Ablation of the murine alpha myosin heavy chain gene leads to dosage effects and functional deficits in the heart.

The alpha-myosin heavy chain (alpha-MyHC) is the major contractile protein expressed in the myocardium of adult mice. We have produced mice carrying a null mutation of alpha-MyHC by homologous recombination in murine ES cells. Homozygous null animals die between 11 and 12 d in utero of gross heart defects, while alpha-MyHC+/- heterozygotes survive and appear externally normal. The presence of a single functional alpha-MyHC+ allele in heterozygous animals results in reduced levels of the transcript and protein as well as fibrosis and alterations in sarcomeric structure. Examination of heart function using a working heart preparation revealed severe impairment of both contractility and relaxation in a subset of the alpha-MyHC+/- animals. Thus, two alpha-MyHC+ alleles are necessary for normal cardiac development, and hemizygosity for the normal allele can result in altered cardiac function.

The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene.

Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.

Genetic and molecular mapping of chromosome region 85A in Drosophila melanogaster.

Chromosome region 85A contains at least 12 genetic complementation groups, including the genes dhod, pink and hunchback. In order to better understand the organization of this chromosomal segment and to permit molecular studies of these genes, we have carried out a genetic analysis coupled with a chromosome walk to isolate the DNA containing these genes. Complementation tests with chromosomal deficiencies permitted unambiguous ordering of most of the complementation groups identified within the 85A region. Recombinant bacteriophage clones were isolated that collectively span over 120 kb of 85A DNA and these were used to produce a molecular map of the region. The breakpoint sites of a number of 85A chromosome rearrangements were localized on the molecular map, thereby delimiting regions of the DNA that contain the various genetic complementation groups.

Embryonic stem cells as a model for cardiogenesis.

Embryonic stem (ES) cells are derived from the inner cell mass of mouse blastocysts. These cells, when placed upon a suitable fibroblast feeder layer, continue to proliferate without overt differentiation and remain totipotent. Cells in this state are competent for gene targeting via homologous recombination. Hence, they hold the possibility of developing defined animal models of human cardiovascular disease. When removed from the feeder layer, ES cells undergo differentiation and development into large, multicellular structures, termed embryoid bodies (EBs). Morphologic, biochemical, and molecular genetic analyses indicate that during EB development some early aspects of cardiogenesis are recapitulated. Thus, EB development in culture is useful for studying certain early cardiogenic events.

On-site, rapid HIV testing with same-day results and counseling.

BACKGROUND: New rapid HIV antibody tests have allowed provision of results and result-specific counseling on the day on initial visit, and have the potential to increase the efficiency of HIV counseling and testing. METHODS: To evaluate the use of rapid testing with same-day results in public clinics, the Single Use Diagnostic System HIV-1 rapid assay was used for a 3-month period at an anonymous testing clinic and a sexually transmitted disease (STD) clinic in Dallas, Texas. Non-reactive rapid test results were reported as HIV-negative. Reactive results were reported as 'preliminary positive'. These procedures were compared with standard testing during a baseline period, with respect to number of clients receiving results and post-test counseling, client satisfaction, counselor acceptance, cost and effectiveness at reducing HIV risk. RESULTS: Rapid testing resulted in an increase in the number of persons learning their serostatus: a 4% increase for uninfected and a 16% increase for infected clients at the Anonymous Testing Clinic; a 210% increase for uninfected patients and a 23% increase for infected patients at the STD clinic. Rapid testing resulted in a cost saving of US$ 11 per test in both the anonymous and STD clinics. Of those previously tested, 88% responded that they preferred the rapid test. In the year following initial HIV test, clients tested with rapid and standard procedures were equally likely to return to the clinic with a new STD (odds ratio, 0.97; 95% confidence interval, 0.7-1.4). CONCLUSIONS: Rapid, on-site HIV testing was feasible, preferred by clients, and, resulted in significant improvement in the number of persons learning their serostatus, without increasing the costs or decreasing the effectiveness of counseling and testing.

The insulin-like growth factor-binding proteins of porcine serum: endocrine and nutritional regulation.

