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Biomedical subjects

J L Roberts

Publications and source records attributed to J L Roberts.

At least 73 records · Page 4Linked to original sources
PubMed1997

Informed consent and parental choice of anesthesia and sedation for the repair of small lacerations in children.

This study investigated the issue of informed consent by surveying parent preferences for local anesthesia and sedation in the repair of small lacerations in their children in the emergency department (ED). Of the 45 ED patients with actual lacerations receiving a suture repair, 11 requested tetracaine-adrenaline-cocaine (TAC), 25 requested infiltrated lidocaine, and 9 were not given a choice (lidocaine administered because of wound proximity to a mucous membrane site). All 45 patients preferred nonsedation over sedation. In 44 of 45 patient cases, parents preferred to be included in the medical decision-making for their children. Of the 94 non-ED cases (interviewed in private offices) with a hypothetical chin laceration, 16 preferred TAC and 78 preferred infiltrated lidocaine. Sixty-seven of 94 preferred nonsedation over sedation. In 89 of 94 patient cases, parents preferred to be included in the medical decision-making for their children. Favorable points of continuous informed consent were presented, with risks, benefits, and alternatives disclosed. From the data presented, the following conclusions were drawn: (1) parents preferred infiltrated local anesthesia more commonly than topical local anesthesia; (2) parents preferred nonsedation over sedation under the clinical circumstances described; (3) parents overwhelmingly preferred to be included in the medical decisions affecting their children.

Anesthesia, Local↗
PubMed1997

Post-transcriptional regulation of the gonadotropin-releasing hormone gene in GT1-7 cells.

GT1-7 cells respond to treatment with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), with an inhibition of transcription of the proGnRH gene and decreases in GnRH mRNA levels. However, the timing of this decrease in GnRH mRNA levels suggests that a decrease in GnRH mRNA stability may be involved in addition to an inhibition of transcription of the proGnRH gene. To address this possibility, we treated GT1-7 cells with 100 nM PMA for 4 h and then monitored GnRH mRNA levels over time after blockade of GnRH gene transcription with DRB. PMA treatment caused GnRH mRNA half-life to decrease from 30 to 11 h. Then, to verify this observation, we examined changes in GnRH mRNA poly (A) tail length, which may be a reflection of mRNA turnover, following treatment of GT1-7 cells with PMA or vehicle for 0, 4, 8 or 24 h. The poly (A) tail was removed from half of the GT1 cytoplasmic RNA sample by digestion with RNase H and the difference in GnRH mRNA size with and without RNase H treatment was determined by Northern hybridization. PMA treatment (4 and 8 h) resulted in a significant decrease in the length of the GnRH mRNA poly (A) tail, consistent with a decrease in GnRH mRNA stability. This finding suggests that GnRH mRNA turnover is inducible by substances such as PMA. Our study indicates that a change in mRNA stability is one of a multiplicity of levels at which GnRH gene expression is regulated.

Blotting, Northern↗
PubMed1997

Endopeptidase EC 3.4.24.15 presence in the rat median eminence and hypophysial portal blood and its modulation of the luteinizing hormone surge.

The endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is widely distributed in a variety of tissues, including the testes, pituitary and the central nervous system. Among its numerous roles in metabolizing and processing biologically-active peptides, the enzyme degrades gonadotropin-releasing hormone (GnRH) by cleaving the central Tyr5-Gly6 bond. The aim of the present studies was to determine whether EP24.15 can modulate the concentrations of GnRH within the hypothalamo-hypophysial portal blood and thereby play a physiological role in reproduction. Our data suggest the presence of immunoreactive EP24.15 in the perivascular space of the median eminence and that this enzyme is secreted into portal blood. We have also shown a physiological role for this enzyme in that an inhibition of its activity with a specific inhibitor augmented the steroid-induced LH increase in ovariectomized rats. The present results suggest that secretory and post-secretory mechanisms are important in shaping the GnRH signal from the central nervous system; GnRH metabolism by EP24.15 may be one such mechanism.

Animals↗
PubMed1997

Characterization of multiple promoters directing tissue-specific expression of the human gonadotropin-releasing hormone gene.

