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Biomedical subjects

J L Roberts

Publications and source records attributed to J L Roberts.

At least 109 records · Page 6Linked to original sources
PubMed1994

Regulation of gonadotropin-releasing hormone gene expression by the excitatory amino acids kainic acid and N-methyl-D,L-aspartate in the male rat.

The glutamate analogs N-methyl-D,L-aspartate (NMA) and kainic acid are involved in the regulation of GnRH and LH release in mammals. It has recently been reported that the increase in GnRH release induced by NMA is accompanied by an increase in GnRH mRNA levels, as measured by in situ hybridization. In the present study we assessed the effects of NMA and kainic acid on cytoplasmic mRNA levels using the more quantitative solution hybridization/RNase protection assay. To address the mechanism responsible for these mRNA changes, we also examined changes in heteronuclear RNA transcripts as a reflection of gene transcription. Adult male rats were implanted with a jugular catheter, and 1-2 days later, NMA (14 mg/kg BW), kainic acid (2 mg/kg BW), both NMA and kainic acid, or saline vehicle were injected through the cannula. Rats were killed 15 min or 1 h later by decapitation, blood samples were collected for RIA of LH, brains were removed, and the preoptic area was dissected and frozen. Cytoplasmic and nuclear RNA were extracted and assayed separately by RNase protection assay. Treatment with NMA or NMA plus kainic acid resulted in significantly elevated cytoplasmic mRNA levels 15 min and 1 h later compared to saline control values, with no differences between the two drug treatments observed. Kainic acid stimulated mRNA levels 1 h, but not 15 min after injection. Nuclear RNA transcripts were unaffected by all drug or vehicle treatments. As nuclear primary transcript levels presumably reflect GnRH gene transcription, and these levels are unaltered, the present study indicates that the regulation of GnRH gene expression by excitatory amino acids occurs at a posttranscriptional level. The increase in cytoplasmic GnRH mRNA levels also does not result from an increased translocation of the relatively large nuclear GnRH mRNA pool into the cytoplasm, because nuclear GnRH mRNA levels are also unchanged. Therefore, the elevation of cytoplasmic mRNA levels after excitatory amino acid treatment is probably due to an increase in mRNA stability.

Animals↗
PubMed1994

c-myc, c-fos, and c-myb gene expression in human pituitary adenomas.

The possible roles of certain oncogenes in the development of pituitary tumors has not been investigated. We have examined the expression of c-myc, c-fos, and c-myb in a number of human pituitary tumors by ribonuclease protection assays, as these oncogenes have been implicated to have roles in the pathogenesis of other human tumors (12, 13, 15, 16). In several tumors examined (9 of 30) c-myc was expressed at levels 4-9 times greater than the level detected in normal postmortem pituitary. Although a larger percentage of negative immunohistochemical-staining tumors overexpressed c-myc, c-myc over-expression was not limited to this group of tumors. c-Fos was overexpressed in 1 of 30 tumors examined at a level 5.8-fold higher than that detected in normal postmortem pituitary. This tumor stained positive for ACTH by immunohistochemistry and was considered highly aggressive, demonstrating invasion beyond the sella turcica; however, when other ACTH-staining and invasive pituitary tumors were examined, no abnormality in the expression of c-fos was detected. In 30 tumors, c-myb was expressed at approximately the same level as that detected in normal postmortem pituitary. We conclude that c-myc is overexpressed in a subgroup of pituitary tumors and that this overexpression occurs broadly among the different groups of immunohistochemical-staining tumors. c-Fos overexpression appears to be much less common in pituitary tumors and does not necessarily correlate with the ability of the tumor to become invasive. c-Myb does not appear to have a role in the pathogenesis of pituitary tumors.

Adenoma↗
PubMed1994

Characterization of a corticotropin-releasing hormone-responsive element in the rat proopiomelanocortin gene promoter and molecular cloning of its binding protein.

