Building Youth Capacity to Address Environmental Health and Justice Concerns in Dearborn, Michigan.

BACKGROUND: Environmental Health Research-to-Action (EHRA) is a community-academic partnership focused on building skills and intergenerational knowledge in environmental health, community science, and policy advocacy to address cumulative exposures in Dearborn, Michigan and nearby communities, primarily through a youth academy. OBJECTIVES: This article outlines our EHRA Youth Academy curriculum with sample recruitment materials, and we describe its beginnings, steering committee (SC), learning objectives, design, implementation, and recommendations from ongoing program evaluation and reflections of the SC. METHODS: In 2018 and 2019, we piloted the EHRA Academy with a total of forty-five fellows (16-18 years old), primarily Arab youth living in or near frontline communities. Fellows participated in a 2-week academy of interactive sessions, including a tour of local industry, participatory mapping, practice using handheld monitors to measure air pollution, and a policy advocacy 101 training. Applying lessons in accessing secondary data and environmental health literacy, fellows then created scientifically-informed materials including infographics and oral presentations for varied audiences. They completed a pre-survey, brief daily surveys, and a post-survey, and reported increased likelihood of advocacy behaviors and knowledge related to all content areas. CONCLUSIONS: In Southeast Dearborn, Michigan, threats to environmental health are constant, and intergenerational community mobilization remains necessary to reduce their adverse effects. Grounded in the principles of community-based participatory research (CBPR) and using high-impact active learning strategies, the EHRA Academy may provide one effective model for centering youth to build community capacity towards environmental justice (EJ).

Hospital admission rates, length of stay, and in-hospital mortality for common acute care conditions in COVID-19 vs. pre-COVID-19 era.

OBJECTIVES: The impact of COVID-19 upon acute care admission rates and patterns are unknown. We sought to determine the change in rates and types of admissions to tertiary and specialty care hospitals in the COVID-19 era compared with pre-COVID-19 era. METHODS: Acute care admissions to the largest tertiary care referral hospital, designated national referral centers for cardiac, cancer and maternity hospital in the State of Qatar during March 2020 (COVID-19 era) and January 2020 and March 2019 (pre-COVID-19 era) were compared. We calculated total admissions, admissions for eight specific acute care conditions, in-hospital mortality rate, and length of stay at each hospital. RESULTS: A total of 18,889 hospital admissions were recorded. A sharp decline ranging from 9% to 75% was observed in overall admissions. A decline in both elective and non-elective surgeries was observed. A decline of 9%-58% was observed in admissions for acute appendicitis, acute coronary syndrome, stroke, bone fractures, cancer, and live births, whereas an increase in admissions due to respiratory tract infections was observed. Overall length of stay was shorter in the COVID-19 period possibly suggesting lesser overall disease severity, with no significant change in in-hospital mortality. Unadjusted mortality rate for Qatar showed marginal increase in the COVID-19 period. CONCLUSIONS: We observed a sharp decline in acute care hospital admissions, with a significant decline in admissions due to seven out of eight acute care conditions. This decline was associated with a shorter length of stay but not associated with a change in in-hospital mortality rate.

[Rhegmatogenous retinal detachment: Topography of breaks and agreement with lincoff's rules]. / Décollement de rétine rhegmatogène : topographie des déhiscences et conformité aux lois de Lincoff.

PURPOSE: To study the topography of retinal breaks and their agreement with Lincoff's rules. MATERIALS AND METHODS: We performed a retrospective descriptive study of patients with recent rhegmatogenous retinal detachments followed on the ophthalmology service of Abass Ndao Hospital from January 2006 through December 2016. Patients with no prior retinal treatment were included. RESULTS: Over 11 years, we reviewed 97 patients with 107 eyes with retinal detachments. The mean age of our patients was 51.7 years, range 23-79 years. There were 69 male patients, for a male:female ratio of 2.46. Refraction revealed that 38.1% of patients were myopes. Fourteen percent (14%) of patients had experienced trauma to the eye with the detachment. The right eye was involved in 54.6% of patients. The onset was insidious in 54.6% of cases and sudden in 23.7% of cases. All patients had decreased visual acuity, associated with a scotoma in 26.8% of cases. Visual acuity was decreased to light perception through 7/10. In 64.9% of cases, Lincoff's rules were observed. DISCUSSION: Lincoff's rules are still relevant for localization of the breaks in retinal detachments. CONCLUSION: Diagnosis of a retinal detachment is an essential step, since it determines the treatment. Lincoff's rules still have a role in finding the retinal break in retinal detachments.

