Signaling pathways in Ras-mediated tumorigenicity and metastasis

The effector domain mutants of oncogenic Ras, V12S35 Ras, V12G37 Ras, and V12C40 Ras were tested for their abilities to mediate tumorigenic and metastatic phenotypes in athymic nude mice when expressed in NIH 3T3 fibroblasts. All mutants displayed comparable tumorigenic properties, but only the mutant that activates the Raf-mitogen-activated protein kinase kinase (MEK)-extracellular regulated kinase (ERK) 1/2 pathway, V12S35 Ras, induced tumors in the experimental metastasis assay. Furthermore, direct activation of the MEK-ERK1/2 pathway in NIH 3T3 cells by mos or a constitutively active form of MEK was sufficient to induce metastasis whereas R-Ras, which fails to activate the ERK1/2 pathway, is tumorigenic but nonmetastatic. The subcutaneous tumors and lung metastases derived from V12S35 Ras-transformed NIH 3T3 cells expressed higher levels of activated ERK1/2 in culture when compared with the parental cellular pool before injection, indicating that selection for cells with higher levels of activated ERK1/2 occurred during tumor growth and metastasis. By contrast, cells explanted from V12G37-Ras or V12C40-Ras-induced tumors did not show changes in the level of ERK1/2 activation when compared with the parental cells. When tumor-explanted cell lines derived from each of the effector domain mutants were passaged one additional time in vivo, all mediated rapid tumor growth, but, again, only cells derived from V12S35 Ras-tumors formed numerous metastatic lesions within the lung. These results show that the metastatic properties of the Ras effector domain mutants segregate, and that, whereas Ras-mediated tumorigenicity can arise independently of ERK1/2 activation, experimental metastasis appears to require constitutive activation of the ERK1/2 pathway.     

Bayesian analysis of twinning and ovulation rates using a multiple-trait threshold model and Gibbs sampling

The Multiple-Trait Gibbs Sampler for Animal Models programs were extended to allow analysis of ordered categorical data using a Bayesian threshold model. The algorithm is based on data augmentation, where a value on the unobserved underlying normally distributed variable (liability) is generated in each round of iteration for each categorical observation. The programs allow analysis of several continuous and ordered categorical traits. Categorical traits can have any number of response levels. Models can be different for each trait. The programs were used to analyze twinning and ovulation rates from a herd of cattle selected for twinning rate at the U.S. Meat Animal Research Center. Data included number of calves born at each parturition for the lifetime of a cow and number of eggs ovulated for several estrous cycles before first breeding as heifers. A total of 6,411 calvings was recorded for 2,087 cows with 83.2% single and 16.8% multiple births. A total of 19,849 ovulations was recorded for 2,332 heifers with 85.2% single and 14.8% multiple ovulations. Mean posterior estimates of heritability and fraction of variance accounted for by permanent environmental effects (PE) were .128 and .103 for twinning rate and .168 and .079 for ovulation rate. Mean posterior estimate of genetic correlation was .808, and correlation of PE effects was .517. Use of a threshold model could allow for more rapid genetic improvement of the twinning herd through improved identification and selection of genetically superior animals because of higher heritability on the underlying scale.     

Role of orlistat in the treatment of obese patients with type 2 diabetes. A 1-year randomized double-blind study

