Biomedical subjects

J L Roberts

Publications and source records attributed to J L Roberts.

At least 55 records · Page 3Linked to original sources

PubMed1998

Incontinentia pigmenti in a newborn male infant with DNA confirmation.

We report on a woman with incontinentia pigmenti (IP), who had two successive term pregnancies. The first pregnancy ended in the birth of a male infant, who is alive and well at 2 years. A second liveborn male had early postnatal distress and died after 1 day of life, after a fulminating clinical course. Polymorphic microsatellite markers, closely linked to the IP gene on the X chromosome, showed that each son inherited a different X chromosome from his mother. Although in most instances IP appears to be prenatally lethal for the male, the phenotype is not completely known. We propose that the neonatal phenotype may be characterized by lethal disturbances in the hematopoietic and immunologic systems.

Adult↗

PubMed1998

Derivatization of progesterone to a neurally active steroid by pituitary neurointermediate lobe.

The melanotrophs of the neurointermediate lobe and peptidergic terminals of the neural lobe are regulated by gamma-aminobutyric acid (GABA) via GABA-A receptors and therefore, may be important sites for the modulatory actions of neurally active steroids. These steroid compounds might be produced peripherally, synthesized de novo in the pituitary, or derivatized from circulating steroids, each pathway having different physiological implications. In the present study, we show that neurointermediate lobe tissue can derivatize progesterone to the neurally active steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one. The neurointermediate lobe was found to be four times as active as anterior pituitary and mediobasal hypothalamus in conversion of progesterone to 3 alpha-hydroxy-5 alpha-pregnan-20-one; mediobasal hypothalamus was relatively more active in the production of the intermediate 5 alpha-pregnan-3,20-dione. The identity of the compounds was confirmed by the method of serial isotopic dilution. We observed rates of synthesis in the neurointermediate lobe consistent with the production of physiologically relevant quantities of 3 alpha-hydroxy-5 alpha-pregnan-20-one from concentrations of progesterone which can occur naturally. In support of these findings, we demonstrate the presence of 3 alpha-hydroxysteroid oxidoreductase in neurointermediate lobe by immunocytochemistry.

3-Hydroxysteroid Dehydrogenases↗

PubMed1998

Finasteride in the treatment of men with androgenetic alopecia. Finasteride Male Pattern Hair Loss Study Group.

BACKGROUND: Androgenetic alopecia (male pattern hair loss) is caused by androgen-dependent miniaturization of scalp hair follicles, with scalp dihydrotestosterone (DHT) implicated as a contributing cause. Finasteride, an inhibitor of type II 5alpha-reductase, decreases serum and scalp DHT by inhibiting conversion of testosterone to DHT. OBJECTIVE: Our purpose was to determine whether finasteride treatment leads to clinical improvement in men with male pattern hair loss. METHODS: In two 1-year trials, 1553 men (18 to 41 years of age) with male pattern hair loss received oral finasteride 1 mg/d or placebo, and 1215 men continued in blinded extension studies for a second year. Efficacy was evaluated by scalp hair counts, patient and investigator assessments, and review of photographs by an expert panel. RESULTS: Finasteride treatment improved scalp hair by all evaluation techniques at 1 and 2 years (P < .001 vs placebo, all comparisons). Clinically significant increases in hair count (baseline = 876 hairs), measured in a 1-inch diameter circular area (5.1 cm2) of balding vertex scalp, were observed with finasteride treatment (107 and 138 hairs vs placebo at 1 and 2 years, respectively; P < .001). Treatment with placebo resulted in progressive hair loss. Patients' self-assessment demonstrated that finasteride treatment slowed hair loss, increased hair growth, and improved appearance of hair. These improvements were corroborated by investigator assessments and assessments of photographs. Adverse effects were minimal. CONCLUSION: In men with male pattern hair loss, finasteride 1 mg/d slowed the progression of hair loss and increased hair growth in clinical trials over 2 years.