Effectiveness, treatment completion and safety of sofosbuvir/ledipasvir and paritaprevir/ritonavir/ombitasvir + dasabuvir in patients with chronic kidney disease: an ERCHIVES study.

BACKGROUND: Chronic kidney disease (CKD) was a relative contraindication to hepatitis C virus (HCV) treatment in the interferon/ribavirin era. AIM: To determine the efficacy, tolerability and safety of sofosbuvir/ledipasvir (SOF/LDV) and paritaprevir/ritonavir/ombitasvir/dasabuvir (PrOD) regimens in persons with CKD. METHODS: We identified persons initiated on a SOF/LDV or PrOD regimen from October 30, 2014 to April 30, 2016. We excluded those with missing HCV genotype or eGFR values. We determined treatment completion and sustained virologic response (SVR) rates, and proportion developing worsening renal function or grade 3/4 haematologic toxicity. RESULTS: Among 13 663 persons on SOF/LDV±ribavirin, 14% and 1% persons had CKD Stage 3 and 4-5 respectively, 67.8% completed treatment, 98.2% achieved SVR. Treatment completion or SVR rates did not decline with advanced CKD or ribavirin administration. Among 3961 persons on PrOD±ribavirin, 9% and 3% persons had CKD Stage 3 and 4-5, respectively, 74.0% completed treatment and 98.2% achieved SVR. A decrease in treatment completion rates was seen in CKD stage 4-5 and those on ribavirin, but this did not impact SVR rates. A >10 mL/min/1.73 m2 drop in eGFR from baseline was observed in 30%-38% of persons with baseline eGFR ≥60 mL/min/1.73 m2 , but in only 0%-6% with CKD4-5. Grade 3/4 anaemia was more frequent in persons with CKD4-5, but ribavirin co-administration did not appear to affect this. CONCLUSIONS: SOF/LDV and PrOD achieved high SVR rates in CKD population. Treatment completion rates were lower than expected. A decline in eGFR and development of anaemia were observed in a substantial proportion of persons, but the clinical implications remain unclear.

Hepatitis B reactivation and outcomes in persons treated with directly acting antiviral agents against hepatitis C virus: results from ERCHIVES.

BACKGROUND: Higher risk of hepatitis B reactivation (HBV-r) has been reported in patients with hepatitis C treated with newer directly acting antiviral agents (DAAs). AIM: To determine the proportion of persons who develop HBV-r and its clinical consequences among DAA treated vs pegylated interferon/ribavirin (PEG/RBV) treated persons. METHODS: We calculated the proportion of persons who developed HBV viral reactivation (HBV-r; new detectable HBV DNA or increase of >1 log10 ); serum alanine aminotransferase flare (>5 times baseline); all-cause mortality and hepatic decompensation in persons treated with a newer DAA regimen or PEG/RBV. Kaplan-Meier curves were used to demonstrate survival and hepatic decompensation by treatment group and HBV-r. RESULTS: In 34 632 persons treated with DAA and 23 475 treated with PEG/RBV, HBV-r rate per 1000 person-years was 30.04 (10.41, 49.67) and 25.42 (95% CI 17.23, 33.62) respectively (P = .8). When stratified by SVR or by baseline HBsAg status, HBV-r was not different between groups. Kaplan-Meier survival curves comparing each regimen stratified by presence or absence of HBV-r did not demonstrate a significant difference in incidence of hepatic decompensation over time. For overall survival, there was no difference between PEG/RBV treated persons with or without HBV-r. For DAA treated persons, those with HBV-r had a shortened survival, though the numbers at risk were small. CONCLUSIONS: HBV-r is relatively uncommon after DAA therapy and not higher than among those treated with a PEG/RBV regimen. The small numbers of persons treated with a DAA regimen who do develop HBV-r have a shortened survival compared to those without HBV-r.

Proton pump inhibitors are associated with accelerated development of cirrhosis, hepatic decompensation and hepatocellular carcinoma in noncirrhotic patients with chronic hepatitis C infection: results from ERCHIVES.

