Using GIS Mapping to Target Public Health Interventions: Examining Birth Outcomes Across GIS Techniques.

With advances in spatial analysis techniques, there has been a trend in recent public health research to assess the contribution of area-level factors to health disparity for a number of outcomes, including births. Although it is widely accepted that health disparity is best addressed by targeted, evidence-based and data-driven community efforts, and despite national and local focus in the U.S. to reduce infant mortality and improve maternal-child health, there is little work exploring how choice of scale and specific GIS visualization technique may alter the perception of analyses focused on health disparity in birth outcomes. Retrospective cohort study. Spatial analysis of individual-level vital records data for low birthweight and preterm births born to black women from 2007 to 2012 in one mid-sized Midwest city using different geographic information systems (GIS) visualization techniques [geocoded address records were aggregated at two levels of scale and additionally mapped using kernel density estimation (KDE)]. GIS analyses in this study support our hypothesis that choice of geographic scale (neighborhood or census tract) for aggregated birth data can alter programmatic decision-making. Results indicate that the relative merits of aggregated visualization or the use of KDE technique depend on the scale of intervention. The KDE map proved useful in targeting specific areas for interventions in cities with smaller populations and larger census tracts, where they allow for greater specificity in identifying intervention areas. When public health programmers seek to inform intervention placement in highly populated areas, however, aggregated data at the census tract level may be preferred, since it requires lower investments in terms of time and cartographic skill and, unlike neighborhood, census tracts are standardized in that they become smaller as the population density of an area increases.

Nutritional value of soybean meal produced from conventional, high-protein, or low-oligosaccharide varieties of soybeans and fed to broiler chicks.

Three experiments were conducted to determine the feeding value to broiler chicks of soybean meal (SBM) produced from high-protein (SBM-HP), low-oligosaccharide (SBM-LO), or conventional (SBM-CV) varieties of soybeans. The 3 SBM contained 54.9, 53.6, and 47.5% CP, respectively. The standardized digestibility (SDD) of amino acids (AA) in the 3 ingredients was measured using a precision-fed rooster assay with cecectomized Single Comb White Leghorn roosters. Results indicated that the SDD of AA was not different among the 3 sources of SBM, with the exception that the SDD of Lys in SBM-HP tended to be greater (P = 0.07) than that in SBM-CV. In the second experiment, a precision-fed rooster assay was used to measure the concentration of TME(n) in each source of SBM. Results indicated that the TME(n) in SBM-HP was greater (P < 0.001) than those in SBM-LO and SBM-CV (3,104 vs. 2,984 and 2,963 kcal/kg of DM). A 14-d growth performance experiment was also conducted using 120 Ross 308 male commercial broiler chicks (mean initial BW = 102.6 g) that were allotted to a completely randomized design. There were 5 chicks/pen and 8 replicate pens/diet. Three corn- and SBM-based diets were formulated based on the data for digestible AA and TME(n) that were measured in the previous experiments. Each source of SBM was used in 1 diet, but because of the greater concentrations of digestible AA in SBM-HP and SBM-LO than in SBM-CV, the inclusion of SBM-HP and SBM-LO were 31.21 and 32.60%, respectively, whereas an inclusion of 38.21% SBM-CV was used. There were no differences among the 3 diets for BW gain or feed efficiency, which indicated that the reduced inclusion rates of SBM-HP and SBM-LO compared with SBM-CV were not detrimental to broiler chick growth performance. It was concluded that, compared with SBM-CV, SBM-HP and SBM-LO are needed in lower concentrations in diets fed to broiler chicks because these 2 sources of SBM have a greater nutritional value than does SBM-CV.

Amino acid digestibility and concentration of digestible and metabolizable energy in soybean meal produced from conventional, high-protein, or low-oligosaccharide varieties of soybeans and fed to growing pigs.

