Phosphatidylinositol 3'-OH kinase and protein kinase A pathways mediate the anti-apoptotic effect of pituitary adenylyl cyclase-activating polypeptide in cultured cerebellar granule neurons: modulation by ethanol

Cerebellar granule neurons cultured in the presence of 5 mm KCl undergo spontaneous apoptosis, which is reduced by exposure to pituitary adenylyl cyclase-activating polypeptide (PACAP). Previous work has suggested roles for the cyclic AMP/PKA and MAP kinase signaling pathways in the anti-apoptotic effect of PACAP. In the present study, the use of specific inhibitors confirmed the role of the cyclic AMP/PKA pathway, and also demonstrated a role for the phosphatidylinositol 3'-OH kinase (PI 3-kinase) neuroprotective pathway in the action of PACAP. Ethanol exposure accelerates the anti-apoptotic effect of PACAP by a mechanism that involves the PKA and PI-3 kinase pathways. The results demonstrate that ethanol can increase neuroprotection induced by PACAP. As previous work has shown that ethanol can increase apoptosis of cerebellar granule neurons by inhibiting the protective effect of agents such as NMDA or IGF-1, the overall effect of ethanol on cerebellar neuron apoptosis during development may reflect the balance between inhibition and enhancement of the actions of various endogenous neuroprotective agents.     

Nicastrin functions as a gamma-secretase-substrate receptor

gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.     

Near-infrared fluorescent RGD peptides for optical imaging of integrin alphavbeta3 expression in living mice

Near-infrared fluorescence optical imaging is a powerful technique for studying diseases at the molecular level in preclinical models. We recently reported that monomeric RGD peptide c(RGDyK) conjugated to the NIR fluorescent dye specifically targets integrin receptor both in cell culture and in living subjects. In this report, Cy5.5-conjugated mono-, di-, and tetrameric RGD peptides were evaluated in a subcutaneous U87MG glioblastoma xenograft model in order to investigate the effect of multimerization of RGD peptide on integrin avidity and tumor targeting efficacy. The binding affinities of Cy5.5-conjugated RGD monomer, dimer, and tetramer for alpha(v)beta(3) integrin expressed on U87MG cell surface were determined to be 42.9 +/- 1.2, 27.5 +/- 1.2, and 12.1 +/- 1.3 nmol/L, respectively. All three peptide-dye conjugates had integrin specific uptake both in vitro and in vivo. The subcutaneous U87MG tumor can be clearly visualized with each of these three fluorescent probes. Among them, tetramer displayed highest tumor uptake and tumor-to-normal tissue ratio from 0.5 to 4 h postinjection. Tumor-to-normal tissue ratio for Cy5.5-conjugated RGD monomer, dimer, and tetramer were found to be 3.18 +/- 0.16, 2.98 +/- 0.05, and 3.63 +/- 0.09, respectively, at 4 h postinjection. These results suggest that Cy5.5-conjugated monomeric, dimeric, and tetrameric RGD peptides are all suitable for integrin expression imaging. The multmerization of RGD peptide results in moderate improvement of imaging characteristics of the tetramer, compared to that of the monomer and dimeric counterparts.     

Prevalence of Undiagnosed Depression among Persons with Hypertension and Associated Risk Factors: A Cross-Sectional Study in Urban Nepal

BackgroundDespite an increasing number of studies exploring prevalence of depression among hypertensive patients in high income countries, limited data is available from low and middle income countries, particularly Nepal. Our aim was to investigate the prevalence of undiagnosed (sub clinical) depression and associated risk factors among hypertensive patients attending a tertiary health care clinic in Nepal.MethodsThe study was based on a cross-sectional study design, with 321 hypertensive patients attending the Out-Patient Department of a central hospital in Nepal. Blood measure was recorded via a mercury column sphygmomanometer. Depression levels were assessed using the Beck Depression Inventory-Ia (BDI) scale. Demographics and risk factors were assessed.ResultThe proportion of participants with undiagnosed depression was 15%. Multivariable analyses demonstrated an increase in BDI scores with increased aging. Approximately a 1 point increase in the BDI score was observed for each additional decade of aging in hypertensive patients. Additional factors associated with increased risk of depression included being female (4.28 point BDI score increase), smoking (5.61 point BDI score increase), being hypertensive with no hypertensive medication (4.46 point BDI score increase) and being illiterate (4.46 point BDI score increase).ConclusionsAmong persons with hypertension in outpatient settings in Nepal, demographic (age, sex, education), behavioural (smoking,) and adherence factors (anti-hypertensive medication) were associated with undiagnosed depression. Screening programs in Nepal may assist early intervention in hypertensive patients with sub clinical depression.     

