A new high frequency polymorphism in the HER-2/neu oncogene in normal tissue and breast tumors.

The HER-2/neu (erbB-2) oncogene, if amplified and/or overexpressed in breast and ovarian cancers, is associated with a poor prognosis. Employing direct DNA sequencing, we have discovered and sequenced an 80 base pair intron from human placenta which contains an A to G polymorphism. This polymorphism lends itself to restriction fragment length polymorphism analysis of the PCR product spanning this intron. All three genotypes, homozygous A, heterozygous, and homozygous G appear in normal control populations and breast tumors. Also, no difference was seen between the polymorphic form found in five breast cancers and the corresponding normal tissue.

Increased hepatic mitochondrial capacity in rats with hydroxy-cobalamin[c-lactam]-induced methylmalonic aciduria.

Treatment of rats with the vitamin B12 analogue hydroxy-cobalamin[c-lactam] (HCCL) impairs methylmalonyl-CoA mutase function and leads to methylmalonic aciduria due to intracellular accumulation of propionyl and methylmalonyl-CoA. Since accumulation of these acyl-CoAs disrupts normal cellular regulation, the present investigation characterized metabolism in hepatocytes and liver mitochondria from rats treated subcutaneously with HCCL or saline (control) by osmotic minipump. Consistent with decreased methylmalonyl-CoA mutase activity, 14CO2 production from 1-14C-propionate (1 mM) was decreased by 76% and 82% after 2-3 wk and 5-6 wk of HCCL treatment, respectively. In contrast, after 5-6 wk of HCCL treatment, 14CO2 production from 1-14C-pyruvate (10 mM) and 1-14C-palmitate (0.8 mM) were increased by 45% and 49%, respectively. In isolated liver mitochondria, state 3 oxidation rates were unchanged or decreased, and activities of the mitochondrial enzymes, citrate synthetase, succinate dehydrogenase, carnitine palmitoyltransferase, and glutamate dehydrogenase (expressed per milligram mitochondrial protein) were unaffected by HCCL treatment. In contrast, activities of the same enzymes were significantly increased in both liver homogenate (expressed per gram liver) and isolated hepatocytes (expressed per 10(6) cells) from HCCL-treated rats. The mitochondrial protein per gram liver, calculated on the basis of the recovery of the mitochondrial enzymes, increased by 39% in 5-6 wk HCCL-treated rats. Activities of lactate dehydrogenase, catalase, cyanide-insensitive palmitoyl-CoA oxidation, and arylsulfatase A in liver were not affected by HCCL treatment. Hepatic levels of mitochondrial mRNAs were elevated up to 10-fold in HCCL-treated animals as assessed by Northern blot analysis. Thus, HCCL treatment is associated with enhanced mitochondrial oxidative capacity and an increased mitochondrial protein content per gram liver. Increased mitochondrial oxidative capacity may be a compensatory mechanism in response to the metabolic insult induced by HCCL administration.

A complex noncoordinate regulation of alpha-lactalbumin and 25 K beta-casein by corticosterone, prolactin, and insulin in long term cultures of normal rat mammary cells.

The concentrations of PRL, corticosterone, and insulin required by long term cultures of normal rat mammary cells to produce alpha-lactalbumin (alpha LA) and the 25,000 mol wt beta-casein were evaluated with a variety of hormone ratios and concentrations. For these studies a double antibody RIA for beta-casein capable of measuring 0.5 ng beta-casein/100 microliter growth media was developed and used along with our previously reported RIA for alpha LA. PRL was active at physiological levels (0.05-0.15 micrograms/ml) and quantitatively stimulated beta-casein more than alpha LA, whereas physiological levels of corticosterone (0.05-0.15 micrograms/ml) quantitatively stimulated alpha LA more than beta-casein. The concentration of corticosterone greatly altered the magnitude of the cells' response to insulin and PRL for alpha LA output by cells from either virginal or midpregnant rats. Insulin also enhanced production of these milk proteins, but very little effect was measured in the physiological range. alpha LA was increased more by insulin than by PRL, and beta-casein was enhanced more by insulin than by corticosterone. Cells from midpregnant rats required less insulin to stimulate beta-casein production than to stimulate alpha LA. Cells from virginal rats required a supraphysiological insulin level to stimulate both beta-casein and alpha LA under these conditions. These cells generally require 5-6 weeks to achieve a steady-state rate of milk protein output. The complexities of our observations help explain some of the conflicting reports in the literature concerning which hormone is of prime importance for quantitatively increasing the synthesis of a particular milk protein, particularly since high hormone levels are often employed and time in culture varies considerably among reports. We conclude that lower levels of all these hormones can and should be used in vitro. Our messenger RNA (mRNA) studies using cloned complementary DNA probes for two rat casein mRNAs show that cells grown for 2 months with hormones contain significant amounts of both alpha- and beta-casein mRNAs. Simultaneous quantification of beta-casein mRNA levels and rates of beta-casein protein production in these long term cell cultures indicated that a substantial portion of their beta-casein protein production is regulated by the amount of its mRNA. This could be controlled by mRNA synthesis and/or mRNA degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria.