Two promoters directing tissue-specific expression of GnRH gene in neuronal and reproductive tissues were characterized by functional analyses of GnRH promoter-luciferase (LUC) constructs in transfected placental cells (JEG) and hypothalamic neuronal cells (GT1-7). Results indicate that the downstream promoter directs the expression in a neuronal cell-specific manner, whereas the upstream promoter is fully active in the nonneural placental cell line. Transfection studies carried out in several tumor cell lines derived from human reproductive tissues verified that the upstream GnRH promoter construct was much more active in directing luciferase expression in reproductive tissue. The use of both upstream and downstream promoters in various human tumor cell lines derived from reproductive tissues were demonstrated by RT-PCR. Our studies also demonstrate that the reproductive tissue-specific messenger RNA transcribed from upstream promoter is capable of directing synthesis of preproGnRH protein. Serial deletion studies localized a cell-specific upstream promoter region that directs reproductive tissue expression. DNase I footprint analysis using nuclear extract obtained from the JEG cells indicated DNA/protein interactions in four specific sequence elements of the upstream promoter region. The interaction between nuclear binding proteins present in the JEG cells (but not the GT1-7 cells) and the four specific sequences in the upstream promoter region was confirmed by gel mobility shift analysis.

Animals↗
PubMed1996

Gonadotropin-releasing hormone and NMDA receptor gene expression and colocalization change during puberty in female rats.

During development, an increase in gonadotropin-releasing hormone (GnRH) release occurs that is critical for the initiation of puberty. This increase is attributable, at least in part, to activation of the GnRH neurosecretory system by inputs from neurotransmitters, such as glutamate, acting via NMDA receptors. We examined changes in GnRH and NMDA-R1 gene expression by RNase protection assay of preoptic area-anterior hypothalamic (POA-AH) dissections of female rats undergoing normal puberty or in which precocious puberty was induced by treatment with the glutamate agonist NMA. GnRH mRNA levels increased significantly throughout normal development; this was accelerated by treatment with NMA. NMDA-R1 mRNA levels increased only between P10 and P20. The acceleration of the elevation in GnRH mRNA levels by NMDA suggests that a stimulation of GnRH gene expression may be a rate-limiting factor for the onset of puberty. This is attributable to a post-transcriptional mechanism because GnRH primary transcript levels, an index of proGnRH gene transcription, were not observed to change during puberty. Alterations in the colocalization of GnRH neurons with the NMDA-R1 subunit during puberty also were assessed immunocytochemically. The percentage of GnRH neurons that double-labeled with NMDA-R1 was 2% in prepubertal rats and 3% in pubertal rats; this increased to 19% in postpubertal rats. Taken together, these studies suggest that an increase in glutamatergic input to GnRH neurons plays a role in the increase in GnRH release and gene expression that occurs at the initiation of puberty.

Animals↗
PubMed1996

Glucocorticoid repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter elements that are recognized by heteromeric complexes containing glucocorticoid receptor.

We have identified two regions of the mouse gonadotropin-releasing hormone (GnRH) promoter, one between -237 and -201 (distal element) and the other between -184 and -150 (proximal element), which are required for glucocorticoid repression in transiently transfected GT1-7 cells. These sequences show no similarity to known positive or negative glucocorticoid response elements (nGREs) and do not function when placed upstream of heterologous viral promoters. The glucocorticoid receptor (GR) does not bind directly to the distal or proximal promoter elements but may participate in glucocorticoid repression of GnRH gene transcription by virtue of its association within multiprotein complexes at these nGREs. Electrophoretic mobility shift assays with GT1-7 nuclear extract demonstrate the presence of GR-containing protein complexes on GnRH nGREs. One protein that co-occupies the distal nGRE in vitro along with GR is the POU domain transcription factor Oct-1. Thus, the tethering of GR to the GnRH distal nGRE, by virtue of a direct or indirect association with DNA-bound Oct-1, could play a role in hormone-dependent transcriptional repression of the GnRH gene. In contrast, Oct-1 does not appear to be a component of the GR-containing protein complex that is bound to the proximal nGRE.

Animals↗
PubMed1996

Multiple transcription start sites for the GnRH gene in rhesus and cynomolgus monkeys: a non-human primate model for studying GnRH gene regulation.

In humans, transcription of the gonadotropin-releasing hormone (GnRH) gene can be initiated at two transcription start sites to produce different GnRH mRNAs. The upstream transcription start site is used only in reproductive tissues and tumors. To determine if a similar pattern of GnRH gene expression exists in non-human primates, we cloned GnRH cDNA from rhesus monkey hypothalamic RNA using reverse transcriptase-polymerase chain reaction (RT-PCR) and the 5' flanking region of the monkey GnRH gene by PCR. A 96% similarity between monkey and human GnRH cDNA was found with 94% similarity in the upstream promoter region. An upstream transcriptional start site, was identified in cynomolgus monkey testicular mRNA, 504 base pairs upstream from the hypothalamic site, which was different from that identified in the human GnRH gene. Various cynomolgus monkey reproductive tissues were found to utilize this upstream transcriptional start site. In contrast, no evidence was found for the use of upstream transcriptional start sites in rat testis or placenta, suggesting that the reproductive tissue specificity of the upstream transcription start site may be a primate specific feature.