Two experiments were conducted to determine AA digestibility and the concentration of DE and ME in 5 sources of soybean meal (SBM). The 5 sources included hexane-extracted SBM produced from high-protein soybeans (SBM-HP) and conventional soybeans (SBM-CONV), and mechanically extruded-expelled SBM produced from high-protein soybeans (EE-SBM-HP), low-oligosaccharide soybeans (EE-SBM-LO), and conventional soybeans (EE-SBM-CONV). Five diets that each contained 1 source of SBM and a N-free diet were used in Exp. 1 to determine AA digestibility in each meal. Twelve growing barrows (initial BW: 67.7 +/- 1.34 kg) were allotted to a replicated 6 x 6 Latin square design with 6 periods and 6 diets in each square. Each period lasted 7 d, and ileal digesta were collected on d 6 and 7 of each period. Results of the experiment showed that the standardized ileal digestibility (SID) of all AA except Trp was similar for SBM-HP and SBM-CONV, but EE-SBM-HP and EE-SBM-LO had greater (P < 0.05) SID of His, Ile, Lys, Thr, and Val than EE-SBM-CONV. The SID of all indispensable AA in EE-SBM-HP was greater (P < 0.05) than in SBM-HP. The SID of Arg, Ile, Leu, and Phe in EE-SBM-CONV was greater (P < 0.05) than in SBM-CONV, but the SID of Trp was also greater (P < 0.05) in SBM-CONV than in EE-SBM-CONV. Experiment 2 was conducted to measure DE and ME in the same 5 sources of SBM as used in Exp. 1. Forty-eight growing barrows (initial BW: 38.6 +/- 3.46 kg) were placed in metabolism cages and randomly allotted to 6 diets with 8 replicates per diet. A corn-based diet and 5 diets based on a mixture of corn and each source of SBM were formulated. Urine and feces were collected during a 5-d collection period, and values for DE and ME in each source of SBM were calculated using the difference procedure. Results showed that the ME in SBM-HP tended to be greater (P = 0.10) than in SBM-CONV (4,074 vs. 3,672 kcal/kg of DM). The ME in EE-SBM-HP also tended to be greater (P = 0.10) than in EE-SBM-CONV and in EE-SBM-LO (4,069 vs. 3,620 and 3,721 kcal/kg of DM), but there was no difference in ME between extracted and extruded-expelled meals. It is concluded that SBM-HP has a greater feeding value than SBM-CONV because of greater concentrations of digestible AA and ME. Likewise, EE-SBM-LO has a greater concentration of most indispensable AA than EE-SBM-CONV, but the concentration of ME is similar in these 2 meals. Results of this experiment also showed that AA digestibility values in extruded-expelled SBM are greater than in hexane-extracted SBM.

Kinematic analysis of upper extremity joint motion in children using posterior walkers.

This study applies an upper extremity model to analyze motion in 25 children with cerebral palsy using posterior walkers. The study indicates that throughout a gait cycle, the shoulders and wrist are in extension and the elbows are flexed. It also reveals that the elbows are the most asymmetrical joint of the upper extremities during walker-assisted ambulation.

The effect of muscle damage on strength and fatigue deficits.

Many studies have reported prolonged force deficits after a bout of resistance training. However there is a dearth of information on the neuromuscular mechanisms underlying these deficits. This study examined whether an acute bout of resistance training had prolonged detrimental effects on muscle activation and excitation-contraction coupling. Two groups of 16 subjects each were tested before resistance exercise and at 1, 3, 5, and 7 days postexercise. A dvnamic group was tested for concentric and eccentric 1 repetition maximum and 3-methylhistidine (3-MH). An isometric group was tested for maximal voluntary contraction, muscle inactivation, relative fatigue, and evoked twitch properties. Both groups experienced similar increases in pain, limb circumference, and decreased range of motion between 1 and 3 days postexercise. Decrements occurred with eccentric strength, maximal voluntary contraction, muscle inactivation, relative fatigue, twitch amplitude, and increases in 3-MH. Although muscle damage-induced characteristics (pain, swelling, range of motion, 3-MH) were not correlated with neuromuscular impairments (muscle activation, force output), disruption of excitation-contraction coupling may have contributed to decrements in fatigue.

Role of transglutaminase II in retinoic acid-induced activation of RhoA-associated kinase-2.

