Search PubMed⌕ Search

Biomedical subjects

H M Goodman

Publications and source records attributed to H M Goodman.

At least 109 records · Page 6Linked to original sources
PubMed1990

Effect of hypophysectomy on growth hormone receptor gene expression in rat tissues.

Two cDNA probes derived from the nucleic acid sequence for the rabbit GH receptor were used to study RNA samples from normal and hypophysectomized (hypox) rat tissues by Northern analysis. Results obtained with a probe that contained a nucleotide sequence corresponding to part of the extracellular domain of the GH receptor indicated that rat liver, gastrocnemius muscle, and epididymal fat each contain a 4.4-kilobase (kb) message and one or more shorter messages that appear to be homologous to the rabbit GH receptor message. The other probe, which contained a nucleotide sequence that corresponds to the intracellular domain of the GH receptor, detected only one 4.4-kb message in these rat tissues. These results suggest that rat tissues may synthesize several forms of the GH receptor, but only one form that contains a region homologous to the intracellular domain of the rabbit liver GH receptor. Hypophysectomy increased the abundance of the 4.4-kb message 5-fold in muscle and reduced it by a factor of 2 in adipose tissue. No significant difference was seen between GH receptor message levels of normal and hypox rat liver when the results were expressed as a fraction of the total RNA. The level of the beta-actin message was also measured in liver, muscle, and fat from normal and hypox rats. No significant differences were found when the message levels in normal rats were compared to those for the corresponding tissue in hypox rats. When normalized to the beta-actin message levels, a significant increase was seen in the relative amount of the GH receptor mRNA in muscle and liver of hypox rats. The increased levels of the GH receptor message in muscle and liver and the simultaneous decreased level in fat suggest that GH receptor synthesis may be regulated selectively in these tissues by hormonal factors that are altered by hypophysectomy.

Adipose Tissue↗
PubMed1990

Refractoriness to the insulin-like effects of growth hormone depends upon calcium.

GH produces an acute but transient insulin-like response in adipocytes that have been deprived of GH for at least 3 h. The insulin-like response is followed by a period of refractoriness during which a second insulin-like response to GH cannot be elicited. These studies were undertaken to evaluate the role of calcium in the insulin-like response and refractoriness. Methionyl human GH (hGH) (100 ng/ml) increased the incorporation of D-[3-3H] glucose into lipid by 50-100% in fat cells that preincubated for 3 h without hormone and usually by less than 10% in fat cells that were made refractory by exposure to 100 ng/ml of hGH in the first hour of incubation. Insulin (100 microU/ml) increased lipogenesis by 3-5-fold whether fat cells were sensitive or refractory to GH. To determine whether calcium plays a role in either the insulin-like response to GH or the refractory phenomenon, we examined the effects of trifluoroperazine (20 or 50 microM) and calmidazolium (1.0 microM) which block calmodulin, as well as verapamil (30 microM), which blocks calcium channels. These agents did not interfere with stimulation of incorporation of D-[3-3H]glucose into lipid by GH (100 ng/ml) or insulin (100 microU/ml) but restored sensitivity to the insulin-like effects of GH in otherwise refractory cells. When freshly isolated, and hence refractory, cells were incubated for 1 h in calcium-free medium that contained 0.5 mM EGTA, GH stimulated lipogenesis by 30% (P less than 0.001) even though the response to insulin was markedly decreased in the absence of calcium. Conversely, when added to sensitive cells in the presence of normal extracellular Ca2+ concentrations during the 4th h of incubation, the calcium ionophore, A23187 (1.0 microM) produced an apparent refractoriness to the insulin-like effect of GH but had no effect on the response to insulin. The data indicate that maintenance of refractoriness to the insulin-like action of GH depends upon a calcium-calmodulin-sensitive process and suggest that, in producing refractoriness, GH may increase availability of intracellular calcium perhaps by increasing the rate of calcium entry into adipocytes. Since changes in responsiveness to GH were not paralleled by changes in responsiveness to insulin, it is suggested that the calcium dependent process responsible for refractoriness must lie at an early step in the signaling pathway.

Adipose Tissue↗
PubMed1990

Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture.

