The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes.

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 microM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 microM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in approximately 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 microM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 microM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 microM), which sensitizes the IP3R to IP3. In cells voltage clamped at -40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.

PHOTOPERTURBATION OF THE (1)A-][-(5)T SPIN EQUILIBRIUM IN AN IRON(II) COMPLEX IN SOLUTION VIA LIGAND-FIELD EXCITATION

Variable-temperature magnetic susceptibility measurements in the solid state of the bis complex of tris(1-pyrazolyl)-methane with Fe(II), [Fe(tpm)2](ClO4)2, suggest the existence of singlet-quintet spin crossover with the singlet isomer largely favored at room temperature. In acetonitrile solution, measurement of the absorption spectrum as a function of temperature reveals a spin equilibrium with the quintet population varying from ca. 6% at 233 K to ca. 30% at 295 K. When the complex in solution is irradiated with a laser pulse at wavelengths within the ligand field absorption band of the singlet isomer, ground-state depletion occurs within the pulse duration followed by fast recovery to the original absorbance level with a time constant of 25 +/- 5ns. The recovery time is virtually independent of temperature over the range +23 to -43-degrees-C, but the signal:noise ratio of the transient signals increases with decreasing temperature. The effect was observable at several monitoring wavelengths spanning the LF and MLCT absorption regions of the complex but only when the irradiation wavelength fell within the LF absorption region. Irradiation within the MLCT band produced no effect other than that of laser pulse scatter. The observations are interpreted in terms of photoperturbation of the singlet-quintet spin state equilibrium, which in this case occurs solely through excitation in the ligand field absorption region of the complex and is the first reported instance of this type for a spin-crossover complex in solution.

Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis.

INTRODUCTION: The dichotomization of non-small cell carcinoma (NSCLC) subtype into squamous (SQCC) and adenocarcinoma (ADC) has become important in recent years and is increasingly required with regard to management. The aim of this study was to determine the utility of a panel of commercially available antibodies in refining the diagnosis on small biopsies and also to determine whether cytologic material is suitable for somatic EGFR genotyping in a prospectively analyzed series of patients undergoing investigation for suspected lung cancer. METHODS: Thirty-two consecutive cases of NSCLC were first tested using a panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, 34betaE12, and a D-PAS stain for mucin, to determine their value in refining diagnosis of NSCLC. After this test phase, two further pathologists independently reviewed the cases using a refined panel that excluded 34betaE12 because of its low specificity for SQCC, and refinement of diagnosis and concordance were assessed. Ten cases of ADC, including eight derived from cytologic samples, were sent for EGFR mutation analysis. RESULTS: There was refinement of diagnosis in 65% of cases of NSCLC to either SQCC or ADC in the test phase. This included 10 of 13 cases where cell pellets had been prepared from transbronchial needle aspirates. Validation by two further pathologists with varying expertise in lung pathology confirmed increased refinement and concordance of diagnosis. All samples were adequate for analysis, and they all showed a wild-type EGFR genotype. CONCLUSION: A panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, and a D-PAS stain for mucin increases diagnostic accuracy and agreement between pathologists when faced with refining a diagnosis of NSCLC to SQCC or ADC. These small samples, even cell pellets derived from transbronchial needle aspirates, seem to be adequate for EGFR mutation analysis.

Transcriptional upregulation of SOCS 1 and suppressors of cytokine signaling 3 mRNA in the absence of suppressors of cytokine signaling 2 mRNA after infection with West Nile virus or tick-borne encephalitis virus.

Suppressors of cytokine signaling (SOCS) proteins are a family of proteins that are able to act in a classic negative feedback loop to regulate cytokine signal transduction. The regulation of the immune response by SOCS proteins may contribute to persistent infection or even a fatal outcome. In this study, we have investigated the induction of SOCS 1-3 after peripheral infection with West Nile virus (WNV) or tick-borne encephalitis virus (TBEV) in the murine model. We have shown that the cytokine response after infection of mice with WNV or TBEV induces an upregulation in the brain of mRNA transcripts for SOCS 1 and SOCS 3, but not SOCS 2. We hypothesize that SOCS proteins may play a role in limiting cytokine responses in the brain as a neuroprotective mechanism, which may actually enhance the ability of neuroinvasive viruses such as WNV and TBEV to spread and cause disease.