Transamidation is a post-translational modification of proteins mediated by tissue transglutaminase II (TGase), a GTP-binding protein, participating in signal transduction pathways as a non-conventional G-protein. Retinoic acid (RA), which is known to have a role in cell differentiation, is a potent activator of TGASE: The activation of TGase results in increased transamidation of RhoA, which is inhibited by monodansylcadaverine (MDC; an inhibitor of transglutaminase activity) and TGaseM (a TGase mutant lacking transglutaminase activity). Transamidated RhoA functions as a constitutively active G-protein, showing increased binding to its downstream target, RhoA-associated kinase-2 (ROCK-2). Upon binding to RhoA, ROCK-2 becomes autophosphorylated and demonstrates stimulated kinase activity. The RA-stimulated interaction between RhoA and ROCK-2 is blocked by MDC and TGaseM, indicating a role for transglutaminase activity in the interaction. Biochemical effects of TGase activation, coupled with the formation of stress fibers and focal adhesion complexes, are proposed to have a significant role in cell differentiation.

Effect of Host Plant Resistance and Reduced Rates and Frequencies of Fungicide Application to Control Potato Late Blight.

Field experiments were conducted during 1998 to 2000 to determine the response of commercial potato cultivars and advanced breeding lines (ABL) differing in susceptibility to foliar late blight (caused by Phytophthora infestans) to reduced rates and frequencies of residual, contact fungicide applications. When environmental conditions were most favorable for the development of late blight, the lowest application rate of the fungicides chlorothalonil or fluazinam (33% of the manufacturers' recommended application rate [MRAR]) gave unsatisfactory control of potato late blight. Under conditions moderately conducive for late blight development, effective control was achieved with 33 to 66% MRAR with either fungicide. The Michigan State University advanced selection, MSG274-3, was the least susceptible ABL tested and, during 1998 to 2000, late blight was effectively managed using reduced rates of fungicides. Application rates of chlorothalonil (33 to 100% MRAR) significantly reduced late blight in the cultivar Snowden (5-day application interval) compared with the nontreated control; whereas, late blight was not effectively controlled in Snowden even at 100% MRAR of chlorothalonil at either 10- or 15-day application intervals in 1999 or 2000. The ABL MSG274-3 was the least susceptible of all cultivars and ABL used in this study, and required minimal chemical protection against late blight. The study demonstrates that ABL with reduced susceptibility to late blight can be managed with reduced fungicide rates and longer application intervals, thus offering more economical control of this disease.

Cardiotrophin-1 increases angiotensinogen mRNA in rat cardiac myocytes through STAT3 : an autocrine loop for hypertrophy.

-Cardiotrophin-1, an interleukin-6-related cytokine, stimulates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and induces cardiac myocyte hypertrophy. In this study, we demonstrate that cardiotrophin-1 induces cardiac myocyte hypertrophy in part by upregulation of a local renin-angiotensin system through the JAK/STAT pathway. We found that cardiotrophin-1 increased angiotensinogen mRNA expression in cardiac myocytes via STAT3 activation. Tyrosine phosphorylation of STAT3 by cardiotrophin-1 treatment resulted in STAT3 homodimer binding to the St-domain in the angiotensinogen gene promoter, which lead to promoter activation in a transient transfection assay. Cardiotrophin-1-induced STAT3 tyrosine phosphorylation and binding to the St-domain were suppressed by AG490, a specific JAK2 inhibitor, which also attenuated cardiotrophin-1-stimulated angiotensinogen promoter activity. Cardiotrophin-1 did not activate the angiotensinogen gene promoter that contained a substitution mutation within the St-domain. Finally, losartan, an angiotensin II type 1 receptor antagonist, significantly attenuated cardiotrophin-1-induced hypertrophy of neonatal rat cardiac myocytes. Angiotensin II is known to induce cardiac myocyte hypertrophy by activating the G-protein-coupled angiotensin II type 1 receptor. Our results suggest that upregulation of angiotensinogen and angiotensin II production contribute to cardiotrophin-1-induced cardiac myocyte hypertrophy and emphasize an important interaction between G-protein-coupled and cytokine receptors.