The rat gene encoding phenylethanolamine N-methyltransferase (PNMT) was cloned and a consensus sequence for a glucocorticoid response element (GRE) was found at -513 bp, 5' to the transcriptional start site. In order to define the function of this element, fusion genes containing the PNMT promoter and a chloramphenicol acetyltransferase (CAT) reporter gene were constructed. These constructs did not express after transfection into any of 7 continuous cell lines, none of which endogenously produce PNMT. A system for transfecting chromaffin cells in primary culture was therefore devised using constructs containing 200 bp of the proenkephalin (ENK) promoter, whose expression characteristics are well known. pENK beta GAL-1, containing the ENK promoter with a lac Z reporter, was introduced into these cells and beta-galactosidase activity was visualized in situ. Approximately 90% of cells transfected were chromaffin; transfection efficiency was 5%. High levels of CAT activity were measured in chromaffin cells transfected with pENKAT12, possessing a CAT reporter. In contrast to tumor cell lines, pENKAT12 induction in these cells by forskolin and phorbol esters did not require a phosphodiesterase inhibitor. In this chromaffin system, both basal and regulated expression of the PNMT fusion genes were detected. Dexamethasone (dex) induced expression of pPNMT3000 and pPNMT900, containing the putative GRE and 3000 bp or 863 bp of PNMT promoter sequence, 4- to 10-fold. Expression of pPNMT300 and pPNMT100, which lack the GRE and contain 273 bp or 99 bp of PNMT promoter sequence, was unaffected by dex. Addition of the PNMT region spanning -490 to -863 bp conferred full dex responsiveness to a thymidine kinase promoter. Deletion of the putative GRE sequence by site-directed mutagenesis abolished the dex response. These data identify the sequence at -513 bp in the rat PNMT gene as a functional, positively acting GRE. Primary cultures of bovine chromaffin cells provide a biologically relevant expression system for transcriptional studies of catecholamine genes and their related neuropeptides.

Animals↗
PubMed1989

Purification and partial characterization of 1-aminocyclopropane-1-carboxylate synthase from tomato pericarp.

1-Aminocyclopropane-1-carboxylate synthase was purified 5000-fold from LiCl-induced tomato fruit slices by conventional and high-performance liquid chromatography. The final preparation was estimated to be between 25% and 50% pure. Two-dimensional gel electrophoresis indicates that 1-aminocyclopropane-1-carboxylate synthase activity is associated with a 45-kDa polypeptide, with a pI of 5.8 +/- 0.2. The enzyme is inactivated both by its substrate, S-adenosyl-L-methionine (AdoMet) and by one of its products, 1-aminocyclopropane-1-carboxylate. Due to the extremely low abundance of the protein it was necessary to scale up the extraction in order to obtain reasonable amounts for sequence analysis. Therefore, 200 kg tomatoes were extracted on semi-industrial scale and 1-aminocyclopropane-1-carboxylate synthase purified. This yielded approximately 150 micrograms enzyme.

Amino Acid Sequence↗
PubMed1989

Insulin regulation of the activity and phosphorylation of branched-chain 2-oxo acid dehydrogenase in adipose tissue.

The activity of the intramitochondrial branched-chain 2-oxo acid dehydrogenase (BCDH), like that of pyruvate dehydrogenase, is regulated, at least in part, by interconversion between the active dephosphorylated enzyme and its inactive phosphorylated form. The stimulatory effect of insulin on BCDH activity was compared with its effect on phosphorylation of the enzyme. Intact tissues were incubated in the presence or the absence of insulin, and then mitochondria were isolated and disrupted before assaying for enzyme activity or estimating the extent of enzyme phosphorylation. Tissues were incubated in either the presence or the absence of leucine, which also stimulated BCDH activity up to 10-fold. Insulin (1 munit/ml) doubled the activity of BCDH in the absence and in the presence of leucine. Together, 1 mM-leucine and insulin appeared to stimulate BCDH activity fully. Phosphorylation of BCDH was estimated indirectly by measuring the incorporation of 32P into phosphorylation sites that remained unesterified after preparing mitochondrial extracts under conditions that preserved the effect of insulin on BCDH activity. Increased incorporation of 32P in these experiments implies decreased phosphorylation in situ when tissues were incubated with insulin and leucine. In the absence of leucine, little incorporation of 32P into BCDH was detected. In the presence of leucine, however, incorporation of 32P into BCDH was markedly increased, and insulin increased 32P incorporation still further. The results support the hypothesis that leucine and insulin both stimulate the activity of BCDH by promoting its dephosphorylation.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1989

Adenocarcinoma of the cervical stump.