BACKGROUND: Proton pump inhibitors are among the most commonly prescribed medications in the United States. Their safety in cirrhosis has recently been questioned, but their overall effect on disease progression in noncirrhotic patients with chronic liver disease remains unclear. AIM: To determine the impact of proton pump inhibitors on the progression of liver disease in noncirrhotic patients with hepatitis C virus (HCV) infection. METHODS: Using the electronically retrieved cohort of HCV-infected veterans (ERCHIVES) database, we identified all subjects who received HCV treatment and all incident cases of cirrhosis, hepatic decompensation and hepatocellular carcinoma. Proton pump inhibitor use was measured using cumulative defined daily dose. Multivariate Cox regression analysis was performed after adjusting univariate predictors of cirrhosis and various indications for proton pump inhibitor use. RESULTS: Among 11 526 eligible individuals, we found that exposure to proton pump inhibitors was independently associated with an increased risk of developing cirrhosis (hazard ratio [HR]: 1.32; 95% confidence interval: [1.17, 1.49]). This association remained robust to sensitivity analysis in which only patients who achieved sustained virologic response were analysed as well as analysis excluding those with alcohol abuse/dependence. Proton pump inhibitor exposure was also independently associated with an increased risk of hepatic decompensation (HR: 3.79 [2.58, 5.57]) and hepatocellular carcinoma (HR: 2.01 [1.50, 2.70]). CONCLUSIONS: In patients with chronic HCV infection, increasing proton pump inhibitor use is associated with a dose-dependent risk of progression of chronic liver disease to cirrhosis, as well as an increased risk of hepatic decompensation and hepatocellular carcinoma.

Neonatal Acute Kidney Injury: A Survey of Neonatologists' and Nephrologists' Perceptions and Practice Management.

BACKGROUND: Neonatal acute kidney injury (AKI) occurs in 40 to 70% of critically ill neonatal intensive care admissions. This study explored the differences in perceptions and practice variations among neonatologists and pediatric nephrologists in diagnostic criteria, management, and follow-up of neonatal AKI. METHODS: A survey weblink was emailed to nephrologists and neonatologists in Australia, Canada, New Zealand, India, and the United States. Questions consisted of demographic and unit practices, three clinical scenarios assessing awareness of definitions of neonatal AKI, knowledge, management, and follow-up practices. RESULTS: Many knowledge gaps among neonatologists, and to a lesser extent, pediatric nephrologists were identified. Neonatologists were less likely to use categorical definitions of neonatal AKI (p < 0.00001) or diagnose stage 1 AKI (p < 0.00001) than pediatric nephrologists. Guidelines for creatinine monitoring for nephrotoxic medications were reported by 34% (aminoglycosides) and 62% (indomethacin) of respondents. Nephrologists were more likely to consider follow-up after AKI than neonatologists (p < 0.00001). Also, 92 and 86% of neonatologists and nephrologists, respectively, reported no standardization or infrastructure for long-term renal follow-up. CONCLUSION: Neonatal AKI is underappreciated, particularly among neonatologists. A lack of evidence on neonatal AKI contributes to this variation in response. Therefore, dissemination of current knowledge and areas for research should be the priority.

Prevalence of Diabetes in an Urban Hospital Emergency Room Serving Predominantly Indigenous Population.

OBJECTIVES: The purpose of this study was to examine the rate of diabetes and pre-diabetes in previously undiagnosed individuals who came to the Detroit Receiving Hospital-Ambulatory Rapid Center (DRH-ARC), which is part of the emergency room. This region has a high minority population with over 83% being African-Americans (AAs). Diabetes screening is especially important for this population because AAs are more prone to develop complications. METHODS: Free diabetes screening was done for all patients coming to the walk in unit of the DRH-ARC. This program was supported by the "Healthy Detroit-Diabetes Initiative". The initiative was developed by the Detroit Receiving Hospital and Wayne State University Physicians Group (WSUPG) administration. RESULTS: A total of 15,971 patients, who did not have a history of diabetes, consented for screening during the period of March 2010 through March 2014. A total of 6,149 (38.5%) patients were found to have HbAlc values in the range of pre-diabetes or diabetes. The prevalence of diabetes increased with age in both men and women. The data showed high prevalence of undiagnosed pre-diabetes and diabetes in this population approaching 31% and 8%, respectively. Among patients with elevated blood pressure 41.2% had abnormal HbAlc values. In contrast, 32% of patients with normal blood pressure had abnormal HbAlc values. CONCLUSION: Continued screening of population at risk for diabetes is essential. Public health awareness programs, such as the Healthy Detroit-Diabetes Initiative should be initiated in similar areas where minority populations are prevalent.

