mrad) and nonirradiated yellow perch fillets stored at 1 C. Shelf life was estimated by organoleptic determinations. Microbiological studies included determination of the effects of irradiation on the total aerobic microbial population, lag phase, and rate of growth. Genera of organisms isolated from fillets through the course of microbial spoilage were identified, and the proteolytic activity of the organisms was determined. Plate counts for fish prior to irradiation showed the presence of approximately 10(6) organisms per g of sample. Irradiation to 0.1 and 0.2 Mrad produced 1.4 and 3 logarithm reductions of the initial count, respectively. Irradiation to 0.1 and 0.2 Mrad approximately doubled the product's shelf life. Organisms initially isolated from the nonirradiated fillets, in order of decreasing number, consisted of Flavobacterium, Micrococcus-Sarcina, Achromobacter-Alcaligenes-Mima, Pseudomonas, Microbacterium, Vibrio, Bacillus, Corynebacterium, Lactobacillus, Brevibacterium, and Aeromonas. By the 6th and 9th days of fillet storage, Pseudomonas and the Achromobacter group were the predominant organisms. All members of the genus Flavobacterium, but not all members of the genus Pseudomonas, were proteolytically active on raw fish juice-agar and skim milk-agar media. The Achromobacter group was found to be nonproteolytic on both media. Residual flora of fillets irradiated to 0.1 and 0.2 Mrad consisted of the Achromobacter group, Lactobacillus, Micrococcus-Sarcina, and Bacillus. Their sequence in predominance, however, varied with dose level. Not all proteolytic bacteria in the fillets were eliminated by 0.1 and 0.2 Mrad; proteolytic Micrococcus-Sarcina survived these treatments.

Developmental regulation of alpha-fetoprotein expression in intestinal epithelial cells of transgenic mice.

The alpha-fetoprotein (AFP) gene is transcribed in most epithelial cells lining the fetal mouse small intestine, but transcription persists in only a subset of enteroendocrine cells representing less than 1% of the total intestinal epithelial cells in the adult. The decrease in AFP expression after birth is mediated in part by a repressor element lying between -838 and -250 bp of the AFP gene. Deletion of this element from AFP minigene constructs results in high-level minigene expression in the intestines of adult transgenic mice. Although high levels of AFP minigene RNA are expressed, the fetal pattern of expression is not maintained upon deletion of the repressor element. Instead, the number of cells in which the minigene is expressed increases from less than 1% to approximately 10% of the epithelial cells in the adult small intestine, and includes the majority of the goblet cells in addition to the enteroendocrine cells. In contrast, the pattern of AFP minigene expression in the enterocytes is unaffected by deletion of the repressor element and continues to decrease in the neonate. These studies indicate that the identified AFP repressor is active specifically in goblet cells. The decrease in AFP expression in the enterocytes may be mediated by a separate cis-acting element that is contained in the AFP minigene construct. Alternatively, it is possible that mature enterocytes lack some of the positive factors required for initiation and maintenance of minigene transcription in the absence of the identified negative element.

The zonal expression of alpha-fetoprotein transgenes in the livers of adult mice.

The developmental regulation of the alpha-fetoprotein (AFP) gene in liver results in high-level expression in the fetus, followed by dramatic transcriptional repression after birth. We have examined the mouse AFP gene for transcriptional control sequences that may be involved in its postnatal repression in liver. We showed previously that removal of a DNA region between positions -250 base pairs (bp) and -838 bp of the AFP gene resulted in the persistence of expression of an AFP minigene in the postpartum liver of transgenic mice (Vacher and Tilghman, Science 250:1732-1735, 1990). This study examines the distribution of these transgene transcripts in liver using in situ hybridization. We show that there is a zonal distribution of minigene transcripts in the adult livers of these animals. Hepatocytes surrounding the central veins express high levels of minigene transcripts, while hepatocytes in the intermediate and portal areas contain few, if any, transcripts. Quantitative RNAse protection analysis shows a decrease in transgene RNA levels after birth, consistent with repression in all but a small subset of hepatocytes. These results indicate that repression in the pericentral hepatocytes is dependent upon the presence of a cis-acting, negative-regulatory domain, which is located between the enhancers and the proximal promoter of the AFP gene. In contrast, this domain is not essential for complete repression of AFP transgenes in the intermediate zone and periportal hepatocytes.

Effect of gamma-irradiation on the microflora of freshwater fish. I. Microbial load, lag period, and rate of growth on yellow perch (Perca flavescens) fillets.

Microbial flora were compared in irradiated and nonirradiated yellow perch fillets. These studies included effects of irradiation on the total microbial population, the lag phase, and rate of growth in this freshwater fishery product. The work was conducted concurrently with sensory and chemical evaluation, and constituted part of an investigation designed to evaluate the effect of substerilization doses (0.3 and 0.6 Mrad) of Co(60) gamma rays on the storage life of yellow perch fillets at 1.0 or 6.0 C. In five storage tests, total plate counts prior to irradiation did not exceed 8.7 x 10(5) per gram of sample; this count was reduced nearly 100% by irradiation with either 0.3 or 0.6 Mrad. Progressively lower maximal bacterial populations and lengthened lag phases were obtained as more radiation was used. The growth rate of the population did not appear to decrease significantly. Microbial data obtained in these studies confirmed the sensory and chemical studies, by indicating that irradiation can significantly extend the refrigerated shelf life of freshwater fish.

Focal upper limb demyelinating neuropathy.

Observations are presented on nine selected patients with chronic upper limb demyelinating neuropathy to illustrate the range of manifestations that may be observed. In three, the involvement was purely motor, in five, mixed motor and sensory and, in one, virtually purely sensory; in seven the symptoms were unilateral and in two bilateral. The presence of reduced nerve conduction velocity and conduction block and the response to treatment in seven of the cases indicate that they represented examples of chronic inflammatory demyelinating polyneuropathy (CIDP) with focal involvement. This was confirmed by nerve biopsy in two cases. The presentation in one patient was accompanied by forearm swelling initially suspected of being a tumour but shown to be due to muscle hypertrophy. This was probably the consequence of recurrent muscle cramps and fasciculation and possibly neuromyotonia. The patient with predominant sensory involvement restricted to the upper limbs demonstrates that sensory CIDP can present focally. In one patient with monomelic motor and sensory involvement, nerve biopsy showed multifocal areas of hypertrophic demyelinating neuropathy distally in the ulnar nerve without inflammatory infiltration. This patient failed to respond to therapy. Response in the others was satisfactory, although one patient with a monomelic motor neuropathy showed a severe deterioration after being given corticosteroids; he subsequently improved with intravenous human immunoglobulin therapy.