Adult↗

PubMed1998

Transcriptional activation of the proopiomelanocortin gene by cyclic AMP-responsive element binding protein.

The proopiomelanocortin (POMC) gene expressed in corticotrophs of the anterior pituitary encodes several biologically active peptides and is primarily under the positive control of hypophysiotropic factors (e.g. corticotropin releasing hormone). Using AtT20 cells as a model, we show that these factors increase levels of POMC primary RNA transcripts representative of a transcriptional activation of the gene. This effect is mimicked by several activators of the cAMP signaling pathway. Inhibition of protein synthesis with cycloheximide did not modify the CRH-induced increase in POMC hnRNA suggesting that these early effects are mediated by preexisting transcription factors. Using a reporter gene containing 706 bp of the POMC promoter region, we observe transcriptional activation with the same compounds, their effects being abolished when protein kinase A (PKA) is inactivated by a dominant inhibitory mutant. Promoter deletion analyses mapped an essential cAMP inducible element within the first exon of the POMC gene. This element (PTRE: TGACTAA) located at nucleotides +41/+47 was shown to bind the cAMP responsive element binding protein (CREB) by gel shift analyses and confers strong transcriptional activation by an expression vector coding a CREB-VP16 activator domain fusion protein. Further, expression of a dominant inhibitory mutant of CREB reduced cAMP stimulated transcription of the full length POMC promoter and the PTRE. Taken together, these results show that the major hypophysiotropic factors stimulate POMC transcription through a signaling cascade that involves PKA and CREB.

Animals↗

PubMed1998

Expression of gonadotropin-releasing hormone (GnRH) gene in human uterine endometrial tissue.

Many peripheral reproductive tissues have been found to contain gonadotrophin-releasing hormone (GnRH) and express the GnRH gene at low levels, presumably because the hormone functions in a paracrine/autocrine fashion. This study was designed to investigate and characterize GnRH gene expression in human endometrial tissue at different stages of the endometrial cycle. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis together with Southern blot assay demonstrated that human endometrial tissue expresses the proGnRH gene. RNA samples from endometrial tissue were analysed with two pairs of oligonucleotide primers. Both gave a doublet 870 bases apart at the expected sizes, indicating that both the upstream and downstream transcriptional start sites of the GnRH gene are used in endometrial tissue and that transcripts with and without intron 1 were produced. Our data also demonstrated that utilization of the two promoters varies with the stage of the endometrial cycle. The largest difference came from the mRNA transcribed from the downstream promoter and without intron 1. This mRNA was expressed at a very low level during the proliferative phase and dramatically increased almost 10-fold (P < 0.01) during the early secretory phase, and subsequently decreased 5-fold during the late secretory stage. The presence of GnRH mRNA in the endometrium, as well as the differential expression of the GnRH gene during the early secretory phase provides physiological evidence that human GnRH may play a paracrine/autocrine function in the human uterus.

Adult↗

PubMed1998

Developmental regulation of V(D)J recombination at the TCR alpha/delta locus.

The T-cell receptor (TCR) alpha/delta locus includes a large number of V, D, J and C gene segments that are used to produce functional TCR delta and TCR alpha chains expressed by distinct subsets of T lymphocytes. V(D)J recombination events within the locus are regulated as a function of developmental stage and cell lineage during T-lymphocyte differentiation in the thymus. The process of V(D)J recombination is regulated by cis-acting elements that modulate the accessibility of chromosomal substrates to the recombinase. Here we evaluate how the assembly of transcription factor complexes onto enhancers, promoters and other regulatory elements within the TCR alpha/delta locus imparts developmental control to VDJ delta and VJ alpha rearrangement events. Furthermore, we develop the notion that within a complex locus such as the TCR alpha/delta locus, highly localized and region-specific control is likely to require an interplay between positive regulatory elements and blocking or boundary elements that restrict the influence of the positive elements to defined regions of the locus.

