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Biomedical subjects

J M Buchanan

Publications and source records attributed to J M Buchanan.

At least 37 records · Page 2Linked to original sources
PubMed1982

Regulation of nucleotide and pentose synthesis in resting and stimulated 3T6 fibroblasts.

A two-step procedure has been used to follow the activation of one metabolic system involved in the return of cells to a proliferative state after resting in a Go state as a result of serum limitation. One feature of the resting state is a limited capacity to synthesize nucleotides. The limitation apparently is in the rate of synthesis of 5-phosphoribosylpyrophosphate from glucose and indirectly in the capacity of the resting cells to turn over the triphosphopyridine nucleotide pair, NADPH:NADP+. A reaction utilizing NADPH is apparently greatly diminished in resting cells and is substantially increased by only brief contact of cells with the hormonal elements in dialyzed calf serum. Insulin together with platelet-derived growth factor can substitute for calf serum. Aside from stimulating the turnover of the pyridine nucleotide coenzyme pair, serum also stimulates the utilization and reformation of ATP, principally from AMP. Among the NADPH-linked reactions that have been examined for their physiological significance in the initiation of growth stimulation are two steps in the conversion of glutamate to proline in the cytoplasm. Pyrroline 5-carboxylate, an intermediate in this metabolic pathway, has been shown to stimulate PRPP synthesis when added to cultures of resting 3T6 cells. Proline, the product of the reduction of this 5-membered heterocycle is also a stimulant of PRPP synthesis. In addition, dehydroascorbic acid is a potent stimulant of PRPP synthesis. As a working hypothesis, we are exploring the role of a series of reactions that form a pyrroline 5-carboxylate/proline cycle operating between the cytoplasm and mitochondria. The net result is the oxidation of NADPH by molecular oxygen to yield NADP+ and water. The NADP+ is then used in the hexose monophosphate pathway for the conversion of glucose to PRPP. We wish to determine whether dehydroascorbate is operating in this cycle as an oxidant of proline in the mitochondria or whether it participates in some other reaction in the cell that redistributes the ratio of NADPH to NADP.

Cell Division↗
PubMed1981

Unbound progesterone receptors are in equilibrium between the nucleus and cytoplasm in cells of the rat uterus.

The classical model for the mechanism of action of steroids holds that unbound receptors for steroids reside exclusively in the cytoplasmic compartment and that they undergo translocation to the nucleus when bound to steroids in a process which is temperature sensitive. We have in the past proposed that unbound receptors for estrogen are in both nucleus and cytoplasm in a state of equilibrium. In the present study we looked at the location of the progesterone receptor using autoradiography and biochemical procedures. Uteri were incubated with [3H]progesterone or [3H]R5020 (dimethyl-19-nor-pregna-4,7-diene-3,20 dione, 17 alpha, 21-[17 alpha-methyl-3H] for 5 min at 4 C. When the tissue was processed for autoradiography, the localization of steroid was nuclear. In contrast, when the tissue was processed using the usual biochemical procedures, all binding activity appeared in the cytoplasm. In addition, when concentrated preparations of homogenized uteri were made, free receptor could be demonstrated in the crude nuclear preparations. We hypothesize that unbound progesterone receptor, like unbound estrogen receptor in the rat uterus, is in both the nucleus and cytoplasm of cells. In addition, we propose that the intracellular distribution of unbound receptors for all steroids is dependent upon the equilibrium conditions present.

Animals↗
PubMed1981

Autoradiographic and biochemical studies of hormone receptor localization.

Autoradiography has been used in the past to locate presumptive receptor systems for a number of steroids. It has provided information in rather complex tissues that could not have been obtained by biochemical procedures. In these studies we made use of autoradiography to redirect our biochemical efforts to study androgen receptors in the primate heart. Castrated-adrenalectomized female rhesus monkeys and baboons were injected with 1 microgram and 5 alpha-dihydro-[1,2,4,5,6,7-3H]-testosterone per kilogram of body weight. The animals were killed 1 hour later and the hearts were removed and processed for autoradiography. A nuclear localization of androgen was found in most of the myocardial cells, but in few if any of the interstitial cells. After reviewing the autoradiographic data, we began a new series of biochemical studies using a new buffer system and unlabeled androgen to stabilize the receptor. This in combination with postlabeled gradients allowed us to demonstrate for the first time 8S binding on sucrose density gradients. We feel that autoradiography can be a useful adjunct to biochemical studies even in "less complex" tissue such as the cardiac muscle.

