Ras signals to the cell cycle machinery via multiple pathways to induce anchorage-independent growth

Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.     

Prevention of ischemically induced neointimal hyperplasia using ex vivo antisense oligodeoxynucleotides

The prevention of genetic diseases through prenatal diagnosis depends to a large extent on the awareness and acceptance of available methods by the public. A national survey was conducted among Greek women in order to explore their attitudes towards and their use of prenatal diagnosis in relation to their lifestyle. The survey was originally addressed to 3000 Greek women 18-65 years of age. Using as a criterion having a child 5 years old or younger, 350 women were eligible for the study. It was noted that 52 per cent of the respondents were adequately informed, while 48 per cent had either superficial knowledge of the subject or no knowledge at all. Amniocentesis was the method that most women were familiar with. The majority said that they were informed by their doctors and the media, and 13 per cent of the participants had prenatal diagnosis during a previous pregnancy. Twenty-two per cent of those who were not tested were over 35 years of age at the time of pregnancy. There was a significant positive correlation between awareness and acceptance of prenatal diagnosis, on the one hand, and the social, educational and financial profile of the women, on the other. Women aware of prenatal diagnosis adhered more closely to a healthy lifestyle and lived a family-centred life.     

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-alpha-factor gene encoding an alpha-factor precursor with a single alpha-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR downward arrowEAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-alpha-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >10(6)-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.     

Absolute copy number and relative change in determinations of human immunodeficiency virus type 1 RNA in plasma: effect of an external standard on kit comparisons

Use of a common set of human immunodeficiency virus type 1 (HIV-1) RNA standards eliminated differences among absolute HIV-1 RNA copy number estimates made with three commercially available assays. The relative changes in the viral RNA levels determined by the commercial assays were similar and were unaffected by the use of a common set of standards.     

Symptoms of depression and changes in body weight from adolescence to mid-life

OBJECTIVE: To investigate the relationship of symptoms of depression to weight changes in healthy individuals of normal weight across a follow-up of over 20 y. PARTICIPANTS AND DESIGN: College students (3885 men and 841 women) were administered a self-report depression measure in the mid-1960s. Their baseline body mass index (BMI) was calculated from their college medical records. Participants were contacted by mail in the late 1980s and asked to report their current height and weight as well as their smoking and exercise habits. Another measure of depressive symptoms was obtained from 3560 individuals at follow-up. Multiple regression models were used to relate changes in weight to depression scores while controlling for background (gender, baseline BMI and the gender by BMI interaction) and behavioral (exercise and smoking) predictors. RESULTS: The relationship between depressive symptoms and body weight change took the form of an interaction with baseline BMI (P < 0.001). Those with high baseline depression scores gained less weight than their nondepressed counterparts if they were initially lean, but more if they were initially heavy. This trend was especially strong in those with high depression scores at both baseline and follow-up. CONCLUSIONS: The findings support the hypothesis that depression exaggerates pre-existing weight change tendencies. This pattern would not have been detected by an examination of main effects alone, illustrating the need to move toward more complicated interactive models in the study of psychological factors and weight.     

Isocitrate lyase localisation in Saccharomyces cerevisiae cells

The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm. This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation. This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted.     

Minimizing pain for liposuction anesthesia

The publication of the complete genome sequence of Helicobacter pylori and the computer analysis of the genes it contains represents an enormous advance in H. pylori research. Besides providing an overview of H. pylori biology, the genome sequence will aid future research and radically alter the research process. In particular, it will enable a 'top down' approach whereby genes are investigated because of their similarity to genes of known function in other organisms. Other advances in biotechnology will allow all H. pylori genes to be studied simultaneously, proteins to be identified quickly, and potential drug targets to be evaluated efficiently. Progress in H. pylori research should now be rapid, and with the research interest and resources focused on it, H. pylori could become the paradigm for post-genomic research.     