Critical role of NMDA but not opioid receptors in the acquisition of fat-conditioned flavor preferences in rats.

Animals learn to prefer flavors associated with the intake of dietary fats such as corn oil (CO) solutions. We previously reported that fat-conditioned flavor preferences in rats were relatively unaffected by systemic treatment with dopamine D1 and D2 antagonsits. The present study examined whether systemic opioid (naltrexone, NTX) or NMDA (MK-801) receptor antagonists altered the acquisition and/or expression of CO-CFP. The CFP was produced by training rats to drink one novel flavor (CS+, e.g., cherry) mixed in a 3.5% CO solution and another flavor (CS-, e.g., grape) in a 0.9% CO solution. In expression studies, food-restricted rats drank these solutions in one-bottle sessions (2 h) over 10 d. Subsequent two-bottle tests with the CS+ and CS- flavors mixed in 0.9% CO solutions occurred 0.5h after systemic administration of vehicle (VEH), NTX (0.1-5 mg/kg) or MK-801 (50-200 µg/kg). Rats displayed a robust CS+ preference following VEH treatment (85-88%) which was significantly though moderately attenuated by NTX (69-70%). The lower doses of MK-801 slightly reduced the CS+ preference; the high dose blocked the CS+ preference (49%) but also markedly reduced overall CS intake. In separate acquisition studies, rats received VEH or NTX (0.1, 0.5, 1mg/kg) or MK-801 (100 µg/kg) 0.5h prior to 1-bottle training trials with CS+/3.5% CO and CS-/0.9% CO training solutions. Additional Limited VEH groups were trained with intakes limited to that of the NTX and MK-801 groups. Subsequent two-bottle CS+ vs. CS- tests were conducted without injections. Significant and persistent CS+ preferences were observed in VEH (77-84%) and Limited VEH (88%) groups. NTX treatment during training failed to block the acquisition of CO-CFP although the magnitude of the CS+ preference was reduced by 0.5 (70%) and 1.0 (72%) mg/kg doses relative to the Limited VEH treatment (88%). In contrast, MK-801 (100 µg/kg) treatment during training blocked the acquisition of the CO-CFP. These data suggest a critical role for NMDA, but not opioid receptor signaling in the acquisition of a fat conditioned flavor preferences, and at best limited involvement of NMDA and opioid receptors in the expression of a previously learned preference.

Hypogonadism in patients with sickle cell disease: central or peripheral?

There is conflicting evidence in the literature on the etiology of hypogonadism in patients with sickle cell disease (SCD). A cross-sectional study was done to determine whether hypogonadism in male patients with SCD is due to primary testicular failure or secondary pituitary/hypothalamic dysfunction and assess the association between hypogonadism and serum ferritin levels. Hormonal assessment for serum concentrations of testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) was done for 34 men with SCD and their charts were reviewed for relevant clinical variables. Eight men (24%) were classified hypogonadal based on their serum testosterone levels. These men have significantly lower LH (p = 0.001) and FSH (p = 0.01) levels than normogonadal men, indicating a central etiology. There was no significant difference between hypogonadal and normogonadal men with respect to ferritin levels (p = 0.71). Our study indicates a central etiology of hypogonadism in patients with SCD. In this small study ferritin level was not significantly related to hypogonadism.

Effects of 3T3 adipocytes on interleukin-6 expression and insulin signaling in L6 skeletal muscle cells.