A corticotropin-releasing hormone (CRH) and cAMP-responsive region (-236/-133) in the rat POMC gene promoter previously reported to confer CRH/cAMP responsiveness to heterologous reporter constructs has been characterized. DNAse footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cells. When these individual DNA elements were separately tested in heterologous reporter constructs for CRH induction, only one element, designated PCRH-RE (POMC CRH responsive element, -171/-160) was found to give strong CRH stimulation (5- to 7-fold). This element appears novel as to the possible binding factors, although it has homology to the mouse metallothionein metal regulatory element. Gel shift analyses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stimulation of retarded nucleoproteins following CRH stimulation, suggesting that the possible binding factor(s) may mediate transcriptional regulation at this site. The activity of PCRH-RE binding protein was inhibited by divalent cations, with Cu2+ and Cd2+ being most effective; Zn2+ had no effect, indicating that this binding factor(s) is functionally distinct from the metallothionein metal regulatory element binding protein. A 2.6 kilobase cDNA clone encoding a protein (PCRH-REB-1) binding to this element was isolated by Southwestern screening of an AtT20 expression library with radiolabeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the sequence corresponding to the entire coding region of the protein (PCRH-REB), which proved to be identical to a recently described DNA binding protein of the replication factor C complex, mRFC140/Mouse Southwestern. Primer extension and Northern blot analysis revealed that the size of the full length mRNA is about 4.9 kilobases. PCRH-REB mRNA expression is not restricted to corticotrophs but is present in a broad tissue distribution as evaluated by reverse transcription polymerase chain reaction analysis. A bacterially expressed beta-galactosidase-PCRH-REB-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive element was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCHR-REB protein sequence presents several interesting motifs: one p-Loop motif (ATP binding site), nine protein kinase A phosphorylation sites (implying a possible role in responding to the CRH-induced cAMP signal), and regions of homology to proteins involved in DNA replication and repair. PCRH-REB is, therefore, a potential transacting factor binding to a major CRH-responsive element in the POMC promoter.

Animals↗
PubMed1993

(2,3)-alpha-Methylenepenams: synthesis and in vitro activity.

A series of alpha-methylene penicillins was synthesized and SAR were studied. The alpha-isomers were found to be chemically reactive and biologically active in contrast to the beta-isomers. In addition, the alpha-isomers have broader spectrum of in vitro activity than the corresponding penicillins. Generally, the alpha-isomers are more active against gram-negative bacteria than the corresponding penicillins, but slightly weaker in potency towards gram-positive organisms.

Anti-Bacterial Agents↗
PubMed1993

CT of congenital and acquired abnormalities of the spleen.

Computed tomography (CT) is an excellent imaging modality for demonstrating the size, shape, and position of the spleen, as well as for depicting intrasplenic pathologic features. However, it has limited value in the diagnosis of splenic involvement by lymphoma. Greater accuracy in diagnosing splenic lymphoma may be obtained by demonstrating adenopathy in the splenic hilum or by showing focal splenic defects, in addition to splenomegaly. In discussing the normal anatomy of the spleen, congenital variations (eg, accessory spleen, polysplenia, asplenia, and wandering spleen), and acquired abnormalities such as those resulting from trauma, infection, infarction, cysts, and neoplasms, this article provides evidence that CT is valuable in showing a wide variety of splenic variations and abnormalities while simultaneously allowing evaluation of the remaining intraabdominal structures. The cases presented demonstrate both the value and limitations of CT in the examination of patients with congenital and acquired abnormalities of the spleen.

Humans↗
PubMed1993

CT in abdominal and pelvic trauma.