Animals↗

PubMed1998

Cooperation among multiple transcription factors is required for access to minimal T-cell receptor alpha-enhancer chromatin in vivo.

To understand the molecular basis for the dramatic functional synergy between transcription factors that bind to the minimal T-cell receptor alpha enhancer (Ealpha), we analyzed enhancer occupancy in thymocytes of transgenic mice in vivo by genomic footprinting. We found that the formation of a multiprotein complex on this enhancer in vivo results from the occupancy of previously identified sites for CREB/ATF, TCF/LEF, CBF/PEBP2, and Ets factors as well as from the occupancy of two new sites 5' of the CRE site, GC-I (which binds Sp1 in vitro) and GC-II. Significantly, although all sites are occupied on a wild-type Ealpha, all sites are unoccupied on versions of Ealpha with mutations in the TCF/LEF or Ets sites. Previous in vitro experiments demonstrated hierarchical enhancer occupancy with independent binding of LEF-1 and CREB. Our data indicate that the formation of a multiprotein complex on the enhancer in vivo is highly cooperative and that no single Ealpha binding factor can access chromatin in vivo to play a unique initiating role in its assembly. Rather, the simultaneous availability of multiple enhancer binding proteins is required for chromatin disruption and stable binding site occupancy as well as the activation of transcription and V(D)J recombination.

Animals↗

PubMed1998

The role of calcium in the transcriptional and posttranscriptional regulation of the gonadotropin-releasing hormone gene in GT1-7 cells.

The role of calcium in the regulation of GnRH gene expression and the mechanism for its effects were examined in the present study. Using the immortalized hypothalamic GT1-7 cell line, which synthesizes and secretes GnRH, we demonstrated by ribonuclease protection assay and Northern blot analysis that these cells respond to treatment with the calcium ionophores ionomycin and A23187 with an inhibition of transcription of the GnRH gene and decreases in GnRH messenger RNA (mRNA) levels. Ionomycin treatment caused the GnRH mRNA half-life to decrease from 25 to 9 h, concomitant with a decrease in mRNA poly(A) tail length, suggesting that ionomycin causes a decrease in GnRH mRNA stability. The ionomycin inhibitory effect on GnRH cytoplasmic mRNA levels was significantly inhibited in the presence of cycloheximide or the RNA synthesis inhibitor 5,6-dichloro-1beta-ribofuranosylbenzimidazole, indicating that novel protein/RNA synthesis is obligatory for this effect. We conclude that an increase in calcium levels caused by ionomycin inhibits GnRH gene expression at multiple levels, including GnRH gene transcription and mRNA stability in GT1-7 cells.

Animals↗

PubMed1998

Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin.

Reduction in the activity of the alpha-melanocyte-stimulating hormone (alpha-MSH) system causes obesity, and infusions of alpha-MSH can produce satiety, raising the possibility that alpha-MSH may mediate physiological satiety signals. Since alpha-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased > 60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to alpha-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.

Animals↗

PubMed1998

Accessory splenectomy in the management of recurrent immune thrombocytopenic purpura.

Although splenectomy for chronic immune thrombocytopenic purpura (ITP) is successful in the majority of patients, recurrence of thrombocytopenia may occur in association with a functional accessory spleen, sometimes years after operation. Identification and resection of the accessory spleen under these circumstances may result in a sustained remission. We describe a case of recurrent thrombocytopenia associated with an accessory spleen occurring 5 years after splenectomy in a young woman with chronic immune thrombocytopenic purpura. Accessory splenectomy was successful in producing a sustained remission.

Adult↗

PubMed1997

Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells.