Posttranscriptional regulation of acute phase serum amyloid A2 expression by the 5'- and 3'-untranslated regions of its mRNA

Human acute-phase serum amyloid A protein (A-SAA) is a major acute phase reactant, the concentration of which increases dramatically as part of the body's early response to inflammation. A-SAA is the product of two almost identical genes, SAA1 and SAA2, which are induced by the pro-inflammatory cytokines, IL-1 and IL-6. In this study, we examine the roles played by the 5'- and 3'-untranslated regions (UTRs) of the SAA2 mRNA in regulating A-SAA2 expression. SAA2 promoter-driven luciferase reporter gene constructs carrying the SAA2 5'-UTR and/or 3'-UTR were transiently transfected into the HepG2 human hepatoma cell line. After induction of chimeric mRNA with IL-1beta and IL-6, the SAA2 5'- and 3'-UTRs were both able to posttranscriptionally modify the expression of the luciferase reporter. The SAA2 5'-UTR promotes efficient translation of the chimeric luciferase transcripts, whereas the SAA2 3'-UTR shares this property and also significantly accelerates the rate of reporter mRNA degradation. Our data strongly suggest that the SAA2 5'- and 3'-UTRs each play significant independent roles in the posttranscriptional regulation of A-SAA2 protein synthesis.

Expedient synthesis of (+)-trans-5-allylhexahydroindolizidin-3-one

Treatment of the bicyclic iminium ion derived from 5-methoxyhexahydroindolizidin-3-one with allyltrimethylsilane gave exclusively the diastereoisomer in which the allyl group was axial. This substrate is a useful precursor to 5-propyl-3-alkylindolizidine alkaloids. (C) 1999 Elsevier Science Ltd. All rights reserved.

Retinopathy in type 1 diabetes mellitus: Major differences between rural and urban dwellers in northwest Ethiopia

<p>Aim: To audit levels of diabetes-related eye disease in Type 1 diabetes mellitus (T1DM) patients in northwest Ethiopia. In particular to establish whether, despite identical clinical goals, major differences between the physically demanding life-style of rural subsistence farmers and the sedentary life-style of urban dwellers would influence the prevalence of diabetes-related eye complications. </p><p>Methods: A robust infrastructure for chronic disease management that comprehensively includes all rural dwellers was a pre-requisite for the investigation. A total of 544 T1DM were examined, representing 80% of all T1DM patients under regular review at both the urban and rural clinics and representative of patient age and gender (62.1% male, 37.9% female) of T1DM patients from this region; all were supervised by the same clinical team. Eye examinations were performed for visual acuity, cataract and retinal changes (retinal photography). HbA1c levels and the presence or absence of hypertension were recorded. </p><p>Results/conclusions: Urban and rural groups had similar prevalences of severe visual impairment/blindness (7.0% urban, 5.2% rural) and cataract (7.3% urban, 7.1% rural). By contrast, urban dwellers had a significantly higher prevalence of retinopathy compared to rural patients, 16.1% and 5.0%, respectively (OR 2.9, p &lt;. 0.02, after adjustment for duration, age, gender and hypertension). There was a 3-fold greater prevalence of hypertension in urban patients, whereas HbA1c levels were similar in the two groups. Since diabetic retinopathy is closely associated with microvascular disease and endothelial dysfunction, the possible influences of hypertension to increase and of sustained physical activity to reduce endothelial dysfunction are discussed.</p>

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala(8)-substituted analogues of GLP-1, (Abu(8))GLP-1 and (Val(8) GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu(8))GLP-1 and (Val(8))GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu(8))GLP-1 and (Val(8))GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val(8))GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu(8))GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val(8))GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala(8) in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val(8))GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Thermotropic Ruthenium(II)-Containing Metallomesogens Based on Substituted 1,10-Phenanthroline Ligands

Imidazo[4,5-f]-1,10-phenanthroline and pyrazino[2,3-f]-1,10-phenanthroline substituted with long alkyl chains are versatile ligands for the design of metallomesogens because of the ease of ligand substitution. Whereas the ligands and the corresponding rhenium(I) complexes were not liquid-crystalline, mesomorphism was observed for the corresponding ionic ruthenium(II) complexes with chloride, hexafluorophosphate, and bistriflimide counterions. The mesophases were identified as smectic A phases by high-temperature small-angle X-ray scattering (SAXS) using synchrotron radiation. The transition temperatures depend on the anion, the highest temperatures being observed for the chloride salts and the lowest for the bistriflimide salts. The ruthenium(II) complexes are examples of luminescent ionic liquid crystals.

WASP-37b: A 1.8 M J Exoplanet Transiting a Metal-poor Star

We report on the discovery of WASP-37b, a transiting hot Jupiter orbiting an m v = 12.7 G2-type dwarf, with a period of 3.577469 ± 0.000011 d, transit epoch T 0 = 2455338.6188 ± 0.0006 (HJD; dates throughout the paper are given in Coordinated Universal Time (UTC)), and a transit duration 0.1304+0.0018 –0.0017 d. The planetary companion has a mass M p = 1.80 ± 0.17 M J and radius R p = 1.16+0.07 –0.06 R J, yielding a mean density of 1.15+0.12 –0.15 ?J. From a spectral analysis, we find that the host star has M sstarf = 0.925 ± 0.120 M sun, R sstarf = 1.003 ± 0.053 R sun, T eff = 5800 ± 150 K, and [Fe/H] = –0.40 ± 0.12. WASP-37 is therefore one of the lowest metallicity stars to host a transiting planet.