Adrenal Glands↗
PubMed1980

The heart is a target organ for androgen.

Autoradiographic and biochemical analyses of the hearts of female rhesus monkeys and baboons indicate that atrial and ventricular myocardial cells contain androgen receptors. Although the specific effects of nuclear uptake and retention of androgen on the function of heart muscle cells are not known, the presence of this receptor suggests that sex steroid hormones may affect myocardial function directly and may explain some of the peculiar differences in heart disease between men and women.

Androgens↗
PubMed1980

Association of the src gene product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts.

We have prepared cytoskeletons from normal and Rous sarcoma virus-transformed cells by extraction with nonionic detergents in a buffered salt solution designed to preserve the structure as it exists in vivo. Virtually all of the phosphoprotein pp60src in the cell is bound to such cytoskeletons. Furthermore, when these cytoskeletons are incubated in situ with [gamma-32P]ATP, pp60src is phosphorylated. Labeling of other apparently transformation-specific cytoskeletons phosphoproteins is also observed. These results directly demonstrate an association between pp60src and elements of the cytoskeleton and suggest that pp60src may exert at least some of its effects as a consequence of its interaction with this cellular framework.

Adenosine Triphosphate↗
PubMed1980

The effects of opiates on androgen binding in the forebrain of the rat.

Many heroin addicts and addicts on methadone treatment exhibit sexual dysfunction. It is believed that the primary site of action of the opiates is the hypothalamic-hypophyseal system. Although it is not known how the opiates might alter the hypothalamic-hypophyseal system, it has recently been suggested that the opiates might mimic the steroids by binding to steroid hormone receptors. To test this hypothesis, in vivo autoradiographic studies and in vitro biochemical studies were conducted to see if the binding of androgen to receptors in the central nervous system would be affected by the opiates. In the autoradiographic studies, animals were treated for 10 days with saline, morphine sulfate (50 mg/kg), or methadone hydrochloride (10 mg/kg) and then injected with 3H-dihydrotestosterone (DHT-0.5 microgram/100 grams body weight). The animals were killed 1 hour later and their brains were removed and processed for autoradiography. No significant effects of opiates on nuclear uptake and retention of androgen by the limbic system were found. In the in vitro binding studies, 3H-DHT (5 X 10(9) M) and 3H-estradiol (5 X 10(-9) M) were incubated with cytosol prepared from brain in the presence of varying concentrations of morphine sulfate (10(-3) - 10(-9) M) and methadone hydrochloride (10(-3) - 10(-9) M). Using three different assay systems (LH-20, hydroxylapatite, and sucrose density gradient centrifugation), no consistent effects of opiates on steroid binding to its receptor could be demonstrated. These data along with previously reported data suggest that the opiates do not induce sexual dysfunction by interacting with steroid receptors.

Animals↗
PubMed1979

Equilibrium: the intracellular distribution of steroid receptors.

Data are presented which suggest that there are unbound receptors for oestrogen in nuclei of the smooth muscle cells of the myometrium. A new model for the distribution of unbound receptors is proposed in which unbound receptor is in equilibrium, partitioned between nucleus and cytoplasm according to the free water content of these intracellular compartments.

Animals↗
PubMed1979

The arthritic in an urban environment.

Ninety-five patients with rheumatoid arthritis or osteoarthrosis were interviewed and questioned in their homes, being randomly selected from 354 outpatients with these diseases. The findings were compared with answers from 30 matched able-bodied controls. The handicapped were mainly female and elderly; over half were severely or appreciably handicapped; nearly one-third lived alone. The average weekly income of these households was pound25, at least pound6 a week less than their age-matched counterparts. Very few subjects lived in adapted or specially designed housing, and half the housing had at least 2 steps to street level. A third of the sample could not walk more than 10 yards (9 m), and only half could walk 100 yards (90 m); yet only one-third had a bus stop, one-quarter a local park, and one-third a local shop within this distance. One-third of the patients could not shop for themselves. Powered wheelchairs were not used. Buses were rarely or never used by nearly half these arthritics because of severe disability. Cars were owned by less than a quarter of the sample, and only by those financially better off. Over a third were unable to get a lift when wanted. Cars ameliorated much of the frustration and lack of social contact caused by physical disability. Adequate financing would enable those with severe disability to engage in normal activities. Better designed buses stopping nearer the homes of handicapped would improve independence for those less severely disabled. Correct siting of mobility housing, shops, community facilities, and parks would be greatly appreciated, as would removal of all unnecessary steps and kerbs.