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunochemical characterization with monoclonal antibodies of three major caseins and alpha-lactalbumin from rat milk.

Rat milk contains at least three major caseins with apparent molecular weights of 41,000 (alpha-casein), 25,000 (beta-casein), and 22,000 (gamma-casein) (estimated in 10% sodium dodecyl sulfate-polyacrylamide gels). These three caseins and alpha-lactalbumin, a major whey protein, were purified from rat milk. The purified caseins and alpha-lactalbumin were used to immunize BALB/c mice, and spleen cells from these mice were hybridized with cells of the mouse myeloma SP-2/0 cell-line. We have isolated a small library of hybridoma cell-lines secreting monoclonal antibodies specific for each of the major caseins and alpha-lactalbumin from rat milk. Antibodies were tested for immunoreactivity with each of the purified milk proteins and with total rat milk proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Some heterogeneity in apparent molecular weight was observed for purified alpha-casein, gamma-casein, and alpha-lactalbumin. Monoclonal antibodies against alpha-casein, gamma-casein, and alpha-lactalbumin recognized all of the molecular weight forms of the antigen for which they were specific. Each monoclonal antibody was specific for one of the caseins or alpha-lactalbumin and did not react with the other caseins or alpha-lactalbumin, suggesting that there is limited structural homology among these proteins. All of the monoclonal antibodies against the rat caseins reacted with components of mouse milk, and the monoclonal antibodies against rat gamma-casein reacted with a component of human milk of apparent molecular weight 27,000. No interspecies reactivity was observed with the antibodies against rat alpha-lactalbumin. These monoclonal antibodies are being used to develop sensitive assays for each of these major rat milk proteins.

Health dimensions of gita.

The author discusses in this paper to what extent 'the Gita' the most authorative source book of Hindu doctorine and ethics has contributed to the cause of a healthy living of mankind.

Normal mammary cells in long term culture. I. development of hormone-dependent functional monolayer cultures and assay of alpha-lactalbumin production.

Mammary cells from normal tissue of virginal, pregnant, and lactating rats have been adapted to long term monolayer culture in plastic culture dishes with retention of hormone-responsive functional activity. The addition of PRL, insulin, and corticosterone resulted in an increase in the proportion of epithelial cells. the development of intracellular lipid droplets, and the ordered aggregations of these cells. In the presence of these hormones, the milk protein, alpha-lactalbumin (a-LA), was secreted into the growth medium at rates of 20-100 ng/mg cellular protein . 24 h. A double antibody RIA for a-LA capable of measuring 0.1 ng a-LA/100 microliter growth medium was developed in our laboratory for these studies. Both intracellular and extracellular a-LA fell below detectability within 2-3 weeks after hormone withdrawal. Intracellular a-LA accounted for less than 3% of the total a-LA accumulated in each culture in 24 h. the production rate of cells continuously given hormones increased 4- to 7-fold over a period of several months in culture, and their output was greater than 100-fold above that of cells not given hormones. These cells were obtained by overnight digestion and dispersion of tissue using selected batches of collagenase in the presence of 5% fetal calf serum. Plating densities of at least 3 X 10(4) cells/cm2 in Minimum Essential Medium supplemented with 14% fetal calf serum were required for optimal functional activity. Despite several months without added hormones, these cultures can retain their hormone responsiveness, since subsequent hormone addition resulted in detectable a-LA production beginning within 7-14 days. Our studies demonstrate for the first time that normal mammary cells can be maintained in a functional hormone-responsive state for extended periods in primary cell culture. These long term cell cultures provide a system with which the effects of these and other hormones on milk production and cell differentiation can be assessed under conditions which minimize the influence of the prior in vivo hormonal millieu. (Endocrinology 108: 573, 1981)

Glucocorticoid regulation of alpha-lactalbumin production in long-term cultures of normal rat mammary cells.

Cultures of normal rat mammary cells have been developed which produce alpha-lactalbumin (a-LA) for several months in response to physiological levels of corticosterone whereas none is measureable without glucocorticoid. In contrast to recent studies in short-term organ cultures, these long-term cell cultures derived from virginal or mid-pregnant rats show no inhibition of a-LA production at high glucocorticoid concentrations. Comparable a-LA production was observed after a few months whether cells were initially cultured into media with various corticosterone levels or initially into high levels of corticosterone for 3 weeks prior to the shift to various lower doses.

Conceptual framework of planning a people-oriented hospital and peripheral health maintenance services.

For development of a people-oriented hospital and primary care facilities in its peripheral health maintenance services, a rethinking of the planning concepts of the roles, attributes and various components, viz. user, provider, institutional, social and financial control is required. The emphasis in discussion of the various elements of the planning process is on need-orientation for the rural disadvantaged and availability of limited resources.

Adrenocorticotropic hormone increases specific proteins of the mitochondrial fraction that are translated inside or outside this organelle in cultured adrenal tumor cells.