Regulation of angiotensinogen gene expression and protein in neonatal rat cardiac fibroblasts by glucocorticoid and beta-adrenergic stimulation.

We previously demonstrated the presence of components for a renin-angiotensin system in fibroblasts cultured from neonatal rat ventricles, the regulation of expression of which has not been studied. Since glucocorticoids and beta-adrenergic stimuli have been implicated in cardiac hypertrophy, and function as regulators of the circulating renin-angiotensin system, we examined the effects of dexamethasone and isoproterenol on angiotensinogen mRNA levels and protein secretion in cultured neonatal rat cardiac fibroblasts. Treatment of cardiac fibroblasts for 8 h with 10 micromol/l isoproterenol or 100 nmol/l dexamethasone increased angiotensinogen mRNA levels by 246 +/- 7% and 1406 +/- 207%, respectively. Over 24 h, dexamethasone and isoproterenol increased angiotensinogen secretion by 148 +/- 32% and 123 +/- 26%, respectively. Angiotensin II, which has been reported to be a positive regulator of angiotensinogen synthesis and secretion in liver, markedly attenuated the effects of dexamethasone and isoproterenol on angiotensinogen mRNA expression and secretion. In the presence of 1 micromol/l angiotensin II, the stimulation in angiotensinogen secretion observed with dexamethasone and isoproterenol was decreased by 62% and 76%, respectively. The negative feedback of angiotensin II on angiotensinogen expression was primarily mediated through the type one angiotensin II (AT1) receptor (IC50 = 0.30 +/- 0.02 nmol/l). In summary, results from this study demonstrate that angiotensinogen mRNA levels and protein secretion in cardiac fibroblasts are positively regulated by glucocorticoid and beta-adrenergic stimulation. In addition, angiotensinogen production by cardiac fibroblasts is under negative feedback control of angiotensin II.

The cardiac renin-angiotensin system: conceptual, or a regulator of cardiac function?

Angiotensin II, the effector peptide of the renin-angiotensin system, regulates cellular growth in response to developmental, physiological, and pathological processes. The identification of renin-angiotensin system components and angiotensin II receptors in cardiac tissue suggests the existence of an autocrine/paracrine system that has effects independent of angiotensin II derived from the circulatory system. To be functional, a local renin-angiotensin system should produce sufficient amounts of the autocrine and/or paracrine factor to elicit biological responses, contain the final effector (angiotensin II receptor), and respond to humoral, neural, and/or mechanical stimuli. In this review, we discuss evidence for a functional cardiac renin-angiotensin system.

Angiotensin II-stimulated induction of sis-inducing factor is mediated by pertussis toxin-insensitive G(q) proteins in cardiac myocytes.

The Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is stimulated by angiotensin II (Ang II) via the type 1 receptor after acute pressure overload in the heart. The purpose of this study was to determine whether activation of the JAK-STAT pathway by Ang II is dependent on G proteins. Ang II (100 nmol/L for 120 minutes) caused formation of sis-inducing factor (SIF) complexes and tyrosine phosphorylation of STAT proteins in neonatal rat ventricular myocytes. The percentage of change in Ang II-stimulated SIF induction was not affected by pertussis toxin (PTX) or GP antagonist-2A, compounds that inhibit activation of G(i) and G(o) proteins. In contrast, GP antagonist-2A, a peptide that selectively inhibits activation of G(q) proteins, completely abolished Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation. Pretreatment of cardiac myocytes with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) activity, decreased Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation in a dose-dependent manner. Chelation of intracellular Ca(2+) with BAPTA-AM did not alter Ang II-stimulated SIF induction. In contrast, pretreatment of cardiac myocytes with Ro-31-8220, a potent and specific inhibitor of protein kinase C (PKC), decreased Ang II-stimulated SIF induction in a dose-dependent manner. Ang II-stimulated SIF induction was abolished in cardiac myocytes after downregulation of PKC by treatment with PMA. From these data, we conclude that Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation is mediated by PTX-insensitive G proteins through a G(q)-PLC-PKC-mediated pathway in neonatal rat ventricular myocytes.

Amplification of angiotensin II signaling in cardiac myocytes by adenovirus-mediated overexpression of the AT1 receptor.