Adult↗
PubMed1978

Multiple forms of sarc gene proteins from Rous sarcoma virus RNA.

In a previous study we were able to identify two proteins of 25,000 and 18,000 daltons that were made from RNA of transforming virions of Rous sarcoma virus (RSV) and that were missing from the translation products of a transformation-defective deletion mutant of RSV. In the present study we have separated RSV virion RNA on sucrose gradients and have determined that the two putative sarc gene products are synthesized as doublets from an mRNA of approximately 18 S. There also appear to be several other sizes of virion mRNA that direct the synthesis of other viral proteins. These data are discussed in terms of the structure of the RSV genome. In addition to the 25,000- and 18,000-dalton doublets, there also is a 60,000-dalton protein whose synthesis is directed by 18S viral RNA from transforming virion of RSV. Peptide mapping has shown that the 60,000- and 25,000-dalton doublet are structurally related. In addition, the use of two-dimensional gel electrophoresis has allowed us to resolve both bands of the 25,000-dalton doublet into several differently charged species.

Amino Acids↗
PubMed1978

Processing of 60,000-dalton sarc gene protein synthesized by cell-free translation.

In this report we show that antiserum prepared against the Mr60,000 transformation-specific antigen of Rous sarcoma virus immunoprecipitates both the Mr60,000 and Mr 25,000 transformation-specific proteins that are synthesized by cell-free translation of virion RNA; however, in the cell-free system the Mr 60,000 protein appears to be synthesized as a precursor that is approximately Mr 2000 larger than the [35S]-methionine-labeled protein immunoprecipitated from Rous sarcoma virus-infected cells. Peptide mapping of the cell-free translation product and of this cellular protein has confirmed that they are structurally related to one another. The addition of membrane vesicles to the reticulocyte lysate system during translation specifically cleaves a Mr 2000 segment from the Mr 60,000 protein so that it comigrates with the cellular species. Secretory proteins and probably at least some integral membrane proteins are synthesized with short hydrophobic signal sequences at their NH2 terminus. Two facts suggest that the segment lost from the Mr 60,000 transformation-specific protein is a signal sequence: (i) the membrane vesicles process the Mr 60,000 protein only during translation, and (ii) the processed protein is sequestered by the vesicles.

Amino Acid Sequence↗
PubMed1977

Initiation of synthesis of messenger RNA of deoxynucleotide kinase by oligoribonucleotides.

The effects of nucleoside triphosphates and oligoribonucleotides on the initiation of synthesis of messenger RNA of the T4 phage-specific enzyme, deoxynucleotide kinase, have been studied. The procedure involved incubation of T4 DNA, purified RNA polymerase from Escherichia coli, and selected nucleotide compounds during a brief period to permit initiation of RNA synthesis. Further initiation was arrested by the addition of ribampicin, and completion of the transcription of the newly initiated RNA was permitted to take place in the presence of the full complement of nucleoside triphosphates. After translation of the messenger RNA into phage-specific enzymes, the measured activities of the latter whe first incubation period. The effectiveness of individual nucleoside triphosphates, when present singly or in combination during the initiation period, was compared to that when all four nucleoside triphosphates were available. ATP alone was extremely effective as an initiator of the synthesis of the messenger RNA for deoxynucleotide kinase. The addition of UTP to ATP not only enhanced the magnitude of initiation but also affected the kinetics of ATP interaction with T4 DNA and RNA polymerase during the initiation period. Several oligoribonucleotides including a series ApA to ApApApA, UpU to UpUpUpU, and the heteropolymers, Ap1pU and ApApApU, were tested as initiators of kinase mRNA synthesis. A sequence of nucleotides in the promoter region of T4 DNA for the deoxynucleotide kinase gene has been proposed as a result of these experiments.

Adenosine Triphosphate↗