Amino Acid Sequence↗
PubMed1996

Randomized multicenter trial comparing synchronized and conventional intermittent mandatory ventilation in neonates.

OBJECTIVE: To compare synchronized intermittent mandatory ventilation (SIMV) and conventional intermittent mandatory ventilation (IMV) in neonates. STUDY DESIGN: Prospective, multicenter, randomized clinical trial. SETTING: Level III neonatal intensive care units at six university or children's hospitals. PATIENTS: Three hundred twenty-seven infants receiving conventional IMV for respiratory distress syndrome, pneumonia, or meconium aspiration pneumonitis were randomly assigned a 7.5 +/- 6 hours of age to either continue with IMV or change to SIMV. Infants assigned to each mode of ventilation had similar birth weight (BW), gestational age, and Apgar scores at birth, and similar oxygenation indexes at randomization. They received similar surfactant therapy and had similar incidence of sepsis, seizures, secondary pneumonia, and necrotizing enterocolitis. In the infants with BW less than 1000 gm, more infants receiving IMV had surgical ligation of their patent ductus arteriosus than did those receiving SIMV (27 vs. 7 %; p = 0.02). ANALYSIS: Data was analyzed overall for all infants and also separately within three BW groups: less than 1000 gm, 1000 to 2000 gm, and more than 2000 gm. The 1000 to 2000 gm BW group was further analyzed in subgroups weighing 1000 to 1499 gm and 1500 to 2000 gm. RESULTS: In all infants, at 1 hour after randomization, the infants receiving SIMV had a lower mean airway pressure than those receiving IMV (8.08 +/- 2.15 vs. 8.63 +/- 2.59; p<0.05), with similar fractions of inspired oxygen and oxygenation indexes. Infants whose BW was 1000 to 2000 gm at 0.5 hour required a lower fraction of inspired oxygen with SIMV than with IMV (0.52 +/- 0.20 vs. 0.62 +/- 0.27; p<0.05) and had better oxygenation at 1 hour, as shown by lower oxygenation indexes with SIMV than with IMV (6.14 +/- 4.17 vs. 9.42 +/- 8.41; p = 0.01). Infants whose BW was 1000 to 2000 gm received a lower number of unit doses of sedative/analgesic drugs per infant during the first 4 days of SIMV than did infants receiving IMV (3.8 +/- 3.4 vs 6.3 +/- 5.5 unit doses; p = 0.02). Infants whose BW was more than 2000 gm had a shorter duration of mechanical ventilation with SIMV than with IMV (median, 72 vs 93 hours; p = 0.02). Three of the forty-six infants receiving IMV but none of the 47 infants receiving SIMV required extracorporeal membrane oxygenation. In the infants with BW less than 1000 gm, fewer infants treated with SIMV required supplemental oxygen at 36 weeks of postconceptional age than did those treated with IMV (47 vs 72%; p<0.05). In 83 infants whose lungs were mechanically ventilated for 14 days or longer, all with BW less than 2000 gm, those treated with SIMV regained their BW earlier than those treated with IMV (median, 21.5 vs 29 days; p<0.01). There were no differences in the rates of death, intraventricular hemorrhage (grades III and IV), air leak, need for pharmacologic paralysis, or need for supplemental oxygen at 28 days. CONCLUSIONS: We found that SIMV was at least as efficacious as conventional IMV, and may have improved certain outcomes in BW-specific groups.

Female↗
PubMed1996

Characterization of gonadotropin-releasing hormone gene transcripts in a mouse hypothalamic neuronal GT1 cell line.