Comparison of the metabolic effects of recombinant human insulin-like growth factor-I and insulin. Dose-response relationships in healthy young and middle-aged adults

The actions of recombinant human insulin-like growth factor-I (rhIGF-I) and insulin were compared in 21 healthy young (24 +/- 1 yr) and 14 healthy middle-aged (48 +/- 2 yr) subjects during 3-h paired euglycemic clamp studies using one of three doses (rhIGF-I 0.2, 0.4, and 0.8 micrograms/kg.min and insulin 0.2, 0.4, and 0.8 mU/kg.min, doses chosen to produce equivalent increases in glucose uptake). In younger subjects, rhIGF-I infusions suppressed insulin by 19-33%, C-peptide by 47-59% and glucagon by 33-47% (all, P < 0.02). The suppression of C-peptide was less pronounced with insulin than with rhIGF-I (P < 0.007). The metabolic responses to rhIGF-I and insulin were remarkably similar: not only did both hormones increase glucose uptake and oxidation in a nearly identical fashion, but they also produced similar suppression of glucose production, free fatty acid levels, and fat oxidation rates. In contrast, rhIGF-I had a more pronounced amino acid-lowering effect than did insulin (P < 0.004). In middle-aged subjects, basal IGF-I levels were 44% lower (P < 0.0001) whereas basal insulin and C-peptide were 20-25% higher than in younger subjects. Age did not alter the response to rhIGF-I. However, insulin-induced stimulation of glucose uptake was blunted in older subjects (P = 0.05). Our data suggest that absolute IGF-I and relative insulin deficiency contribute to adverse metabolic changes seen in middle age.     

Chromosomal aberrations in soft tissue tumors. Relevance to diagnosis, classification, and molecular mechanisms

In recent years, significant progress has been made in identifying characteristic chromosomal rearrangements associated with several solid tumor types, notably sarcomas, a relatively rare subset of human cancer. Most sarcomas analyzed have been found to be characterized by recurrent chromosome translocations that are specific to histological types. We have reviewed published reports of chromosomal aberrations in benign and malignant soft tissue tumors and found an incidence of specific translocations in these neoplasms that ranged from 20% to 93% within histological tumor types. Identification of recurrent chromosomal abnormalities in benign tumors has resulted in a reappraisal of the general concept that benign tumors have a normal (diploid) chromosome constitution. The variety of recurrent changes present in the different tumor types attests to the cytogenetic diversity inherent in these tumors. The chromosomal rearrangements in each of the tumor types were unique and did not correspond to cancer-associated aberrations known from other solid or hematopoietic malignancies. Cytogenetics thus provides an essential adjunct to diagnostic surgical pathology in the case of malignant soft tissue tumors, which often present substantial diagnostic challenges. In addition, it represents another approach to determine the histogenetic origin of some tumors and identifies sites of gene deregulation for molecular analysis. Indeed, recent molecular analyses of several sarcoma-associated translocations have identified novel genes and novel mechanisms of their dysregulation.     

Glucose metabolism in photoreceptor outer segments. Its role in phototransduction and in NADPH-requiring reactions

Glucose metabolism in the photoreceptor rod outer segment produces both ATP (GTP) and NADPH to support phototransduction and NADPH-requiring processes in this organelle. Glycolysis in isolated bovine rod outer segments produces 44.0 +/- 6.4 nmol of ATP/min/mg of protein or 5.7 mM ATP/min. This rate of ATP production is more than sufficient to maintain the basal rate of cGMP synthesis (0.86 mM cGMP/min) in the dark requiring 1.7 mM ATP/min. Following photoexcitation, the 4.5-fold increase in the turnover of cGMP requires an ATP synthesis rate of up to 7.7 mM ATP/min (Ames, A., Walseth, T. F., Heyman, R. A., Barad, M., Graeff, R. M., and Goldberg, N. D. (1986) J. Biol. Chem. 261, 13034-13042). Under these conditions the rate of ATP production by glycolysis as measured in isolated rod outer segments is not sufficient for the regeneration of cGMP. Additional energy is most likely provided by the phosphocreatine shuttle which transports high energy phosphate groups in the form of creatine phosphate from the rod inner segment to the rod outer segment for conversion to ATP. The hexose monophosphate pathway in bovine rod outer segments can produce up to 39.8 +/- 2.2 nmol of NADPH/min/mg of protein. This rate of NADPH production is sufficient to support both the reduction of retinal to retinol (1.2 +/- 0.2 nmol of NADPH/min/mg of protein) following the photobleaching of rhodopsin and glutathione reduction (1.1 +/- 0.1 nmol of NADPH/min/mg of protein) for the protection of rod outer segments from oxidative damage. These studies provide insight into the contribution of anaerobic glycolysis and the hexose monophosphate pathway in providing energy and nucleotides for phototransduction and other outer segment processes.