OBJECTIVE: Central adiposity and inflammation play key roles in the development of insulin resistance through the effects of pro-inflammatory adipokines such as IL-6, but the effect of infiltrating adipocytes in skeletal muscle tissues is not known. Communications between muscle cells and fat cells may contribute to the inflammatory response associated with insulin resistance. METHODS: In this study we used a co-culture system of skeletal muscle (L6) and adipocyte (3T3-L1) cell lines to study expression of the inflammatory cytokine IL-6 and changes in insulin signaling. This model could mimic the adipocytes infiltrating myocytes that is commonly seen in obese patients. RESULTS: When plated alone the L6 cells express IL-6 mRNA and secrete IL-6 protein, both of which are increased when the cells are challenged with the bacterial lipopolysaccharide (LPS). In contrast, the 3T3-L1 cells had very little expression of IL-6 mRNA or protein. Co-culture of 3T3-L1 pre-adipocytes with L6 cells, at a density ratio of 1:10, respectively, increased IL-6 expression significantly and decreased insulin-stimulated Akt phosphorylation. To examine the role of IL-6 in insulin sensitivity we incubated the L6 cells with IL-6. A brief challenge of L6 cells with IL-6 enhanced insulin-stimulated Akt phosphorylation. In contrast, incubation of the L6 cells with IL-6 for 96h markedly decreased insulin-stimulated Akt phosphorylation. CONCLUSION: The enhanced IL-6 mRNA expression and IL-6 release in L6 myocytes co-cultured with 3T3-L1 cells indicate an important interaction between adipocytes and myocytes. This observation may shed some light on the long-standing enigma of obesity-induced insulin resistance where infiltration of the skeletal muscle by preadipocytes/adipocytes is evident.

Involvement of thyrotropin-releasing hormone receptor, somatostatin receptor subtype 2 and corticotropin-releasing hormone receptor type 1 in the control of chicken thyrotropin secretion.

Thyrotropin or thyroid-stimulating hormone (TSH) secretion in the chicken is controlled by several hypothalamic hormones. It is stimulated by thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), whereas somatostatin (SRIH) exerts an inhibitory effect. In order to determine the mechanism by which these hypothalamic hormones modulate chicken TSH release, we examined the cellular localization of TRH receptors (TRH-R), CRH receptors type 1 (CRH-R1) and somatostatin subtype 2 receptors (SSTR2) in the chicken pars distalis by in situ hybridization (ISH), combined with immunological staining of thyrotropes. We show that thyrotropes express TRH-Rs and SSTR2s, allowing a direct action of TRH and SRIH at the level of the thyrotropes. CRH-R1 expression is virtually confined to corticotropes, suggesting that CRH-induced adrenocorticotropin release is the result of a direct stimulation of corticotropes, whereas CRH-stimulated TSH release is not directly mediated by the known chicken CRH-R1. Possibly CRH-induced TSH secretion is mediated by a yet unknown type of CRH-R in the chicken. Alternatively, a pro-opiomelanocortin (POMC)-derived peptide, secreted by the corticotropes following CRH stimulation, could act as an activator of TSH secretion in a paracrine way.

N-glycosylation of CRF receptor type 1 is important for its ligand-specific interaction.

The corticotropin-releasing factor (CRF) receptor type 1 (CRFR1) contains five potential N-glycosylation sites: N38, N45, N78, N90, and N98. Cells expressing CRFR1 were treated with tunicamycin to block receptor glycosylation. The nonglycosylated receptor did not bind the radioligand and had a decreased cAMP stimulation potency in response to CRF. To determine which of the polysaccharide chain(s) is/are involved in ligand interaction, the polysaccharide chains were deleted using site-directed mutagenesis of the glycosylation consensus, N-X-S/T. Two sets of mutations were performed for each glycosylation site: N to Q and S/T to A, respectively. The single mutants Q38, Q45, Q78, Q90, Q98, A40, A47, A80, A92, and A100 and the double mutants A40/A47 and A80/A100 were well expressed, bound CRF, sauvagine (SVG), and urotensin-I (UTS-I) with a normal affinity, and increased cAMP accumulation with a high efficiency. In contrast, the combined mutations A80/A92/A100, A40/A80/A92/A100, and A40/A47/A80/A92/A100 had low levels of expression, did not bind the radioligand, and had a decreased cAMP stimulation. These data indicate the requirement for three or more polysaccharide chains for normal CRFR1 function.

Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) binding to their common receptor stimulates second messenger accumulation, receptor phosphorylation, and internalization. LLC-PK(1) cells expressing a green fluorescent protein-tagged PTH/PTHrP receptor show time- and dose-dependent receptor internalization. The internalized receptors colocalize with clathrin-coated pits. Internalization is stimulated by PTH analogs that bind to and activate the PTH/PTHrP receptor. Cell lines expressing a mutant protein kinase A regulatory subunit that is resistant to cAMP and/or a mutant receptor (DSEL mutant) that does not activate phospholipase C internalize their receptors normally. In addition, internalization of the wild-type receptor and the DSEL mutant is stimulated by the PTH analog [Gly(1),Arg(19)]hPTH-(1-28), which does not stimulate phospholipase C. Forskolin, IBMX, and the active phorbol ester, phorbol-12-myristate-13-acetate, did not promote receptor internalization or increase PTH-induced internalization. These data indicate that ligand-induced internalization of the PTH/PTHrP receptor requires both ligand binding and receptor activation but does not involve stimulation of adenylate cyclase/protein kinase A or phospholipase C/protein kinase C.

An oxidation resistant radioligand for corticotropin-releasing factor receptors.

The methionine residues in Tyr-corticotropin-releasing factor (CRF) and Tyr-sauvagine radioligands are subject to oxidation, which renders them biologically inactive. Therefore [Tyr(0,) Gln(1,) Leu(17)]sauvagine (YQLS), in which the methionine was replaced with leucine was synthesized and labeled with (125)Iodine using chloramine-T. Mass spectroscopy revealed that chloramine-T-treatment did not oxidize YQLS. (125)I-YQLS bound with high affinity to cells expressing the murine CRF receptor 1 (CRFR1), CRF receptor 2 (CRFR2), and the mouse brain regions known to express both CRF receptors. (125)I-YQLS chemically cross-linked to CRFR1. In conclusion, (125)I-YQLS is oxidation-resistant, high affinity radioligand that can be chemically cross-linked to the CRF receptors.

Juxtamembrane region of the amino terminus of the corticotropin releasing factor receptor type 1 is important for ligand interaction.

The functional properties of the amino terminus (NT) of the corticotropin releasing factor (CRF) receptor type 1 (R1) were studied by use of murine (m) CRFR1 and rat (r) parathyroid hormone (PTH)/parathyroid hormone-related peptide receptor (PTH1R) chimeras. The chimeric receptor CXP, in which the NT of mCRFR1 was annealed to the TMs of PTH1R, and the reciprocal hybrid, PXC, bound radiolabeled analogues of sauvagine and PTH(3--34), respectively. Neither hybrid bound radiolabeled CRF or PTH(1--34). CRF and PTH(1--34) weakly stimulated intracellular cAMP accumulation in COS-7 cells transfected with PXC and CXP, respectively. Thus the NT is required for ligand binding and the TMs are required for agonist-stimulated cAMP accumulation. Replacing individual intercysteine segments of PXC with their mCRFR1 counterparts did not rescue CRF or sauvagine radioligand binding or stimulation of cAMP accumulation. Replacement of residues 1--31 of mCRFR1 with their PTH1R counterparts resulted in a chimeric receptor, PEC, which had normal CRFR1 functional properties. In addition, a series of chimeras (F1PEC--F6PEC) were generated by replacement of the NT intercysteine residues of PEC with their PTH1R counterparts. Only F1PEC, F2PEC, and F3PEC showed detectable CRF and sauvagine radioligand binding. All of the PEC chimeras except F5PEC increased cAMP accumulation. These data indicate that the Cys(68)(-)Glu(109) domain is important for binding and that the Cys(87)(-)Cys(102) region plays an important role in CRFR1 activation.

Characterization of three corticotropin-releasing factor receptors in catfish: a novel third receptor is predominantly expressed in pituitary and urophysis.

The present study reports the isolation of three complementary DNA (cDNA) clones encoding distinct subtypes of CRF receptors from the diploid catfish (cf) species, Ameiurus nebulosus. The first clone encodes a 446-amino acid protein (cfCRF-R1) that is highly homologous to mouse (m) CRF-R1 (93% identical). The cfCRF-R1 messenger RNA is highly expressed in the brain, and its distribution pattern correlates well with that of mammalian CRF-R1, except for weak expression in the pituitary. When transiently expressed in COS-7 cells, cfCRF-R1 bound CRF, urotensin I, and sauvagine with similar affinities. The second full-length cDNA, which was cloned from catfish heart, encodes a 406-amino acid protein that showed homology to murine CRF-R2 (88%) and when expressed in COS-7 cells preferentially bound sauvagine. The highest level of cfCRF-R2 expression was observed in the heart. The third full-length cDNA clone, which encodes a 428-amino acid protein, is structurally closer to cfCRF-R1 (85%) than to cfCRF-R2 (80%). This novel CRF receptor (cfCRF-R3) bound CRF with a 5-fold higher affinity than urotensin I and sauvagine and was expressed in the pituitary gland, urophysis, and brain. The presence of three different CRF receptors, each with distinct tissue distribution and ligand binding properties, suggests a complex CRF/urotensin I system.