Two morphologically distinct subpopulations of GT1-7 cells have been characterized and examined for their responsiveness to glucocorticoids. Type I cells have a neuronal phenotype, extending many lengthy processes, and express neuronal, but not glial, markers. Type II cells show weaker or negative immunostaining for neuronal markers and exhibit fewer processes. The effect of glucocorticoids on gonadotropin-releasing hormone (GnRH) secretion and gene expression was compared in type I and type II GT1-7 cells. For secretion studies, cells were attached to Cytodex beads and perifused with control medium or medium containing dexamethasone (dex). The high level of GnRH secreted by type I cells was slightly enhanced in the presence of dex, whereas dex rapidly and profoundly decreased the already low level of GnRH secreted by type II cells. Immunocytochemistry for GnRH showed dark reaction product in the cell bodies and processes of type I cells and little or no immunoreactivity in type II cells. Both the endogenous mouse GnRH mRNA and the transcriptional activity of a mouse GnRH promoter luciferase reporter gene plasmid were suppressed to a greater extent in type II cells than in type I. In electrophoretic mobility shift assays, there was no difference between type I and type II nuclear extracts in the pattern of protein-DNA complexes formed on two previously identified negative glucocorticoid response elements located at -237 to -201 and -184 to -150 bp of the mouse promoter. Both cell types contained glucocorticoid receptors (GR) by Western blot analysis. Cytosols from type I or type II cells were incubated with [3H]dex to obtain GR binding parameters. Binding data were consistent with a one-site model for dex binding in each case. Small differences in Kd (1.7 nM, type I; 3.1 nM, type II) or Bmax (approximately 3600 sites/cell, type I; approximately 1800 sites/cell, type II) were not likely to account for the differential sensitivity to dex treatment. In conclusion, nuclear alterations in type II cells leading to greater transcriptional susceptibility to dex, coupled with low GnRH storage levels, may be reflected in exquisite sensitivity of GnRH secretion to glucocorticoid repression. This represents the first example of a steroid hormone acting directly on GnRH-producing cells to alter GnRH secretion.

Animals↗

PubMed1997

Thiol activation of endopeptidase EC 3.4.24.15. A novel mechanism for the regulation of catalytic activity.

Endopeptidase EC 3.4.24.15 (EP24.15) is a thermolysin-like metalloendopeptidase involved in the regulated metabolism of a number of neuropeptides. Unlike other thermolysin-like peptidases EP24.15 displays a unique thiol activation, a mechanism that is not clearly understood. In this study we show that both recombinant and tissue-derived EP24.15 are activated up to 8-fold by low concentrations (0.1 mM) of dithiothreitol. Additionally, under non-reducing conditions, recombinant and native EP24.15 forms multimers that can be returned to the monomeric form by reduction. We have also shown that competitive inhibitor binding occurs only to the monomeric form, which indicates that catalytic site access is restricted in the multimeric forms. Through systematic site-directed mutagenesis we have identified that cysteine residues 246, 253, and possibly 248 are involved in the formation of these multimers. Furthermore, both a double mutant (C246S/C253S) and a triple mutant (C246S/C248S/C253S) are fully active in the absence of reducing agents, as measured by both inhibitor binding and hydrolysis. The formation and disruption of disulfide bonds involving these cysteine residues may be a mechanism by which EP24.15 activity is regulated through changes in intra- and extracellular redox potential.

Amino Acid Sequence↗

PubMed1997

Regulation of T cell receptor delta gene rearrangement by CBF/PEBP2.

