Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+ entry

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca(2+) concentration, resulting from enhanced capacitative Ca(2+) entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cAMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK; 10 muM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 200 muM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH), and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA), because the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulation of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP, downregulated TRPC3 expression in IPAH PASMC and FSK-mediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.     

Forearm blood flow follows work rate during submaximal dynamic forearm exercise independent of sex.

To test the hypothesis that sex influences forearm blood flow (FBF) during exercise, 15 women and 16 men of similar age [women 24.3 +/- 4.0 (SD) vs. men 24.9 +/- 4.5 yr] but different forearm muscle strength (women 290.7 +/- 44.4 vs. men 509.6 +/- 97.8 N; P < 0.05) performed dynamic handgrip exercise as the same absolute workload was increased in a ramp function (0.25 W/min). Task failure was defined as the inability to maintain contraction rate. Blood pressure and FBF were measured on separate arms during exercise by auscultation and Doppler ultrasound, respectively. Muscle strength was positively correlated with endurance time (r = 0.72, P < 0.01) such that women had a shorter time to task failure than men (450.5 +/- 113.0 vs. 831.3 +/- 272.9 s; P < 0.05). However, the percentage of maximal handgrip strength achieved at task failure was similar between sexes (14% maximum voluntary contraction). FBF was similar between women and men throughout exercise and at task failure (women 13.6 +/- 5.3 vs. men 14.5 +/- 4.9 ml.min(-1).100 ml(-1)). Mean arterial pressure was lower in women at rest and during exercise; thus calculated forearm vascular conductance (FVC) was higher in women during exercise but similar between sexes at task failure (women 0.13 +/- 0.05 vs. men 0.11 +/- 0.04 ml.min(-1).100 ml(-1).mmHg(-1)). In conclusion, the similar FBF during exercise was achieved by a higher FVC in the presence of a lower MAP in women than men. Still, FBF remained coupled to work rate (and presumably metabolic demand) during exercise irrespective of sex.     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Adoptive transfer of T<Subscript>reg</Subscript> depleted autologous T cells in advanced renal cell carcinoma

Purpose

CD4⁺CD25⁺ regulatory T (T_reg) cells are present in increased numbers in patients with advanced cancer and CD25⁺ T cell depletion potentiates tumour immunity in animal models. The aim of this study was to assess the feasibility and safety of adoptive transfer of CD25⁺ depleted autologous T cells in patients with advanced renal cell carcinoma and to examine resulting changes in lymphocyte subsets.

Patients and methods

Six patients with advanced renal cell carcinoma underwent leukapheresis followed by conditioning chemotherapy with cyclophosphamide and fludarabine. The autologous leukapheresis product was depleted of CD25⁺ cells using CliniMACS^® System then re-infused into the patient.

Results

Efficient CD25⁺ depletion from all leukapheresis products was achieved and 0.55–5.87 × 10⁷/kg CD3⁺ cells were re-infused. Chemotherapy related haematological toxicity was observed, but blood counts recovered in all patients allowing discharge after a mean inpatient stay of 21 days. One patient subsequently developed a rapidly progressive neurological syndrome. A transient reduction in CD25⁺ subset was noted in the peripheral blood of 5 out of 6 patients with evidence of increased T cell responses to PHA in 4 out of 6 patients. One patient showed increased specific proliferative responses to the tumour associated antigen h5T4 coinciding with the nadir of T_reg cells.

Conclusions

Given the transient nature of the reduction in CD25⁺ subset and the observed toxicity there is a need to explore further strategies to improve the safety and efficacy of this approach. Nevertheless, the results provide proof of concept in potentiation of tumour antigen T cell responses when T_reg cell levels are depleted.

     

Two-dimensional diffusion of amphiphiles in phospholipid monolayers at the air-water interface.

Steady-state and time-resolved fluorescence spectroscopy has been used to examine lateral diffusion in dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and dimyristoyl-L-alpha-phosphatidylcholine (DMPC) monolayers at the air-water interface, by studying the fluorescence quenching of a pyrene-labeled phospholipid (pyrene-DPPE) by two amphiphilic quenchers. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the DMPC lipid matrix for all measured surface pressures and only in the liquid-expanded (LE) phase of the DPPC monolayer. Time-resolved fluorescence decays for pyrene-DPPE in DMPC and DPPC (LE phase) in the absence of quencher were best described by a single-exponential function, also suggesting a homogeneous distribution of pyrene-DPPE within the monolayer films. Addition of quencher to the monolayer film produced nonexponential decay behavior, which is adequately described by the continuum theory of diffusion-controlled quenching in a two-dimensional environment. Steady-state fluorescence measurements yielded lateral diffusion coefficients significantly larger than those obtained from time-resolved data. The difference in these values was ascribed to the influence of static quenching in the case of the steady-state measurements. The lateral diffusion coefficients obtained in the DMPC monolayers were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression.     

Membrane lipids and ethanol tolerance in the mouse. The influence of dietary fatty acid composition

Mice of the TO Swiss strain received diets containing different amounts of saturated or unsaturated fat throughout life. These diets produced characteristic changes in cardiac phospholipid fatty acid composition, but produced no significant differences in fatty acid composition of phospholipids from a crude membrane fraction of brain. When littermates of these animals were exposed to ethanol vapour in an inhalation chamber it was observed that mice which had received a diet high in saturated fat lost the righting reflex at an estimated concentration of ethanol in blood higher than that required for mice receiving a control diet, or a diet rich in polyunsaturated fat. Analysis of the brain membrane fraction from those animals which had received ethanol revealed that mice receiving the highly saturated fat diet now had a significantly greater proportion of saturated fatty acids in brain membrane phospholipids. These results are discussed in relation to the hypothesis that brain membrane lipid composition may influence the behavioural response to ethanol.     

Temperature acclimation has no effect on ryanodine receptor expression or subcellular localization in rainbow trout heart

In cardiomyocytes, ryanodine receptors (RYRs) mediate Ca²⁺-induced Ca²⁺-release (CICR) from the sarcoplasmic reticulum (SR) during excitation–contraction (e–c) coupling. In rainbow trout heart, the relative importance of CICR increases with cold-acclimation. Thus, the aim of this study was to investigate the effect of temperature acclimation (4, 11 and 18°C) on RYR intracellular localization and expression density. We used immunocytochemistry to assess intracellular localization in ventricular myocytes and Western blotting to assess RYR expression in both atrial and ventricular tissue. In ventricular myocytes, RYRs were localized peripherally in transverse bands aligning with sarcomeric m-lines and centrally around mitochondria and the nucleus. Localization did not change with temperature acclimation. RYR expression was also unaffected by temperature acclimation. The localization of RYRs at the m-line is similar to neonatal mammalian cardiomyocytes. We suggest this positioning is indicative of myocytes which rely predominantly on transsarcolemmal Ca²⁺-influx, rather than CICR, during e–c coupling.