Several studies have shown that insulin-like growth factor-binding proteins (IGF-BPs) modify IGF activity. To investigate their role in regulating growth, the number and size of IGF-BPs in porcine serum and the role of nutritional and endocrine factors in controlling their relative abundance were determined. IGF-BPs were analyzed by ligand blotting; sera were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then the separated proteins were transferred to nitrocellulose filters and probed with [125I]IGF-I or -II. The band intensities of various forms of IGF-BPs were quantified by scanning densitometry. Fetal and postnatal sera contained six IGF-BPs of 220,000, 43,000, 39,000, 34,000, 29,000, and 24,000 mol wt (Mr). The band intensity of all forms of IGF-BPs increased with advancing gestational age. Specifically, the intensities of the 43,000, 39,000, 34,000, 29,000, and 24,000 Mr IGF-BP bands were 2.8-, 2.7-, 4.3-, 4.4-, and 3.1-fold higher, respectively, in fetal plasma at 110 than at 45 days gestation. In fetal plasma the 34,000 and 29,000 Mr forms predominated, whereas postnatally, the 43,000 and 39,000 Mr IGF-BPs predominated. Fasting of newborn pigs for 24 h reduced the intensity of the 43,000, 39,000, 34,000, and 24,000 Mr forms to 11.5%, 7.2%, 69.8%, and 5.2% of control levels, respectively. However, the 29,000 Mr IGF-BP was 1.8-fold higher in fasted pig serum than in that of fed controls. The band intensities of the 34,000 and 29,000 Mr forms were increased in postnatal animals after hypophysectomy. In contrast, fetal decapitation resulted in a preferential decrease in only the 34,000 Mr form, which was reduced by 30% compared to that in age-matched controls. These studies indicate that porcine serum contains six IGF-BPs that can be detected by ligand blotting. The level of each of these proteins increases with advancing gestational age, although the increases are not uniform, suggesting that the proteins may be regulated differentially. In the postnatal animal both endocrine and nutritional factors modulate the levels of IGF-BPs by distinct controlling mechanisms.

A quality assurance program for human immunodeficiency virus seropositivity screening of dried-blood spot specimens.

Epidemiologic projects have been implemented for using dried-blood spot (DBS) specimens routinely collected on filter paper from neonates as a seroepidemiologic resource to monitor seroprevalence of human immunodeficiency virus (HIV) among childbearing women. To ensure the quality of the data base produced from the national and other epidemiologic surveys, a quality assurance program was developed to interact with all the neonatal screening laboratories. The focus of the Centers for Disease Control's quality assurance program for HIV seropositivity testing of neonatal blood specimens is to maintain a national program to produce, certify, and provide external quality control materials as DBSs on filter paper, to monitor the filter paper matrix, to operate an external performance surveillance program, and to provide other special and consultative services. The dried-blood control and surveillance materials are certified by rigorous testing for accuracy, stability, and homogeneity. Preliminary results from the first performance evaluation of screening laboratories indicated excellent performance.

Managing the electronic NIH-guide for grants and contracts.

This article describes the implementation of a suite of computer programs to manage and provide access to a database containing the electronic documents that constitute the NIH-Guide that is distributed by the NIH on a weekly basis. The software consists of a management program that reads, processes, and stores the incoming documents and performs erratum updates on existing documents; an alerting program that sends selected information to users who have registered their information needs; a viewer that can be used on the local computer to read these documents; and a World-Wide-Web (WWW) server that can distribute the guide to computers that run WWW client software. The design of the documentation annotations, the management software, and the WWW server are expected to constitute valuable models for similar projects in the future.

Female genital mutilation. Female circumcision. Who is at risk in the U.S.?

Female genital mutilation/female circumcision (FGM/FC) refers to a group of traditional practices that involve partial or total removal of the external female genitalia or other injury to the female genital organs for cultural, religious, or other non-therapeutic reasons. These practices are usually performed by a nonmedical practitioner in the home or other nonclinical setting. Complications occurring immediately after the practice as well as those encountered months and years afterward can result in disability or premature death. In 1996 Congress directed the Department of Health and Human Services to develop estimates of the prevalence of women and girls with or at risk for FGM/FC in the United States. This paper reports those estimates, as derived by the Centers for Disease Control and Prevention, which showed that in 1990 there were an estimated 168,000 girls and women living in the United States with or at risk for FGM/FC.

Counseling and testing for HIV prevention: costs, effects, and cost-effectiveness of more rapid screening tests.

New rapid human immunodeficiency virus (HIV) antibody tests permit many individuals to receive test results and appropriate counseling at one clinic visit. Because currently used tests require significant time for processing, all individuals must return for a second visit for test results and counseling. Since return rates for the second visit are low, the more rapid tests present an opportunity to improve the efficiency of HIV counseling and testing. The authors compared the costs and effectiveness of the currently used counseling and testing procedure and a streamlined procedure made possible by the new, more rapid screening tests. When test-positive clients are given preliminary screening test results, the rapid procedure is more cost-effective than the current procedure. Since over 90% of the clients in most clinics will test negative, the rapid counseling and testing procedure allows the vast majority of clients to be counseled and tested and to receive their results and posttest counseling in one visit. However, in the case where the goal of HIV counseling and testing is to focus only on infected individuals, if information regarding a positive result from the rapid screening test is not given to clients at the initial visit before a confirmatory test is performed, then the rapid counseling and testing procedure is not more cost-effective than the current procedure.