In addition to its stimulatory effects on steroidogenesis, adrenocorticotropic hormone (ACTH) also has a trophic action on the adrenal cell. This is manifested in part by increases in the levels of key mitochondrial steroidogenic enzymes. The mechanism by which this trophic action of ACTH occurs has been studied in monolayer cultures of mouse adrenal cortical tumor cells. ACTH treatment of these cells stimulates the relative incorporation of amino acids into at least eight specific proteins in mitochondrial preparations. Two of these ACTH-responsive proteins are among the nine major adrenal polypeptides that fulfill the criteria of mitochondrial translation products: (i) their synthesis in intact cells is specifically resistant to inhibition by cycloheximide yet uniquely sensitive to chloramphenicol and (ii) they are synthesized in vitro by isolated mitochondria. The other six ACTH-responsive proteins are within the much larger category of mitochondrial proteins that are synthesized on cytoplasmic ribosomes. One of the proteins synthesized in the cytoplasm electrophoretically comigrates with purified beef adrenodoxin reductase and another with beef adrenodoxin. These findings indicate that ACTH regulates the synthesis (and turnover, or both) of specific mitochondrial proteins that are synthesized inside as well as outside the mitochondria of these adrenal cells.

Regulation of mitochondrial ribosomal RNA synthesis in yeast. I. In search of a relaxation of stringency.

Studies were undertaken to determine if mitochondrial rRNA synthesis in yeast is regulated by general cellular stringent control mechanism. Those variables affecting the relaxation of a cycloheximide-induced stringent response as a result of medium-shift-down or tyrosine limitation include: 1) the stage of cell growth, 2) carbon source, 3) strain differences and, 4) integrity of the cell wall. The extent of phenotypic relaxation decreased or was eliminated entirely in a strain dependent manner as cells entered stationary phase of growth or by growth of cells on galactose or in osmotically stabilized spheroplast cultures. Cytoplasmic and mitochondrial RNA species were extracted from regrowing spheroplast cultures subjected to different experimental regimens and analyzed by electrophoresis on 2.5% polyacrylamide gels. Relative rates of synthesis were determined in pulse experiments and normalized by double-label procedures to longterm label material. Tyrosine starvation was found to inhibit synthesis of the large and small rRNA species of both cytoplasmic and mitochondrial rRNAs to about 5-20% of the control values. Chloramphenicol inhibits mitochondrial and cytoplasmic rRNA synthesis to 60-80% of control; however, chloramphenicol addition does not relax the stringent inhibition of either class of rRNAs. Cycloheximide addition results in 70-80% inhibition of synthesis of both cellular speceis of rRNAs. As noted above, cycloheximide does not relax the stringent response of cytoplasmic rRNA synthesis in spheroplasts, and also does not relax the stringent inhibition of mitochondrial rRNA synthesis. From these studies, we conclude that both cytoplasmic and mitochondrial rRNA synthesis share common control mechanisms related to regulation of protein synthesis by shift-down or amino acid limitation.

A study of combustibility of wastes of two hospitals for determining disposal by incineration.

Determination of quantity, quality and characteristics of the wastes of two hospitals located in the same sector of a class B city was undertaken to review the present dumping method of disposal. On an average 1,424.71 and 224.6 kgs. of five types of wastes as per the moisture content were found daily for seven days in these two hospitals of 1000 general beds, and 600 long stay beds for chest tuberculosis and other Cardiothoracic diseases respectively. The wastes of first hospital had higher combustibility due to its sprawling nature and activities for general patients than that of the second multistoreyed hospital treating special cases. A combustibility analysis of hospital wastes would help to decide whether to adopt incineration for disposal.

Isolation, characterization, and stability of the 30S ribosomal RNA complex from HeLa cells.

The HeLa 30S rRNA molecule (historically designated 28S rRNA) can be dissociated into two components, a 7S rRNA and a large rRNA component which we call 29S rRNA. To evaluate conformational differences between the 30S rRNA complex and the isolated 29S rRNA component of the complex, viscosity, sedimentation velocity, circular dichroism, and ultraviolet absorption measurements with the two species were performed. Sedimentation equilibrium studies were also carried out with the 30S rRNA complex. In addition, the kinetics of the reaction which dissociates the 30S rRNA complex were characterized. The removal of glycogen-like molecules by cetyltrimethylammonium bromide prescipitation of the rRNA and the preequilibration of rRNA with solvent by Sephadex column chromatography were found to be essential for reproducibility. The s20,2o values for the 30S rRNA complex and the isolated 29S rRNA were determined from the experimental data obtained at various rRNA concentrations as 29.89 plus or minus 0.40 and 29.09 plus or minus 0.14, respectively. The corresponding intrinsic viscosity values were 74 plus or minus 5 and 67 plus or minus 5 cm3/g, respectively. The optical properties of the 30S rRNA and 29S rRNA were not significantly different. These results indicate that there is no significant conformational difference between 30S rRNA and 29S rRNA under the conditions studied. We conclude from the sedimentation equilibrium data that the molecular weight of 30S rRNA is 2.1 x 10-6. From the kinetic data, the 30S rRNA dissociation appears to be an irreversible, cooperative, and ionic strength dependent reaction which at an ionic strength of 0.051 has an activation enthalpy of 123.5 kcal/mol and an activation entropy of 0.21 kcal/(mol deg).