Computed tomography (CT) is valuable in the evaluation of the abdomen and pelvis in victims of blunt trauma and stab wounds to the back if their condition is stable or if results of their physical examination are unreliable (eg, due to altered mental status). The appearance of blood at CT depends on window width, hematocrit, physical state of blood, use of intravenous contrast material, and attenuation of adjacent organs. Rapid infusion of contrast material improves visualization of hematoma. CT is most useful in evaluating injuries to the spleen, liver, pancreas, kidney, duodenum, and small and large bowel. Although CT can be accurate in demonstrating lacerations and hematoma, radiologists must beware of pitfalls in the CT diagnosis of splenic injuries (normal lobulation or cleft within the spleen, elongation of the left hepatic lobe, adjacent unopacified bowel loop, previous splenic infarct), hepatic injuries (beam-hardening artifact from adjacent ribs, air-contrast level in the stomach), and pancreatic injuries (streak artifacts from nasogastric tubes, folding of the pancreas, misinterpretation of adjacent structures, pancreatic contusion or hematoma, intrinsic elasticity of the pancreas). Delayed scanning or scanning with slow infusion rates may also obscure hematoma. Knowledge of the various appearances of the posttraumatic abdomen and pelvis on CT scans is essential for prompt and effective treatment of patients.

Abdominal Injuries↗
PubMed1993

Ovine anterior pituitary proopiomelanocortin gene expression is not increased by ACTH secretagogues in vitro.

Recent reports have demonstrated that the secretion of ACTH from sheep anterior pituitary primary cultures is markedly stimulated by arginine vasopressin (AVP) but not by CRF, and that AVP-stimulated ACTH secretion is potentiated by CRF. It has also been reported that AVP increases total ACTH content (secreted plus intracellular ACTH), suggesting that AVP stimulates POMC biosynthesis in the ovine anterior pituitary. These observations differ from the rat, in which CRF is the most potent of the ACTH-releasing factors and the only ACTH secretagogue which stimulates POMC gene expression and biosynthesis. The second messenger pathways which mediate CRF- and AVP-stimulated ACTH release (protein kinase A and protein kinase C, respectively) are the same in sheep and rat corticotrophs. The present studies were undertaken to determine if ovine POMC gene expression, unlike the rat POMC gene, is stimulated by AVP via the protein kinase C pathway. A 295 base pair portion of the ovine POMC gene was isolated using polymerase chain reaction and sequenced. Ovine POMC messenger RNA (mRNA) levels were quantitated using this partial complementary DNA clone in a solution hybridization/nuclease protection assay with cytoplasmic RNA from sheep anterior pituitary primary cultures which had been treated with various combinations of ACTH secretagogues or with glucocorticoids for 18 h. Treatment with AVP, alone or with CRF, greatly increased total and secreted ACTH levels; however, the amount of POMC mRNA in these cells was not significantly increased. Treatments which stimulated secretion to a lesser extent and did not alter total ACTH levels (CRF alone, cAMP alone, or with phorbol ester) were associated with a decrease in POMC mRNA levels relative to untreated cells. Glucocorticoid treatment decreased both total ACTH and POMC mRNA levels. Taken together, the data demonstrate a lack of secretagogue-induced stimulation of POMC mRNA levels concomitant with increased total ACTH levels, an unexpected result given the close association between secretion of POMC-derived peptides and POMC gene expression in other mammalian corticotroph systems.

Adrenocorticotropic Hormone↗
PubMed1993

Identification of a major up-stream transcription start site for the human progonadotropin-releasing hormone gene used in reproductive tissues and cell lines.

Previous studies suggested that GnRH gene transcripts in human tissues may be derived from an upstream transcriptional start site in addition to the well characterized hypothalamic start site. To resolve this issue we characterized the transcriptional start sites of the human GnRH gene in a human placental tumor cell line (JEG) and a human breast tumor cell line (MDA). Using primer extension and reverse transcription-polymerase chain reaction (RT-PCR) assay, we identified a discrete upstream transcriptional start site 579 bases up-stream from the hypothalamic site in both JEG and MDA cell lines. The up-stream start site lacks the TATA and CAAT elements often present in RNA polymerase-II promoters, but contains the sequence GGTCTTGCT located 84 bases 5' to the up-stream start site similar to other genes that lack TATA/CAAT boxes. RT-PCR quantitation shows that the up-stream start site is the major transcriptional start site, representing 74% of the cytoplasmic transcripts in JEG cells and 67% in MDA cells. Supporting this observation, transfection assay using a human GnRH promoter/luciferase reporter gene construct containing only the up-stream transcription start site has a higher level of transcriptional activity than the human GnRH promoter/luciferase reporter construct containing only the down-stream start site. A high relative abundance (approximately 45%) of total GnRH mRNAs were also found in the nucleus of both cell lines, which did not appear to be a consequence of the nuclear/cytoplasmic fractionation procedure. To determine if this upstream start site was used in normal GnRH-expressing human tissues, we analyzed RNA from a variety of postmortem/surgical procedure tissue samples. RT-PCR analysis together with Southern blot analysis demonstrated the presence of GnRH mRNA in human pituitary, cerebral cortex, testes, ovary, and mammary gland for the first time as well as verified GnRH gene expression in hypothalamus and placenta. The up-stream transcriptional start site is used only in reproductive tissues, such as placenta, testes, ovary, and mammary gland, suggesting tissue-specific regulation at this site.