We have analyzed transgenic mice carrying versions of a human T cell receptor (TCR)-delta gene minilocus to study the developmental control of VDJ (variable/diversity/joining) recombination. Previous data indicated that a 1.4-kb DNA fragment carrying the TCR-delta enhancer (E(delta)) efficiently activates minilocus VDJ recombination in vivo. We tested whether the transcription factor CBF/PEBP2 plays an important role in the ability of E(delta) to activate VDJ recombination by analyzing VDJ recombination in mice carrying a minilocus in which the deltaE3 element of E(delta) includes a mutated CBF/PEBP2 binding site. The enhancer-dependent VD to J step of minilocus rearrangement was dramatically inhibited in three of four transgenic lines, arguing that the binding of CBF/PEBP2 plays a role in modulating local accessibility to the VDJ recombinase in vivo. Because mutation of the deltaE3 binding site for the transcription factor c-Myb had previously established a similar role for c-Myb, and because a 60-bp fragment of E(delta) carrying deltaE3 and deltaE4 binding sites for CBF/PEBP2, c-Myb, and GATA-3 displays significant enhancer activity in transient transfection experiments, we tested whether this fragment of E(delta) is sufficient to activate VDJ recombination in vivo. This fragment failed to efficiently activate the enhancer-dependent VD to J step of minilocus rearrangement in all three transgenic lines examined, indicating that the binding of CBF/PEBP2 and c-Myb to their cognate sites within E(delta), although necessary, is not sufficient for the activation of VDJ recombination by E(delta). These results imply that CBF/PEBP2 and c-Myb collaborate with additional factors that bind elsewhere within E(delta) to modulate local accessibility to the VDJ recombinase in vivo.

Animals↗

PubMed1997

Protein synthesis-dependent and -independent mechanisms for the regulation of GnRH RNA transcript levels in GT1 cells.

The cellular mechanism for the suppression of GnRH gene expression by the phorbol ester PMA was investigated in GT1 cells. The protein synthesis inhibitor cycloheximide decreased GnRH primary transcript levels, indicating a protein synthesis requirement for basal GnRH transcription. PMA decreased GnRH primary transcript levels even in the presence of cycloheximide, indicating that the PMA suppression of GnRH gene transcription is protein synthesis-independent. In contrast, the PMA-inhibitory effect on GnRH cytoplasmic mRNA levels was significantly reduced or inhibited in the presence of cycloheximide or RNA synthesis inhibitors given within 4 h of PMA, suggesting a protein/RNA synthesis-dependent mechanism for the regulation of GnRH mRNA levels by PMA. Thus, the mechanism for the PMA inhibition of GnRH primary transcript is mediated through a protein and RNA synthesis-independent mechanism, while the decrease in GnRH mRNA levels occurs through a mechanism that involves the induction of new RNA and protein synthesis that happens within 4 h of PMA administration.

Animals↗

PubMed1997

Developmental regulation of VDJ recombination by the core fragment of the T cell receptor alpha enhancer.

The role of T cell receptor alpha enhancer (E alpha) cis-acting elements in the developmental regulation of VDJ recombination at the TCR alpha/delta locus was examined in transgenic mice containing variants of a minilocus VDJ recombination substrate. We demonstrate that the 116-bp T alpha 1,2 core enhancer fragment of the 1.4-kb E alpha is sufficient to activate the enhancer-dependent step of minilocus rearrangement, and that within T alpha 1,2, intact binding sites for TCF/LEF and Ets family transcription factors are essential. Although minilocus rearrangement under the control of the 1.4-kb E alpha initiates at fetal day 16.5 and is strictly limited to alpha beta T cells, we find that rearrangement under the control of T alpha 1,2 initiates slightly earlier during ontogeny and occurs in both gamma delta and alpha beta T cells. We conclude that the core fragment of E alpha can establish accessibility to the recombinase in developing thymocytes in vivo in a fashion that is dependent on the binding of TCF/LEF and Ets family transcription factors, but that these and other factors that bind to the E alpha core cannot account for the precise developmental onset of accessibility that is provided by the intact E alpha. Rather, our data suggests a critical role for factors that bind E alpha outside of the core T alpha 1,2 region in establishing the precise developmental onset of TCR alpha rearrangement in vivo.

Animals↗

PubMed1997

Prostaglandin F2alpha is required for NMDA receptor-mediated induction of c-fos mRNA in dentate gyrus neurons.