OBJECTIVE: Obesity is an important risk factor for type 2 diabetes. Weight loss in patients with type 2 diabetes is associated with improved glycemic control and reduced cardiovascular disease risk factors, but weight loss is notably difficult to achieve and sustain with caloric restriction and exercise. The purpose of this study was to assess the impact of treatment with orlistat, a pancreatic lipase inhibitor, on weight loss, glycemic control, and serum lipid levels in obese patients with type 2 diabetes on sulfonylurea medications. RESEARCH DESIGN AND METHODS: In a multicenter 57-week randomized double-blind placebo-controlled study, 120 mg orlistat or placebo was administered orally three times a day with a mildly hypocaloric diet to 391 obese men and women with type 2 diabetes who were aged > 18 years, had a BMI of 28-40 kg/m2, and were clinically stable on oral sulfonylureas. Changes in body weight, glycemic control, lipid levels, and drug tolerability were measured. RESULTS: After 1 year of treatment, the orlistat group lost 6.2 +/- 0.45% (mean +/- SEM) of initial body weight vs. 4.3 +/- 0.49% in the placebo group (P < 0.001). Twice as many patients receiving orlistat (49 vs. 23%) lost > or = 5% of initial body weight (P < 0.001). Orlistat treatment plus diet compared with placebo plus diet was associated with significant improvement in glycemic control, as reflected in decreases in HbA1c (P < 0.001) and fasting plasma glucose (P < 0.001) and in dosage reductions of oral sulfonylurea medication (P < 0.01). Orlistat therapy also resulted in significantly greater improvements than placebo in several lipid parameters, namely, greater reductions in total cholesterol, (P < 0.001), LDL cholesterol (P < 0.001), triglycerides (P < 0.05), apolipoprotein B (P < 0.001), and the LDL-to-HDL cholesterol ratio (P < 0.001). Mild to moderate and transient gastrointestinal events were reported with orlistat therapy, although their association with study withdrawal was low. Fat-soluble vitamin levels generally remained within the reference range, and vitamin supplementation was required in only a few patients. CONCLUSIONS: Orlistat is an effective treatment modality in obese patients with type 2 diabetes with respect to clinically meaningful weight loss and maintenance of weight loss, improved glycemic control, and improved lipid profile.     

Altered inotropic response of endothelin-1 in cardiomyocytes from rats with isoproterenol-induced cardiomyopathy

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone alpha-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369-377). Retrieval depends on the HDEL sequence; the alpha-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.     

Impact of a children's health insurance program on newly enrolled children

CONTEXT: Although there is considerable interest in decreasing the number of US children who do not have health insurance, there is little information on the effect that health insurance has on children and their families. OBJECTIVE: To determine the impact of children's health insurance programs on access to health care and on other aspects of the lives of the children and their families. DESIGN: A before-after design with a control group. The families of newly enrolled children were interviewed by telephone using an identical survey instrument at baseline, at 6 months, and at 12 months after enrollment into the program. A second group of families of newly enrolled children were interviewed 12 months after the initial interviews to form a comparison sample. SETTING: The 29 counties of western Pennsylvania, an area with a population of 4.1 million people. SUBJECTS: A total of 887 families of newly enrolled children were randomly selected to be interviewed; 88.3% agreed to participate. Of these, 659 (84%) responded to all 3 interviews. The study population consists of 1031 newly enrolled children. The children were further classified into those who were continuously enrolled in the programs. The 330 comparison families had 460 newly enrolled children. MAIN OUTCOME MEASURES: The following access measures were examined: whether the child had a usual source of medical or dental care; the number of physician visits, emergency department visits, and dentist visits; and whether the child had experienced unmet need, delayed care, or both for 6 types of care. Other indicators were restrictions on the child's usual activities and the impact of being insured or uninsured on the families. RESULTS: Access to health care services after enrollment in the program improved: at 12 months after enrollment, 99% of the children had a regular source of medical care, and 85% had a regular dentist, up from 89% and 60%, respectively, at baseline. The proportion of children reporting any unmet need or delayed care in the past 6 months decreased from 57% at baseline to 16% at 12 months. The proportion of children seeing a physician increased from 59% to 64%, while the proportion visiting an emergency department decreased from 22% to 17%. Since the comparison children were similar to the newly enrolled children at enrollment into the insurance programs, these findings can be attributed to the program. Restrictions on childhood activities because of lack of health insurance were eliminated. Parents reported that having health insurance reduced the amount of family stress, enabled children to get the care they needed, and eased family burdens. CONCLUSIONS: Extending health insurance to uninsured children had a major positive impact on children and their families. In western Pennsylvania, health insurance did not lead to excessive utilization but to more appropriate utilization.     

Dysregulation through the NF-kappaB enhancer and TATA box of the human immunodeficiency virus type 1 subtype E promoter