Effect of fasting neonatal piglets on blood hormone and metabolite profiles and on skeletal muscle metabolism.

A pair of weight-matched piglets was identified at birth in each of six litters. One member of the pair was allowed to suckle for 24 h while the other was fasted. Serum concentrations of insulin, thyroxine (T4), somatomedin-C (RIA) and somatomedin-like activity (bioassay) were non-detectable or lower in non-suckled piglets when compared with suckled piglets. The converse situation was found for serum growth hormone (GH) levels, i.e., GH levels were higher (P less than .01) in non-suckled piglets. Triiodothyronine (T3) concentration was not influenced by nutritional status. Serum concentrations of fructose and of free fatty acids were not influenced by fasting (P greater than .05), but glucose and triacylglycerol serum levels were lower (P less than .01, P less than .05, respectively) in the non-suckled group. The biceps femoris muscle of the non-suckled group weighed less (P less than .05) and contained less RNA (P less than .01) and protein (P less than .07), but DNA and dry matter were similar between groups. Rates of glucose oxidation to CO2 (with [1-14C] glucose tracer, P less than .01; with [6,14C] glucose tracer, P less than .09) and incorporation into glycogen (P less than .01) and of palmitate esterification into triacylglycerol (P less than .01) were reduced by fasting. However, the rates of glycolytic flux and of palmitate oxidation to CO2 (expressed per mg protein) were not affected by fasting. The rate of glucose incorporated into glycogen was reduced (P less than .05) by the presence of palmitate in the muscle of suckled piglets. A dose-dependent decrease in somatomedin-like activity with increased concentration of serum from non-suckled piglets in the cartilage disc bioassay suggested that the production of growth inhibitors accompanied the loss of nutrient availability. The biceps femoris muscle responded to the fast by conserving fatty acid for oxidation as an energy source rather than for incorporation into triacylglycerol or phospholipid. The reduction in glucose oxidation to CO2, but maintenance of glycolytic flux rates, may be important to production of lactate, which can be used by the brain and heart as an energy source. It was suggested that the metabolic adaptations of pig skeletal muscle to the fasted state may be critical to survival.

Blood flow in the arm under brachial plexus anaesthesia.

Changes in blood blow and skin temperature have been measured in the arms of twenty patients under brachial plexus anaesthesia. A rise in each was noted in every patient. The patients were subdivided into two groups of ten. The anaesthetic administered to the patients of one group contained adrenaline (1 in 100,000), and the patients in the other group received the same anaesthetic but with no added adrenaline. The rise in flow volume, flow velocity and temperature was greater in the group which received adrenaline. The difference in change of flow volume was significant (P less than 0.05), the difference in temperature rise highly significant (P less than 0.01), but the difference in change of flow velocity was not significant.

Women living long, living well: community driven women's health priorities.

The Office on Women's Health of the Department of Health and Human Services led an effort to seek feedback on women's health issues. The response showed that women in communities nationwide focused less on individual organ-specific health issues than on broader strategies they thought were critical to improving and sustaining women's health programs. This article summarizes the result of those discussions as expressed in the "Women Living Long, Living Well" framework.

Physiological response of mature rats to replacement of dietary fat with a fat substitute.

The effects of replacing dietary fat with a fat substitute on food intake, body composition and lipid metabolism were examined in rats. Female Sprague-Dawley rats (250 g) were fed diets containing between 2 and 63% of energy as fat for 64 d. Inclusion of a substitute resulted in diets of different fat content but similar texture. When 10% corn oil (21% kJ-fat diet) was replaced with the substitute supplemented with linoleic acid (2% kJ-fat diet), rats increased food intake so that there was no effect on energy intake, body weight, body composition or serum lipid profile. Rats fed a diet containing 10% corn oil and 30% Crisco vegetable shortening (63% kJ-fat diet) became obese and hyperinsulinemic. When half (51% kJ-fat diet) or all (30% kJ-fat diet) of the Crisco was replaced with the fat substitute, the rats increased food intake and were fatter than controls but less obese than rats fed the 63% kJ-fat diet. Hepatic lipid oxidation and ketone synthesis were proportional to the percentage of dietary energy as fat. Adipocyte de novo lipid synthesis was inhibited by 51% kJ-fat and 63% kJ-fat diets. Partial or total replacement of Crisco prevented the hyperinsulinemia observed in 63% kJ-fat rats, suggesting a protective effect against the development of insulin resistance with diet-induced obesity.