Sixteen women with adenocarcinoma of the cervical stump were treated over a 15-year period. The median survivals of 40 months for stage IB and 17 months for stages II and III were significantly worse compared with those for patients treated for cervical adenocarcinoma of the intact uterus or squamous carcinoma of the cervical stump. The poor results were due to both local and distant failure. Implications regarding tumor radiosensitivity and adjuvant therapy in these high-risk patients are discussed.

Adenocarcinoma↗
PubMed1989

Adenocarcinoma of the uterine cervix: prognostic factors and patterns of recurrence.

Survival data, prognostic factors, and patterns of recurrence were analyzed for 70 women with adenocarcinoma of the uterine cervix treated between 1968 and 1982. The 5-year survival rates for stages I, II, and III-IV were 82, 90, and 38%, respectively. Control of pelvic tumor was achieved in 82, 80, and 12.5% of cases of stage I, II, and III-IV disease, respectively. When radiation therapy techniques alone were employed, pelvic control was achieved in 100% of stage I and 75% of stage II cases. Tumor grade was an important prognostic factor in stage I disease, with 92% of patients with grade 1 and 2 lesions surviving 5 years, in contrast to 68% of patients with grade 3 lesions (P less than 0.05, log rank test).

Adenocarcinoma↗
PubMed1989

Coregulation of dihydrofolate reductase and thymidylate synthase in overproducer cell lines of wild carrot.

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities are associated with a 285,000 molecular weight enzyme complex in carrot (Daucus carota L.). Selection for methotrexate (MTX) resistance by stepwise increase of the concentration of MTX results in a high frequency adaptation to MTX with little or no significant increase in DHFR activity. However, when as a second step following MTX selection a specific inhibitor of TS, 5-fluoro-2-deoxyuridine was used, DHFR overproducer lines were obtained. The overproduction phenotype of the lines was almost completely lost after 8 weeks of growth in the absence of selection pressure. Although DHFR and TS are independent gene products, their activities increase in proportion ( approximately 20-fold) in the overproducer lines. This strongly suggests that DHFR and TS are not only functionally and physically linked in the same enzyme complex, but also are coregulated. These cell lines resemble the MTX-induced DHFR overproducer amplified cell lines of mammalian origin in their mode of selection, high frequency of appearance, elevated enzyme activity, and increased specific mRNA levels.

Journal Article↗
PubMed1989

Different growth hormone-receptor interactions mediate insulin-like and lipolytic responses of rat adipose tissue.

The GH receptor in adipocytes is a glycoprotein that has a half-life of less than 1 h. After 2 h of treatment with the alkaloid swainsonine, which interferes with carbohydrate processing, virtually all of the GH receptors on the surface of adipocytes are replaced with receptors whose carbohydrate side-chains are incomplete. We examined the effects of swainsonine on the responsiveness of adipose tissue to GH to determine whether these receptors, which bind GH normally, retain biological competence. In the concentration range of 100-300 ng/ml human (h) GH rapidly evokes insulin-like responses in adipose tissue or adipocytes that have been deprived of GH for at least 3 h. hGH, at concentrations ranging from 1-10 ng/ml, also increases lipolysis after a delay of at least 2 h. Pretreatment with 50 micrograms/ml swainsonine failed to influence insulin-like responsiveness to hGH, as judged by increased glucose oxidation, but nearly completely abolished the lipolytic response. Pretreatment with swainsonine, however, did not reduce lipolysis in response to isoproterenol, suggesting that signal transmission rather than the lipolytic apparatus per se had been affected. To determine whether the same receptors mediate lipolytic and insulin-like responses, the binding properties of hGH were compared to those of Da1, a chemically modified form of hGH, whose insulin-like potency is reduced relative to its lipolytic potency. Da1 and hGH were equipotent in promoting lipolysis and had an ED50 of about 3 ng/ml, but hGH was at least 6 times as potent as Da1 in promoting glucose oxidation (ED50 of 65 vs. 400 ng/ml). Scatchard plots of both Da1 and hGH binding data were linear, consistent with a single class of binding sites whose affinity for hGH was about 3.5 times higher for hGH than Da1. hGH and Da1 both produced half-maximal stimulation of glucose oxidation when about 90% of the GH receptors were occupied. In contrast, half-maximal lipolysis was produced by Da1 when 8% of GH receptors were occupied, but 21% occupancy was required for a similar effect of hGH. If a subclass of GH receptors mediates lipolysis, it is likely to comprise 10% or less of the total receptor population.