The global diversity of human immunodeficiency virus type 1 (HIV-1) genotypes, termed subtypes A to J, is considerable and growing. However, relatively few studies have provided evidence for an associated phenotypic divergence. Recently, we demonstrated subtype-specific functional differences within the long terminal repeat (LTR) region of expanding subtypes (M. A. Montano, V. A. Novitsky, J. T. Blackard, N. L. Cho, D. A. Katzenstein, and M. Essex, J. Virol. 71:8657-8665, 1997). Notably, all HIV-1E isolates were observed to contain a defective upstream NF-kappaB site and a unique TATA-TAR region. In this study, we demonstrate that tumor necrosis factor alpha (TNF-alpha) stimulation of the HIV-1E LTR was also impaired, consistent with a defective upstream NF-kappaB site. Furthermore, repair of the upstream NF-kappaB site within HIV-1E partially restored TNF-alpha responsiveness. We also show, in gel shift assays, that oligonucleotides spanning the HIV-1E TATA box displayed a reduced efficiency in the assembly of the TBP-TFIIB-TATA complex, relative to an HIV-1B TATA oligonucleotide. In transfection assays, the HIV-1E TATA, when changed to the canonical HIV-1B TATA sequence (ATAAAA-->ATATAA) unexpectedly reduces both heterologous HIV-1B Tat and cognate HIV-1E Tat activation of an HIV-1E LTR-driven reporter gene. However, Tat activation, irrespective of subtype, could be rescued by introducing a cognate HIV-1B TAR. Collectively, these observations suggest that the expanding HIV-1E genotype has likely evolved an alternative promoter configuration with altered NF-kappaB and TATA regulatory signals in contradistinction with HIV-1B.     

The role of dopaminergic and serotonergic mechanisms in the development of swimming ability of young rats

Neonatal swimming behavior was studied after a single subcutaneous injection of L-dopa methyl ester (50 mg/kg; 200 mg/kg) apomorphine (0.1 mg/kg; 1.0 mg/kg), DL-amphetamine (0.5 mg/kg; 10 mg/kg), haloperidol (0.5 mg/kg; 1.0 mg/kg), L-tryptophan (50 mg/kg; 100 mg/kg), methysergide (1.0 mg/kg; 5.0 mg/kg) as well as intraventricular injection of 100 microgram 6-OHDA. 1-, 3-, 5- and 7-day-old rats were placed into a temperature-controlled aquarium (37 degrees C) and the pattern of motor coordination, latency time to swimming (LTS) and the number of foreleg strokes for 10 s (FS) were measured. When compared to the physiological saline-injected controls, rats that received L-dopa showed a striking increase of FS at all ages but the most striking improvement of motor coordination was found in newborn rats. On day 1 both doses of DL-amphetamine induced increases in FS and improvement of motor coordination, whereas apomorphine failed to show any effect at this age. On days 3, 5 and 7 low doses of DL-amphetamine and apomorphine increased the FS. However, high doses resulted in a decrement in swimming performance. Haloperidol impaired swimming on day 1 but produced a significant increase of FS on days 5 and 7. Neonatal injection of 6-OHDA delayed development of motor coordination, reduced FS and increased LTS. On days 3, 5 and 7 high doses of L-tryptophan elicited an increase of FS, while high doses of methysergide caused significant impairment of performance. It is suggested that the brain rapidly converts the administered L-dopa to dopamine during the first week of life and there appears to be a strong dependent relationship between the pattern of motor coordination and the amount of available dopamine in the developing brain.     

Field experinence of the molluscicide "Frescon" in the control of fascioliasis (author''s transl)

The desipramine-sensitive uptake (neuronal uptake) of 14C-bretylium and 14C-bethanidine into the rabbit aortic adventitial layer from 3 X 10(-6) M solutions increased with time during a 20 min incubation. For both compounds a neuronal uptake of 50 pmol/50 mg wet weight adventitia was associated with 10% block of the contractile response to field stimulation at 16 Hz and 150 pmol/50 mg with 60% block. The concentration of blocking agents inside the neuron at 50% blockade was calculated to be 260 X 10(-6) M, an 87-fold increase over the medium. The bretylium in the neuron decreased by 50% during 20 min washout, and bethanidine by 29%. Desipramine when added to the bath 20 min following the addition of the blocking agents led to a loss of bretylium but not of bethanidine from the adventitia. Desipramine had little or no effect on the uptake, washout or disposition of either blocking agent in the media-intimal layers. The data indicate that bretylium has a greater propensity than bethanidine to be lost from the neurons; however, it appears to be recycled back through the membrane via the amine pump more readily than bethanidine. The fact that conservative calculations indicate that the neuronal membrane slowly established a concentration of the blocking agents within the neuron that is known to produce rapid local anesthesia when topically applied to adrenergic nerve trunks and which approaches a concentration needed to inhibit sensory endings suggests that local anesthesia may play a role in the mechanism of neuron blockade.