The transcription factors SP1 and MAZ regulate expression of the parathyroid hormone/parathyroid hormone-related peptide receptor gene.

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor regulates extracellular calcium concentrations and is therefore important for mineral homeostasis. ROS 17/2.8 cells, a rat osteoblast-like osteosarcoma cell line, express the PTH/PTHrP receptor and provide a good model for examining the transcriptional regulation of its gene. The rat PTH/PTHrP receptor gene has two promoters, U1 and U3, which were shown to be important for its expression. Using extracts from ROS 17/2.8 cells, we have demonstrated two regions (termed FP1 and FP2) of nuclear protein/DNA interaction within promoter sequences previously shown to be important for the activity of the U3 promoter. Nuclear extracts from rat 2 fibroblasts, which do not express the PTH/PTHrP receptor, produced one site of protein/DNA interaction which was found at a position on the promoter identical to the position of FP1 produced by a ROS 17/2.8 nuclear extract. Mutation of these two sites of protein/DNA interaction resulted in reduced U3 promoter activity. Furthermore, we have demonstrated that the transcription factors SP1 and MAZ regulate U3 promoter expression and have shown their functional significance using mutational analysis. These data demonstrate that SP1 and MAZ bind to the PTH/PTHrP receptor promoter and that they are involved in cell-specific expression of its gene product.

Prospective evaluation of deep topical fornix nerve block versus peribulbar nerve block in patients undergoing cataract surgery using phacoemulsification.

We compared the efficacy of deep topical fornix nerve block anaesthesia (DTFNBA) versus peribulbar nerve block in patients undergoing cataract surgery using phacoemulsification. We studied 120 patients, allocated randomly to two groups. Group 1 (n = 60) received peribulbar block with 5 ml of a 1:1 mixture of 0.5% plain bupivacaine and 2% lidocaine supplemented with hyaluronidase 300 i.u. ml-1. Group 2 received DTFNBA with placement of a sponge soaked with 0.5% bupivacaine deep into the conjunctival fornices for 15 min. No sedation was given to either group. Analgesia was assessed by the reaction to insertion of the superior rectus suture and by questioning during the procedure. A three-point scoring system was used (no pain = 0, discomfort = 1, pain = 2). Scoring was repeated at keratotomy, hydrodissection and hydrodelineation, phacoemulsification, irrigation and aspiration, and at intraocular lens insertion. If the patient's pain score was 0 or 1, no further action was taken. If the pain score at any stage of the operation was 2, intracameral injection of 1% preservative-free lidocaine was given. One patient in Group 2 needed intracameral lidocaine at the stage of phacoemulsification (P > 0.05) and four experienced discomfort at irrigation and aspiration (P = 0.043). We conclude that DTFNBA may be a useful needle-free anaesthetic technique in patients undergoing cataract surgery using phacoemulsification.

Role of asparagine-linked oligosaccharides in the function of the rat PTH/PTHrP receptor.

The receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP) is a G-protein-coupled receptor with four potential sites for N-linked glycosylation. The contribution of the oligosaccharide moieties to cell surface expression, ligand binding, and signal transduction was investigated. Site-directed mutagenesis of the rat PTH/PTHrP receptor cDNA was performed at single or combination of the four potential glycosylation sites to determine the effect of the putative carbohydrate chains on the activities of the receptor. The results revealed that all four potential N-glycosylation sites in the PTH/PTHrP receptor are glycosylated. Receptors missing a single or multiple glycosylation consensus but with at least one intact glycosylation site expressed sufficiently and functioned normally. In contrast, the nonglycosylated receptor, in which all four glycosylation sites were mutated, is deficient in these functions. These data indicate important roles for N-linked glycosylation in PTH/PTHrP receptor functions.