Low levels of AT1 receptor can make studying the growth-related signal transduction events mediated by this angiotensin II receptor in cardiac myocytes technically difficult. The purpose of the present study was to establish whether an adenovirus expression system could be used to increase the number of plasma membrane AT1 receptors in neonatal rat ventricular myocytes, thereby amplifying the signaling pathways activated by this receptor. Cardiac myocytes infected with adenovirus expressing the AT1 receptor exhibited increased ligand binding. The overexpressed receptor appeared to function like the endogenous receptor, in regard to agonist-induced internalization, as well as coupling to MAPK activation and protein tyrosine phosphorylation events. In addition, adenovirus-mediated overexpression of the AT1 receptor resulted in the amplification of angiotensin II intracellular signaling. In conclusion, adenovirus-mediated overexpression of angiotensin II receptors appears to be a useful strategy for studying the signal transduction events activated by this hormone in cardiac myocytes and for unraveling the molecular means by which this receptor type couples to a hypertrophic pattern of growth and gene expression.

Paracrine actions of cardiac fibroblasts on cardiomyocytes: implications for the cardiac renin-angiotensin system.

Conditioned medium of cardiac fibroblasts was found to induce protein synthesis and signal transduction events rapidly, and to increase angiotensinogen messenger RNA (mRNA) levels in neonatal rat ventricular myocytes. Within 4 hours, fibroblast-conditioned medium (FCM) stimulated protein synthesis in cardiac myocytes, independent of the contractile state, and induced marked increases within 24 hours in total protein content. Endothelin- released by cardiac fibroblasts was not responsible for the stimulation of protein synthesis. FCM rapidly activated signal transduction events in cardiac myocytes associated with hypertrophic stimuli, including: (1) increased tyrosine phosphorylation of several prominent protein bands; (2) mitogen-activated protein kinases (ERK 1 and ERK 2); and (3) protein kinase C. Finally, FCM caused an increase at 8 hours in angiotensinogen mRNA levels of cardiac myocytes, whereas no effect was observed on mRNA levels for renin or the type 1 angiotensin II receptor (AT1). Our results suggest that cardiac fibroblasts produce a factor that rapidly activates cardiac myocyte growth through a membrane receptor that couples to conventional signal transduction pathways.

Highly corrected submicrometer grid patterning on curved surfaces.

A compact holographic projector system was built and tested. This projection system offers a practical approach for making a highly corrected mesh or grid pattern on curved surfaces. The pattern can range in size from multimicrometer to submicrometer dimensions and be recorded in either positive or negative photoresist. Standing-wave interference patterns in the form of a diverging close-packed lattice of either hexagonal or square rodlike intensity maxima extending outward from a point or a locus of points are produced by multiple-beam holography that involves the combination of a holographic diffraction grating and a hypercomatic focusing objective.

Highly corrected close-packed microlens arrays and moth-eye structuring on curved surfaces.

The fabrication of near-micrometer-sized close-packed coherent microlens arrays on spheric or aspheric surfaces has been accomplished by use of a compact holographic projector system that was developed for producing multimicrometer down to submicrometer grid patterning on curved surfaces. The microlens arrays, which can be utilized as moth-eye relief structures, are formed in a photoimageable bisbenzocyclobutene polymeric resin by a photolytic process involving standing-wave interference patterns from the holographic projector system. Because of absorption, each integral microlenslet of the finished arrays possesses a near-paraboloid contour. The trajectories of the meridional rays from each microlenslet can be optimized to intersect at either a single point or a locus of points.

Phosphorylation of the angiotensin II (AT1A) receptor carboxyl terminus: a role in receptor endocytosis.