We have characterized the nuclear and cytoplasmic RNA transcripts derived from the gonadotropin releasing hormone (GnRH) gene in a mouse hypothalamic neuronal GT1 cell line. Analyses of nuclear GnRH RNA precursors present in the GT1 cells by RNase protection assay show that there is no particular order of intron excision, suggesting the existence of multiple processing pathways. A similar pattern is observed in mouse preoptic area-anterior hypothalamus (POA-AH). In GT1 cells, approximately 5% of the total GnRH RNA transcripts are found in the nucleus. In contrast, in the POA-AH of mice, nuclear transcripts comprise 40% of the total GnRH transcripts. Thus the GT1 cells, while similar in overall GnRH RNA processing to mouse hypothalamic GnRH neurons, do not exhibit the high abundance of nuclear GnRH RNA transcripts seen in the rodent GnRH neuron in vivo. Quantitative analysis of the nuclear RNA species shows that the GnRH primary transcript comprises more than 90% of the total nuclear GnRH mRNA precursors in both GT1 cells and mouse POA-AH and thus GnRH processing intermediates account for fewer than 10% of these precursors. Using these probes, we have examined changes in GnRH primary transcript expression in GT1-7 cells. In the presence of RNA synthesis inhibitors, the half-life of the GnRH primary transcript was found to be quite short, approximately 18 min, suggesting that the level of primary transcript would reflect levels of GnRH gene transcription. When GT1-7 cells are treated with the phorbol ester PMA (phorbol, 12-myristate, 13-acetate) for 1 h, GnRH primary transcript levels decrease by approximately 70%. Supporting the hypothesis that GnRH primary transcript is a good indicator of GnRH gene transcription is the finding that 1 h of PMA treatment results in a similar (approximately 50%) decrease in GnRH gene transcription, as assayed by nuclear run-on assay. Our observation that GT1 cells resemble mouse hypothalamic GnRH neurons in their pattern of intron excision and in the ratio of primary transcript to other nuclear transcripts emphasizes the utility of these cells for studying the regulation of GnRH gene expression in this immortalized hypothalamic cell line.

Animals↗
PubMed1996

CT of abdominal and pelvic trauma.

CT is the diagnostic study of choice at most institutions in the evaluation of hemodynamically stable, blunt abdominal trauma. It is highly specific and sensitive in the detection and definition of the extent of most intra-abdominal and pelvic injuries. The clinical examination is the most important factor in the decision regarding whether surgery should be undertaken. However, a progressive and steady trend toward the nonoperative management of solid parenchymal injury is occurring, partly because of the ability of CT to define the extent of injury and to follow its healing over time. Clinical management, CT findings, and the utility of these findings are presented and discussed for each organ system injury within the abdomen and pelvis.

Abdominal Injuries↗
PubMed1995

Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development.

Males with X-linked severe combined immunodeficiency (XSCID) have defects in the common cytokine receptor gamma chain (gamma c) gene that encodes a shared, essential component of the receptors of interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. The Janus family tyrosine kinase Jak3 is the only signaling molecule known to be associated with gamma c, so it was hypothesized that defects in Jak3 might cause an XSCID-like phenotype. A girl with immunological features indistinguishable from those of XSCID was therefore selected for analysis. An Epstein-Barr virus (EBV)-transformed cell line derived from her lymphocytes had normal gamma c expression but lacked Jak3 protein and had greatly diminished Jak3 messenger RNA. Sequencing revealed a different mutation on each allele: a single nucleotide insertion resulting in a frame shift and premature termination in the Jak3 JH4 domain and a nonsense mutation in the Jak3 JH2 domain. The lack of Jak3 expression correlated with impaired B cell signaling, as demonstrated by the inability of IL-4 to activate Stat6 in the EBV-transformed cell line from the patient. These observations indicate that the functions of gamma c are dependent on Jak3 and that Jak3 is essential for lymphoid development and signaling.

Amino Acid Sequence↗
PubMed1995

Characterization and regulation of upstream transcripts of the murine proopiomelanocortin gene.

Upstream transcription start sites in the murine POMC gene were detected at -48, -113, and -134 by primer extension analysis of RNA derived from a murine pituitary (AtT20) cell line. By RNase protection assay, dexamethasone treatment of AtT20 cells was found to reduce levels of transcripts initiated from the major transcription start site, but had a less prominent effect levels of transcripts initiated upstream. Conversely, treatment with 8-bromo-cAMP was found to induce levels of POMC gene transcripts from the major start site and from upstream start sites at -113 and -134, but reduced transcript levels initiated at a start site at -48. Treatment with the phorbol ester PDBU had no significant effects on levels of transcripts originating from any of the transcription start sites. Chronic treatments with these agents did not alter the proportion of upstream transcripts to the total level of POMC mRNA.

Animals↗
PubMed1995

Substrate specificity differences between recombinant rat testes endopeptidase EC 3.4.24.15 and the native brain enzyme.

We have characterized and compared the substrate specificity of affinity-purified recombinant rat testes endopeptidase EC 3.4.24.15 (EP 24.15) with that reported for the isolated brain enzyme. Of the peptides tested, only bradykinin, dynorphin A1-8, and neurotensin were efficiently cleaved by the recombinant enzyme (kcat/Km = 3.0, 2.8 and 0.5 x 10(5) M-1sec-1, respectively); other peptides considered substrates of EP 24.15 (gonadotropin-releasing hormone, substance P, somatostatin and angiotensin) were not metabolized. The enzyme was inhibited by metal ion chelators and thiol-reactive agents, as well as a specific EP 24.15 inhibitor (N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate), thus confirming the enzyme as a thiol-dependent metalloendopeptidase. The observed discrepancies in substrate specificity of the recombinant testicular and the isolated brain enzymes may result from tissue-specific forms and/or post-translational modifications of EP 24.15.