Activation of NMDA receptors has been linked to a diversity of lasting physiological and pathological changes in the mammalian nervous system. The cellular and molecular mechanisms underlying permanent modifications of nervous system structure and function after brief episodes of neuronal activity are unknown. Immediate-early genes (IEGs) have been implicated in the conversion of short-term stimuli to long-term changes in cellular phenotype by regulation of gene expression. The intracellular signaling pathways coupling activation of receptors at the cell surface with induction of IEGs in the nucleus are incompletely understood. NMDA produces a striking increase in the IEG c-fos in dentate gyrus (DG) neurons in vitro; this induction is dependent, in part, on the arachidonic acid cascade. Here we show that NMDA receptor activation triggers the synthesis of the prostaglandins PGF2alpha and PGE2, but not PGD2, in rat cerebral cortical neurons in vitro. We further demonstrate that PGF2alpha, but not PGE2 or PGD2, is necessary but not sufficient for NMDA induction of c-fos mRNA in DG neurons. These findings provide insight into the molecular events coupling activation of the NMDA receptor with regulation of the IEG c-fos and identify the diffusable messenger PGF2alpha as obligatory for NMDA receptor-mediated transcription of a nuclear IEG.

Animals↗

PubMed1997

Regulation of gonadotropin-releasing hormone gene expression in vivo and in vitro.

The pulsatile release of gonadotropin-releasing hormone (GnRH) into the portal vasculature is responsible for the maintenance of reproductive function. Levels of GnRH decapeptide available for this process can be regulated at transcriptional, posttranscriptional, and posttranslational levels. In the immortalized neuronal GT1 cell lines which synthesize and secrete GnRH, regulation of GnRH biosynthesis has been studied using activators of the protein kinase A (PKA), protein kinase C (PKC), and calcium second messenger systems. These substances, while stimulating GnRH release, cause a universal inhibition of all biosynthetic indices measured to date, including decreases in transcription of the proGnRH gene, GnRH mRNA levels, mRNA stability, and translational efficiency. In contrast, in the animal, the mechanism for the regulation of GnRH gene expression appears to be primarily posttranscriptional, since changes in GnRH mRNA levels often occur in the absence of changes in GnRH primary transcript levels an index of GnRH gene transcription. For example, GnRH mRNA levels increase in response to stimulation with glutamate analogs, while GnRH primary transcript levels are unchanged. However, parallel changes in GnRH mRNA and primary transcript have been observed on proestrus prior to the LH/GnRH surge, suggesting that the regulation of GnRH mRNA levels in vivo involves a complex interplay of transcriptional and posttranscriptional processes.

Animals↗

PubMed1997

Human severe combined immunodeficiency: genetic, phenotypic, and functional diversity in one hundred eight infants.

OBJECTIVE: To determine the relative frequencies of the different genetic forms of severe combined immunodeficiency (SCID) and whether there are distinctive characteristics of the particular genotypes. STUDY DESIGN: The demographic, genetic, and immunologic features of 108 infants with SCID who were treated consecutively at Duke University Medical Center were analyzed. RESULTS: Eighty-nine subjects were boys and 19 were girls; there were 84 white infants, 16 black infants, and 8 Hispanic infants. Forty-nine had X-linked SCID with mutations of common cytokine receptor gamma chain (gamma c), 16 had adenosine deaminase (ADA) deficiency, 8 had Janus kinase 3 (Jak3) deficiency, 21 had unknown autosomal recessive mutations, 1 had reticular dysgenesis, 1 had cartilage hair hypoplasia, and 12 (all boys) had SCID of undetermined type. Deficiency of ADA caused the most profound lymphopenia; gamma c or Jak3 deficiency resulted in the most B cells and fewest natural killer (NK) cells; NK cells and function were highest in autosomal recessive and unknown types of SCID. CONCLUSIONS: Different SCID genotypes are associated with distinctive lymphocyte characteristics. The presence of NK function in ADA-deficient, autosomal recessive, and unknown type SCIDs, and low NK function in a majority of gamma c and Jak3 SCIDs indicates that some molecular lesions affect T, B, and NK cells (gamma c and Jak3), others primarily T cells (ADA deficiency), and others just T and B cells.

Adenosine Deaminase↗