Animals↗
PubMed1993

Haploidentical bone marrow stem cell transplantation in human severe combined immunodeficiency.

From May 1992 to March 1993, 50 infants with severe combined immunodeficiency (SCID) were given bone marrow transplants at Duke University Medical Center. None received chemotherapy for conditioning or for graft-versus-host disease (GVHD) prophylaxis. Forty-one received haploidentical parental marrow depleted of T cells by soybean lectin and sheep red blood cell resetting, and nine received HLA-identical marrow. Forty (80%) survived from 1 week to almost 11 years posttransplantation, including nine of nine (100%) HLA-identical marrow recipients and 31 of 41 haploidentical recipients. T-cell function was present within 2 weeks after transplantation of unfractionated HLA-identical marrow, but not until 3 to 4 months after T-cell-depleted haploidentical marrow stem cells. All 37 patients who are more than 4 months posttransplantation have good T-cell function, and all but one have 100% donor T cells. B-cell function developed slowly or not at all in some recipients of haploidentical marrow. Fourteen (four HLA-identical and 10 haploidentical recipients) have some donor B cells; 19 patients are receiving intravenous immune globulin (IVIG) therapy.

B-Lymphocytes↗
PubMed1992

Beta-adrenergic stimulation of cFOS via protein kinase A is mediated by cAMP regulatory element binding protein (CREB)-dependent and tissue-specific CREB-independent mechanisms in corticotrope cells.

Catecholamines stimulate proopiomelanocortin (POMC) gene expression in corticotrope cells, but the molecular mechanisms of these effects are not known. While beta-adrenergic receptors stimulate the protein kinase A (PKA) system, the POMC promoter does not have classical cAMP-response elements (CREs). Therefore, we investigated the induction of the c-fos protooncogen, previously shown to increase POMC transcription in AtT20 cells. In this corticotrope-derived cell line, we show that activation of beta-receptors with isoprenaline (Iso) induces a transient rise in c-fos mRNA levels. Gel mobility shift assays with a labeled AP1 consensus sequence (TGACTCA) showed induction of specific binding activity after Iso treatment. Cotransfection experiments with dominant inhibitory PKA mutants and reporter genes containing c-fos promoter sequences showed that c-fos induction by Iso is entirely dependent on a functional PKA activity. Furthermore, we show that beta-receptor induction of c-fos in corticotrophs is mediated by at least two distinct cAMP-responsive sequences. cAMP regulatory element binding (CREB)-dependent induction is observed on the CRE located at -60 bp on the c-fos promoter. A region located in the vicinity of the dyad symetry element (-290) is also found to mediate tissue-specific cAMP induction. Transcriptional activation by this site, although sensitive to PKA antagonism, is not blocked by CREB mutants.

Animals↗
PubMed1992

Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation and reversal of hypogonadism in mutant mice.