Amino Acid Sequence↗
PubMed1995

Disruption of intrathymic CD4-Ia interactions on immature CD4+CD8+ thymocytes results in diminished TCR expression on mature CD8+ T cell progeny.

Cell surface TCR expression by developing thymocytes is actively regulated during ontogeny. Whereas most immature CD4+CD8+ thymocytes express low levels of TCR alpha beta, mature CD4+ and CD8+ thymocytes express significantly higher levels of surface TCR. Low TCR expression on CD4+CD8+ thymocytes is due, at least in part, to inhibitory signals generated by CD4 interactions with MHC class II ligands in the thymus. In the present study we wished to determine whether levels of TCR expressed on mature thymocytes were also influenced by CD4-Ia interactions that had occurred on their CD4+CD8+ precursors. To do so, we examined TCR expression on mature CD8+ T cells from animals in which CD4-ligand interactions had been disrupted experimentally by in vivo administration of anti-CD4 mAb or by targeted disruption of an MHC class II gene. We found that TCR expression was significantly diminished on all mature CD8+ T cells from both anti-CD4 mAb-treated mice and MHC class II-deficient animals. These results demonstrate that TCR expression by mature CD8+ T cells, as well as that of immature CD4+CD8+ thymocytes, is regulated by CD4-mediated signals acting on CD4+CD8+ thymocytes. Because the effects of disrupting CD4-Ia interactions on TCR expression by CD8+ T cells were independent of TCR specificity, these findings directly support the concept that CD4-CD8+ T cells arise from precursor CD4+CD8+ cells.

Animals↗
PubMed1995

TCR alpha-CD3 delta epsilon association is the initial step in alpha beta dimer formation in murine T cells and is limiting in immature CD4+ CD8+ thymocytes.

The present study has examined the molecular events leading to formation of alpha beta dimers in normal murine thymocytes and mature T cells. We demonstrate that TCR assembly proceeds by initial association of TCR alpha with CD3 delta epsilon proteins and by association of TCR beta with CD3 gamma epsilon proteins to form alpha delta epsilon and beta gamma epsilon trimers; these trimers then associate to form alpha delta epsilon-beta gamma epsilon complexes, within which alpha-beta disulfide bond formation occurs. We also show that TCR-associated protein (TRAP) associates uniquely with CD3 gamma epsilon pairs and that formation of beta gamma epsilon trimers occurs subsequent to TRAP dissociation. Importantly, we document that the assembly step that is quantitatively limiting in CD4+ CD8+ thymocytes is the initial association of TCR alpha with CD3 delta epsilon chains, which appears necessary to protect nascent TCR alpha proteins from accelerated degradation within the ER of immature thymocytes.

Animals↗
PubMed1995

Hyperadrenocorticism and food restriction-induced life extension in the rat: evidence for divergent regulation of pituitary proopiomelanocortin RNA and adrenocorticotropic hormone biosynthesis.

The increased diurnal elevation of plasma corticosterone (B) induced by food restriction (FR) may play a role in the life span extension of FR. We investigated whether FR alters adrenocorticotropic hormone (ACTH) and proopiomelanocortin (POMC) mRNA levels in plasma and anterior pituitary (AP), since these molecules both regulate and can be suppressed by B. Measurements were made in 3-month-old male Fischer 344 rats that had been ad libitum (AL) or FR (60% of AL calories) since 6 weeks of age. Plasma B was 2-fold higher in FR rats in the PM samples, but did not differ in AM samples. By contrast, plasma ACTH did not differ in the PM samples of FR and AL rats and was 20% lower in AM samples (p < .05) of FR rats. AP content of ACTH was 50% lower in FR rats in both AM and PM samples (p < .01). In contrast, AP contents of POMC and mRNA, primary transcript, and processing intermediate were not reduced in FR rats, and PM content of POMC primary transcript was elevated in FR rats (p < .05). The reduced pituitary and plasma ACTH of FR rats may be the consequence of their elevated plasma B levels. This study also suggests that factors other than elevated ACTH account for FR-induced hyperadrenocorticism. These results also indicate that POMC mRNA and ACTH biosyntheses are differentially regulated in FR rats.

Adrenocorticotropic Hormone↗