The Impact of Dyspepsia on Symptom Severity and Quality of Life in Adults with Headache

Background

Dyspepsia and headache frequently co-exist, but the clinical implication of this association is uncertain. We planned to examine the prevalence and impact of dyspepsia in adults with headache.

Methods

A cross-sectional study was conducted in a secondary care setting. Clinical, psychological and health-related quality of life (HRQOL) data were compared between subjects with headache and controls (non-headache subjects). The impact of dyspepsia was analysed further in subjects with headache alone.

Results

280 subjects (93 cases with headache and 187 matched controls) were recruited. The following baseline characteristics of subjects were as follows: mean age 45.0±17.3 years, 57.0% females and ethnic distribution—Malaysian = 45 (48.4%), Chinese n = 24 (25.8%) and Indians n = 24 (25.8%). Headache sub-types among cases with headache were as follows: tension-type headache (TTH) n = 53 (57.0%) and migraine n = 40 (43.0%). Dyspepsia was more prevalent in cases with headache compared to controls (25.8% vs 12.8%, p = 0.011), and headache was independently associated with dyspepsia (OR 2.75, 95% CI 1.39–5.43). Among cases with headache, there was a trend towards a higher prevalence of dyspepsia in those with migraine (27.5%) compared to TTH (24.5%). Subjects with headache and dyspepsia, compared to those with headache alone, had a greater severity of headache symptoms (63.67±22.85 mm vs 51.20 ±24.0 mm VAS, p = 0.029). Overall HRQOL scores were lower in headache subjects with dyspepsia (EQ-5D summary score 0.82±0.18 vs 0.90 ±0.16, p = 0.037 and EQ-5D VAS 62.08±17.50 mm vs 72.62 ±18.85 mm, p = 0.018), compared to those without dyspepsia.

Conclusion

Dyspepsia is associated with more severe headache symptoms and results in a lower HRQOL in patients with headache.     

An Integrated Computational/Experimental Model of Lymphoma Growth

Non-Hodgkin''s lymphoma is a disseminated, highly malignant cancer, with resistance to drug treatment based on molecular- and tissue-scale characteristics that are intricately linked. A critical element of molecular resistance has been traced to the loss of functionality in proteins such as the tumor suppressor p53. We investigate the tissue-scale physiologic effects of this loss by integrating in vivo and immunohistological data with computational modeling to study the spatiotemporal physical dynamics of lymphoma growth. We compare between drug-sensitive Eμ-myc Arf-/- and drug-resistant Eμ-myc p53-/- lymphoma cell tumors grown in live mice. Initial values for the model parameters are obtained in part by extracting values from the cellular-scale from whole-tumor histological staining of the tumor-infiltrated inguinal lymph node in vivo. We compare model-predicted tumor growth with that observed from intravital microscopy and macroscopic imaging in vivo, finding that the model is able to accurately predict lymphoma growth. A critical physical mechanism underlying drug-resistant phenotypes may be that the Eμ-myc p53-/- cells seem to pack more closely within the tumor than the Eμ-myc Arf-/- cells, thus possibly exacerbating diffusion gradients of oxygen, leading to cell quiescence and hence resistance to cell-cycle specific drugs. Tighter cell packing could also maintain steeper gradients of drug and lead to insufficient toxicity. The transport phenomena within the lymphoma may thus contribute in nontrivial, complex ways to the difference in drug sensitivity between Eμ-myc Arf-/- and Eμ-myc p53-/- tumors, beyond what might be solely expected from loss of functionality at the molecular scale. We conclude that computational modeling tightly integrated with experimental data gives insight into the dynamics of Non-Hodgkin''s lymphoma and provides a platform to generate confirmable predictions of tumor growth.     