The molecular mechanism of angiotensin II type I receptor (AT1) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr332Lys333Met334Ser335Thr336Leu337 Ser338) within the carboxyl terminus of the AT1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys325, which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated 32P incorporation into the AT1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr332, Ser335, Thr336, and Ser338, previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar(1)Ile8-Ang II, which paradoxically causes internalization of wild-type AT1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT1A receptors and receptors mutated within the Thr332-Ser338 region were compared. The four putative phosphorylation sites (Thr332, Ser335, Thr336, and Ser338) were substituted with either neutral [alanine (A)] or acidic amino acids [glutamic acid (E) and aspartic acid (D)], the former to prevent phosphorylation and the latter to reproduce the acidic charge created by phosphorylation. Wild-type AT1A receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t1/2 2.3 min; maximal level of internalization (Ymax) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t1/2 2.7 min, Ymax 76.3%; S335D, t1/2 2.4 min, Ymax 76.7%; T336E, t1/2 2.5 min, Ymax 78.2%; S338D, t1/2 2.6 min, Ymax 78.4%). While acidic amino acid substitutions may simply be not as structurally disruptive as alanine mutations, we interpret the tolerance of a negative charge in this region as suggestive that phosphorylation may permit maximal internalization. Substitution of all four residues to alanine produced a receptor with markedly reduced internalization kinetics (T332A/S335A/T336A/S338A, t1/2 10.1 min, Ymax 47.9%), while endocytosis was significantly rescued in the corresponding quadruple acidic mutant (T332E/S335D/T336E/S338D, t1/2 6.4 min, Ymax 53.4%). Double mutation of S335 and T336 to alanine also diminished the rate and extent of endocytosis (S335A/T336A, 3.9 min, Ymax 69.3%), while the analogous double acidic mutant displayed wild type-like endocytotic parameters (S335D/T336E, t1/2 2.6 min, Ymax 77.5%). Based on the apparent rescue of internalization by acidic amino acid substitutions in a region that we have identified as a site of Ang II-induced phosphorylation, we conclude that maximal endocytosis of the AT1A receptor requires phosphorylation within this serine/threonine-rich segment of the carboxyl terminus.

Half-Fourier RARE MR cholangiopancreatography: experience in 300 subjects.

PURPOSE: To determine prospectively the clinical applications and diagnostic accuracy of half-Fourier rapid acquisition with relaxation enhancement (RARE) magnetic resonance (MR) cholangiopancreatography (MRCP) in a large patient population. MATERIALS AND METHODS: Breath-hold, heavily T2-weighted half-Fourier RARE MRCP was performed in 265 patients with suspected pancreaticobiliary disease and in 35 control patients without symptoms or signs referrable to the biliary tract or pancreatic duct. MRCP findings were correlated with those at direct cholangiography, pathologic examination, cross-sectional imaging, and clinical follow-up. RESULTS: Diagnostic MRCP examinations were obtained in 299 (99.7%) subjects. MRCP yielded an accuracy of 100% in determining the presence of pancreaticobiliary disease, the presence and level of biliary obstruction, and obstruction due to bile duct calculi. The accuracy of MRCP and MR imaging in determining the presence and level of malignant obstruction was 98.2%. MRCP obviated endoscopic retrograde cholangiopancreatography (ERCP) by excluding choledocholithiasis in patients with acute pancreatitis (n = 13) and nonspecific abdominal pain (n = 82). In patients with sclerosing cholangitis and acquired immunodeficiency syndrome cholangiopathy, MRCP depicted the biliary tract as clearly as did ERCP (n = 9). After failed ERCP, MRCP delineated the pancreaticobiliary tract and helped determine therapeutic options (n = 27). CONCLUSION: Half-Fourier RARE MRCP enables accurate evaluation of pancreaticobiliary disease and obviates ERCP in some patients.

alpha-Thrombin inhibits signal transducers and activators of transcription 3 signaling by interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor in CCL39 cells.