Adipose Tissue↗
PubMed1989

Glucocorticoid regulation of human growth hormone expression in transgenic mice and transiently transfected cells.

A mouse metallothionein-I/human growth hormone fusion gene was microinjected into fertilized mouse eggs, the embryos were implanted into pseudopregnant foster mothers, and the offspring analysed. Five of twenty-six mice born after one series of injections contained from one to eight copies of the fusion gene stably integrated into their genomes and had human growth hormone in their serum. When several of these transgenic mice and transgenic offspring were treated with glucocorticoids, serum growth hormone levels were elevated from 1.5- to 6.3-fold. A fourfold induction in fusion gene mRNA in the liver of one of the five mice was also observed after treatment with glucocorticoids. When the fusion gene was transiently transfected into mouse L cells, dexamethasone caused a three- to fourfold induction of fusion gene mRNA and secreted human growth hormone. A deletion analysis of regulatory elements required for inducibility in L cells shows that DNA sequences responsible for the observed inductions are located within the transcribed region of the human growth hormone gene. However, a previously described glucocorticoid receptor binding site in the first intron of the gene is not required for response to the hormone.

Animals↗
PubMed1988

Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription.

Synthesis of the endogenous opioid precursor, proenkephalin, is regulated by neurotransmitters and membrane depolarization. These events act through second messenger dependent signal transduction pathways via a short inducible DNA enhancer to regulate transcription of the proenkephalin gene. Two DNA elements located within this enhancer are essential for the transcriptional response to cAMP and phorbol ester. Inactivation of either element by mutation or by alteration of their stereospecific alignment eliminates inducible enhancer activity. The promoter distal element, ENKCRE-1, in the absence of a functional adjacent ENKCRE-2 element, has no inherent capacity to activate transcription. However, in the presence of a functional ENKCRE-2 element, this element synergistically augments cAMP and phorbol ester inducible transcription. The promoter proximal element, ENKCRE-2, is essential for both basal and regulated enhancer function. Four different protein factors found in HeLa cell nuclear extracts bind in vitro to the enhancer region. ENKTF-1, a novel enhancer binding protein, binds to the DNA region encompassing ENKCRE-1. The transcription factors AP-1 and AP-4 bind to overlapping sites spanning ENKCRE-2, and a fourth transcription factor, AP-2, binds to a site immediately downstream of ENKCRE-2. The binding of ENKTF-1 to mutant ENKCRE-1 sequences in vitro correlates with the in vivo inducibility of the mutant elements suggesting that ENKTF-1 acts in combination with factors that recognize the ENKCRE-2 domain to regulate cAMP inducible transcription. Together, the two DNA elements, ENKCRE-1 and ENKCRE-2 and the protein factors with which they interact, play a critical role in the transduction and reception of signals transmitted from cell surface receptors to the proenkephalin nuclear transcription complex.

Cyclic AMP↗
PubMed1988

Intron existence predated the divergence of eukaryotes and prokaryotes.

Nucleotide sequences for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) from Arabidopsis thaliana were determined. Comparison of nucleotide sequences indicates that the divergence of chloroplast and cytosolic GAPDH genes preceded the divergence of prokaryotes and eukaryotes. In addition, some intron-exon junctions are conserved among GapB, GapC, and chicken GAPDH genes. These results provide evidence at the molecular level to support the idea that introns existed before the divergence of prokaryotes and eukaryotes.