GT1 is an immortalized cell line that synthesizes and secretes the neurohormone gonadotropin-releasing hormone (GnRH). We have placed these cells into the brains of adult mutant hypogonadal (hpg) mice, which lack a functional GnRH gene, to determine whether such cells could differentiate in situ and support gonadal development. Immunocytochemical detection of GnRH revealed that these cells migrated widely in the central nervous system and elaborated axonal processes which on rare occasion projected to the normal target, the median eminence. Using a battery of antibodies, we demonstrated that these cells could cleave the GnRH precursor and that the amidated decapeptide as well as other cleavage products were present. The presence of biologically active material and its appropriate secretion were further documented by gonadal growth in both males and females. The morphological differentiation of the GT1 cells correlated with the density of cells injected. Those remaining within the injection site and/or forming a tumor retained a simple, rounded or fibroblastic appearance. Those cells that migrated into the host away from such tumors assumed the simple fusiform shape of normal GnRH neurons with dendrites extending from one or both poles. When cell density was drastically reduced a much more complex dendritic arbor was elaborated. These data suggest that such cell lines can be useful in reversing genetic defects and in studying such processes as GnRH neuronal migration, axonal targeting, and cytological differentiation.

Animals↗
PubMed1992

Expression of Mls determinants in mice exhibiting the severe combined immunodeficiency (scid) mutation or X-linked immunodeficiency (xid) defect.

While Ig+ B cells appear to be the principal cell type expressing immunogenic minor lymphocyte stimulatory (Mls) determinants, both T cells and B cells are capable of mediating deletion of developing Mls-reactive thymocytes. In addition, levels of mouse mammary tumor proviral transcripts are increased after B or T cell stimulation, and expression of functional Mls determinants is augmented by activation of B cells. These findings suggest Mls determinants are present on B and T lymphocytes, and that activation of B and T cells augments Mls expression. In the present study, we wished to determine whether B and T cells were required for expression of Mls determinants by examining mice with severe combined immunodeficiency (SCID) containing no detectable Ig+ B cells or TCR+ T cells, as well as animals that expressed the X-linked immunodeficiency (xid) defect and lacked a subset of mature B cells. We found Mlsa-reactive V beta 6hi T cells were deleted from thymi of male (CBA/NxAKR/J)F1 xid mice, and that spleen cells from these animals stimulated anti-Mlsa mixed lymphocyte responses by unprimed B10.BR spleen T cells. In addition, Mlsc-reactive V beta 3hi AKR/J thymocytes and spleen T cells were deleted in AKR/J----SCID bone marrow chimeras, and spleen cells from SCID mice stimulated proliferation by an Mlsc-specific T cell clone. These results demonstrate that both xid mice and SCID animals express Mls determinants that mediate deletion of developing, Mls-responsive thymocytes and stimulate proliferation of mature, Mls-reactive T cells. Hence, mature B cells and T cells are not essential for Mls expression.

Animals↗
PubMed1992

Positive correlation between proopiomelanocortin and tyrosine hydroxylase mRNA levels in the mediobasohypothalamus of ovariectomized rats: response to estradiol replacement and withdrawal.

We have investigated putative dopaminergic regulation of opiomelanotropinergic activity in the arcuate/periarcuate mediobasohypothalamus (MBH) by assessing the changes in MBH tyrosine hydroxylase (TH; rate-limiting enzyme in catecholamine synthesis) and proopiomelanocortin (POMC; opiomelanotropin precursor) mRNA levels under conditions in which endogenous tuberinfundibular dopaminergic activity exhibits marked changes. Adult Sprague-Dawley rats were sacrificed at 09.00 and 15.00 h, and individual MBH POMC and TH cytoplasmic mRNA levels were simultaneously quantified by multiplex solution hybridization-RNase protection assay with protected fragments separated by polyacrylamide gel electrophoresis. In ovariectomized (OVX) rats treated for 3 days with low-dose estradiol (E2) implants (resulting in 18 +/- 4 pg E2/ml serum), the MBH levels of POMC and TH mRNAs were approximately 17 and 31% lower than those measured in OVX controls, respectively. In OVX rats implanted for 20 days with larger E2 implants (99 +/- 9 pg E2/ml serum), POMC and TH mRNA levels were approximately 29 and 41% lower than in OVX controls, respectively. Additional groups were exposed to the higher E2 dose for 20 days and then killed 10 or 20 days after removal of the E2 implant. In these rats, POMC mRNA levels rebounded to the same level seen in OVX controls, while TH mRNA levels even exceeded control values by 22-27%. TH and POMC mRNA levels did not change significantly between 09.00 and 15.00 h, except 10 days after removal of the E2 implants, when 09.00 h POMC mRNA levels were higher than the 15.00 h levels. MBH POMC and TH mRNA levels were positively correlated with each other within individual animals. This correlation is maintained when both POMC and TH mRNA levels are suppressed in response to both 3-day low-dose and 20-day high-dose E2 treatment. However, although rat MBH opiomelanotropinergic and tuberoinfundibular dopaminergic mRNA biosynthesis thus appear to be positively correlated, the coregulation or functional interactions of these two neuronal systems remain to be determined.