We recently demonstrated that, in rat aortic smooth muscle cells, alpha-thrombin stimulated Stat3/SIF-A (signal transducers and activators of transcription 3/sis-inducing factor-A) activity [G. J. Bhat et al. (1997) Hypertension 29(Pt. 2), 356-360]. In the present study, we observed that exposure of CCL39 cells (a Chinese hamster lung fibroblast cell line) to alpha-thrombin resulted in a time-dependent decrease in basal SIF-A activity. We hypothesized that the decrease in basal SIF-A was due to the initiation of an inhibitory pathway, following alpha-thrombin exposure. To test this hypothesis, we determined if alpha-thrombin would inhibit Stat3 and SIF-A activation by interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF). In support of this hypothesis, alpha-thrombin inhibited the Stat3/SIF-A response induced by all the above cytokines. The inhibition by alpha-thrombin was concentration dependent, was sensitive to hirudin, and was mimicked by the thrombin receptor agonist peptide. The inhibition did not require the activation of phorbol 12-myristate 13-acetate-sensitive isoforms of protein kinase C and was reversed by pretreatment with the mitogen-activated protein kinase kinase 1 (MAPKK1 or MEK1) inhibitor PD98059. Inhibitory cross talk between alpha-thrombin and IL-6 was also observed in MRC-5 cells, a fibroblast cell line derived from human lung tissue. Thus, we identify a novel alpha-thrombin inhibitory pathway which, acting through a MAPKK1-dependent mechanism, blocks IL-6-, LIF-, and CNTF-induced Stat3/SIF-A activation. This inhibitory cross talk may provide an important regulatory function to modulate gene transcription by these cytokines, during immune and inflammatory responses.

Functional role for the angiotensin II receptor (AT1A) 3'-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins.

We demonstrate a functional role for the 3'-untranslated region (3'-UTR) of the angiotensin II (Ang II) receptor subtype AT1A mRNA in Chinese hamster ovary (CHO-K1) cells by stably transfecting the coding region of the receptor gene with or without the 845 bp 3'-UTR. Two cell lines expressing similar levels of cell-surface receptors (with 3'-UTR, Bmax=571 fmol/mg protein; without 3'-UTR, Bmax=663 fmol/mg protein) were used in the present study. Both cell lines expressed high-affinity receptors (with 3'-UTR, Kd=0.83 nM; without 3'-UTR, Kd=0.82 nM), and binding studies with 125I-labelled Ang II in the presence of GTP[S] demonstrated that both coupled to heterotrimeric G-proteins. Despite these similarities, significant differences were observed for receptor-mediated cell signalling pathways. In cells without the 3'-UTR, Ang II stimulated an increase in cAMP accumulation (11-fold above control) and in cells with the 3'-UTR no stimulation was observed, which was consistent with previous observations in most endogenous Ang II receptor (AT1)-expressing cells. Activation of cAMP by Ang II in cells without the 3'-UTR correlated with an inhibition of DNA synthesis, determined by [3H]thymidine incorporation. Ang II-mediated responses were blocked by EXP3174, a selective non-peptide receptor antagonist. We also observed differences in the transient profiles of intracellular calcium between cells with and without the 3'-UTR in response to Ang II. In cells with the 3'-UTR, a sustained level of intracellular calcium was observed after Ang II stimulation, whereas cells without the 3'-UTR displayed a full return to basal level within 50 s of Ang II treatment. Even though the expressed exogenous gene is under the control of a constitutively expressing promoter (cytomegalovirus promoter), Northern-blot analysis revealed a considerably greater accumulation of AT1A mRNA in cells without the 3'-UTR compared with cells with the 3'-UTR. Analysis of the decay rate of the AT1A mRNA in cells with and without the 3'-UTR revealed that the normally unstable AT1A receptor mRNA became highly stable by removing its 3'-UTR, identifying a role for the 3'-UTR in mRNA destabilization. Interestingly, both cells express similar levels of receptors at the cell surface, suggesting that the 3'-UTR is also involved in the efficient translation and/or translocation of the receptor protein to the plasma membrane. We hypothesized that these 3'-UTR-mediated functions of the receptor are regulated by RNA-binding proteins. To identify possible RNA-binding proteins for the AT1A 3'-UTR, cellular extracts were prepared from parental CHO-K1 cells and 3'-UTR-binding assays, electrophoretic mobility-shift assays and UV crosslinking studies were performed. A major cellular protein of 55 kDa was identified, which specifically interacted with the 3'-UTR. Our data suggest that the 3'-UTR of the AT1A can control specific receptor functions, perhaps via selective recognition of the 3'-UTR by RNA-binding proteins.