Animals↗
PubMed1988

Transamination and oxidation of leucine and valine in rat adipose tissue.

Leucine was oxidized by rat adipose tissue at a rate which was not limited by the activity of branched chain amino acid transaminase since high concentrations (10 mM) of [1-14C]leucine and its transamination product, alpha-keto[1-14C]isocaproate, were oxidized at similar rates. Despite the apparent abundance of transaminase activity, however, [1-14C]valine was oxidized at only 10 to 25% of the rate of its transamination product, alpha-keto[1-14C]isovalerate. The net rate at which [1-14C] valine was transaminated by intact tissues was estimated as the sum of the rates of 14CO2 production and alpha-ketoiso[1-14C]valerate release into the medium. Transamination did not limit the rate of valine oxidation since valine was transaminated 3 times as fast as it was oxidized. The rate of valine transamination increased 18-fold when its concentration was raised 100-fold, but the fraction of [1-14C]valine oxidized to 14CO2 remained constant over the range of incubation conditions studied. The oxidation/transamination ratio for leucine was also constant and exceeded the oxidation/transamination ratio for valine unless valine oxidation was stimulated, either by the addition of glucose or leucine. Stimulation of valine oxidation did not increase its transamination but reduced the rate at which alpha-ketoisovalerate was released from the tissue. The faster oxidation of alpha-ketoisocaproate than of alpha-ketoisovalerate may be due to the activation of branched chain alpha-keto acid dehydrogenase by alpha-ketoisocaproate, but the alpha-keto acid oxidation rates do not fully account for the faster transamination of leucine than of valine.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1988

Differential light regulated expression of nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenase in Nicotiana tabacum.

When tobacco (Nicotiana tabacum) plants were transferred from the dark to continuous white light, the steady-state mRNA levels transcribed from the nuclear genes encoding chloroplast (GapA and GapB) glyceraldehyde-3-phosphate dehydrogenase increased at least 30- to 50-fold, while the mRNA level for the cytosolic enzyme (GapC) increased only 10-fold. Kinetic analyses show that the rates of mRNA accumulation for GapA and GapB are identical reaching steady-state levels after 24-48 h in light. In contrast, mRNA accumulation for the GapC gene shows a completely different kinetic pattern, accumulating much faster than that of GapA and GapB. These results suggest that expression of GapC and GapA/B genes are controlled by different light regulated mechanisms and nuclear run-on analyses suggest that these effects are primarily due to increased transcription.

Journal Article↗
PubMed1988

A new locus (NIA 1) in Arabidopsis thaliana encoding nitrate reductase.

We have isolated two nitrate reductase genes and their corresponding cDNAs from Arabidopsis thaliana. Sequences of the two cDNAs, when compared to a sequence of a barley cDNA clone, confirm their identity as nitrate reductase clones and show that they are closely related. The two genes have been mapped using restriction fragment length polymorphisms; gNR2 is close to the previously identified chl-3 locus and is probably identical to it, while gNR1 maps to a new locus (NIA1) on chromosome 1, near gl-2.

Amino Acid Sequence↗
PubMed1988

Sequence determination of the mumps virus HN gene.

The hemagglutinin-neuraminidase protein (HN) of mumps virus was purified by immunoaffinity chromatography and fragmented by the combined action of CNBr and trypsin. The resulting peptides were separated by HPLC and sequenced by automated Edman degradation. Using this HN-specific amino acid sequence data, a degenerate oligonucleotide was produced and subsequently used to screen a mumps virus cDNA library to isolate HN-specific clones. The complete nucleotide sequence of the HN gene was determined. The monocistronic HN mRNA is approximately 1900 nucleotides long and encodes a single open reading frame of 582 amino acids. The HN protein has a unique hydrophobic stretch of 19 amino acids at its N-terminus that apparently anchors the protein in the viral envelope. A comparison of the mumps virus HN protein sequence with the sequences of the other known paramyxovirus HNs indicates that mumps virus is most closely related to SV-5, followed in decreasing order by NDV, parainfluenza virus 3, and Sendai virus.

Amino Acid Sequence↗