Amino Acid Isomerases↗
PubMed1992

Proopiomelanocortin gene expression in a distinct population of rat spleen and lung leukocytes.

Existence of proopiomelanocortin (POMC) messenger RNA (mRNA) and related peptides in extrapituitary sites has been demonstrated in immune cells, although the particular type of immune cell has been the source of considerable debate. Specifically, double labeling studies have shown that POMC peptide expressing cells in the spleen represent a subpopulation of red pulp macrophages, while splenic lymphocyte areas are POMC negative. In addition, it has also been reported that peripheral blood leukocytes express the POMC gene. Using a sensitive solution hybridization technique with a POMC exon-1 RNA probe, we detected 70 +/- 20 fg and 65 +/- 5 fg POMC mRNA per microgram total RNA in whole spleen and lung, respectively, approximately 20,000-fold lower concentrations than found in the neurointermediate lobe of the pituitary. The presence of nuclease protected full length exon-1 bands, rather than the 5' truncated POMC RNAs seen in many nonpituitary tissues, indicates transcription initiation at the normal pituitary POMC promoter site in lung and spleen. In order to localize POMC gene expression in these tissues we employed an in situ hybridization method. There was an intense signal in a small population of large mononuclear cells scattered throughout the splenic red pulp and lung parenchyma. In the lung, these cells were concentrated in the periarteriolar zone in a manner suggestive of migration from the intravascular lumen. These cells had a histomorphology suggestive of monocyte-macrophages. POMC mRNA was undetectable in the splenic white pulp and bronchus-associated lymphoid tissue, indicating an absence of POMC gene expression in splenic and lung lymphocytes. Immunocytochemical studies suggested that POMC-positive cells made up a subpopulation of cells expressing the rat monocyte-macrophage markers ED1 and ED2. Similarly, the distribution of Jenner-Giemsa stained monocyte-macrophages appeared to overlap with POMC positive cells. Studies with anti-rat beta-endorphin antisera revealed scattered cells in the splenic red pulp and lung parenchyma, suggesting that the POMC mRNA is translated in these cells. In summary, POMC mRNA is expressed in a small population of monocyte-macrophage-like cells in lung and spleen but not in lymphocytes in these tissues.

Animals↗
PubMed1992

Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor.

GnRH plays a pivotal role in the reproductive system, and GnRH analogs have wide therapeutic applications ranging from the treatment of prostatic cancer to infertility. Determination of the predicted structure of the GnRH receptor (GnRHR) would illuminate the mechanisms of receptor activation and regulation and allow directed design of improved GnRH analogs. We report the cloning of a cDNA representing the mouse GnRHR and confirm its identity using Xenopus oocyte expression. Injection of sense RNA transcript leads to the expression of a functional, high affinity GnRHR. Expression of the GnRHR using gonadotrope cell line RNA, however, is blocked by an antisense oligonucleotide. In situ hybridization in the rat anterior pituitary reveals a characteristic GnRHR distribution. The nucleotide sequence encodes a 327-amino acid protein which has the seven putative transmembrane domains characteristic of G protein-coupled receptors, but which lacks a typical intracellular C-terminus. The unusual structure and novel potential regulatory domain of the GnRHR may explain unique aspects of its signal transduction and regulation.

Amino Acid Sequence↗