Purification and functional analysis of protein kinase G-1α using a bacterial expression system

3',5' Cyclic guanosine monophosphate (cGMP)-dependent protein kinase G-1α (PKG-1α) is an enzyme that is a target of several anti-hypertensive and erectile dysfunction drugs. Binding of cGMP to PKG-1α produces a conformational change that leads to enzyme activation. Activated PKG-1α performs important roles both in blood vessel vasodilation and in maintaining the smooth muscle cell in a differentiated contractile state. Recombinant PKG-1α has been expressed and purified using Sf9-insect cells. However, attempts at purifying full length protein in a soluble and active form in prokaryotes have thus far been unsuccessful. These attempts have been hampered by the lack of proper eukaryotic protein folding machinery in bacteria. In this study, we report the successful expression and purification of PKG-1α using a genetically engineered Escherichia coli strain, Rosetta-gami 2(DE3), transduced with full-length human PKG-1α cDNA containing a C-terminal histidine tag. PKG-1α was purified to homogeneity using sequential nickel affinity chromatography, gel filtration and ion exchange MonoQ columns. Protein identity was confirmed by immunoblot analysis. N-terminal sequencing using Edman degradation demonstrated that the purified protein was full length. Analysis of enzyme kinetics, using a nonlinear regression curve, identified that, at constant cGMP levels (10μM) and varying ATP concentrations, PKG-1α had a maximal velocity (V(max)) of 5.02±0.25pmol/min/μg and a Michaelis-Menten constant (K(m)) of 11.78±2.68μM ATP. Recent studies have suggested that endothelial function can be attenuated by oxidative and/or nitrosative stress but the role of PKG-1α under these conditions is unclear. We found that PKG-1α enzyme activity was attenuated by exposure to the NO donor, spermine NONOate, hydrogen peroxide, and peroxynitrite but not by superoxide, suggesting that the attenuation of PKG-1α activity may be an under-appreciated mechanism underlying the development of endothelial dysfunction in a number of cardiovascular diseases.     

PCR markers for selection of adult plant leaf rust resistance in barley (Hordeum vulgare L.)

Adult plant resistance (APR) is considered potentially more durable for controlling barley leaf rust than seedling Rph (Resistance to Puccinia hordei) genes. A major gene for adult plant resistance to barley leaf rust has been mapped to the telomere region of chromosome 5HS. PCR-based molecular markers were developed for saturation of this region based on previously mapped simple sequence repeat, restriction fragment length polymorphism and Diversity Arrays Technology markers. In addition, defence gene homologue (DGH) and wheat expressed sequence tags mapped in specific bins were used to develop new PCR markers. Seventeen PCR-based markers were mapped to the short arm of chromosome 5H in 292 doubled haploid lines from a cross of Pompadour × Stirling, in which seven markers were mapped within 5 cM of the APR gene. The closest linked marker was about 0.7 cM from the APR gene. The wheat deletion bin map together with defence gene homologues was demonstrated to be an efficient tool for development of new molecular markers associated with the disease resistance gene. Four DGH markers were associated with the APR gene. The new molecular markers are a useful tool for marker-assisted selection of the APR gene and provided a better understanding of the molecular mechanism for leaf rust resistance.     

Right heart structural changes are independently associated with exercise capacity in non-severe COPD

Background

Pulmonary hypertension (PH) occurs frequently and results in functional limitation in advanced COPD. Data regarding the functional consequence of PH in less severe COPD are limited. Whether echocardiographic evidence of right sided heart pathology is associated with functional outcomes in patients with non-severe COPD is unknown.

Methods

We evaluated pulmonary function, six minute walk distance, and echocardiography in 74 consecutive patients with non-severe COPD. We performed multivariable linear regression to evaluate the association between right heart echocardiographic parameters and six minute walk distance adjusting for lung function, age, sex, race, and BMI.

Main Results

The mean six minute walk distance was 324±106 meters. All subjects had preserved left ventricular (LV) systolic function (LV ejection fraction 62.3%±6.1%). 54.1% had evidence of some degree of diastolic dysfunction. 17.6% of subjects had evidence of right ventricular enlargement and 36.5% had right atrial enlargement. In univariate analysis RV wall thickness (β = −68.6; p = 0.002), log right atrial area (β = −297.9; p = 0.004), LV mass index (β = −1.3; p = 0.03), E/E'' ratio (β = −5.5; p = 0.02), and degree of diastolic dysfunction (β = −42.8; p = 0.006) were associated with six minute walk distance. After adjustment for co-variables, the associations between right atrial area (log right atrial area β = −349.8; p = 0.003) and right ventricular wall thickness (β = −43.8; p = 0.04) with lower six minute walk distance remained significant independent of forced expiratory volume in one second (FEV1). LV mass index, E/E'' ratio, and degree of diastolic dysfunction were not independent predictors of six minute walk distance.

Conclusion

In patients with non-severe COPD right sided cardiac structural changes are associated with lower six minute walk distance independent of lung function. These findings may indicate that echocardiographic evidence of pulmonary hypertension is present in patients with non-severe COPD and has important functional consequences.