Separation and characteristics of antidiuretic factors from the corpora cardiaca of the migratory locust

Summary

Corpora cardiaca (CC) of the migratory locust contain two antidiuretic (AD) factors increasing the fluid reabsorption of isolated recta over a 5-h period.

They are contained in storage lobes (SL) and glandular lobes (GL) of the CC. They differ in size and can be separated from each other in different ways (extraction, dialysis, electrophoresis).

The AD factor contained in GL of the CC is stable at 4°C and quickly destroyed at 100°C. It stimulates the fluid reabsorption in a dose-dependent manner. Its release into the haemolymph seems to be controlled by octopamine, which is probably synthesized in the lateral neurosecretory areas of the brain.     

Structure of the NH2-terminal variable region of cardiac troponin T determines its sensitivity to restrictive cleavage in pathophysiological adaptation

We previously reported that the NH(2)-terminal variable region of cardiac troponin T (cTnT) is removed by restrictive μ-calpain cleavage in myocardial ischemia-reperfusion [24]. Selective removal of the NH(2)-terminal variable region of cTnT had a compensatory effect on myocardial contractility [25]. Here we further studied this posttranslational modification under pathophysiological conditions. Thrombin perfusion of isolated mouse hearts and cardiomyocytes induced the production of NH(2)-terminal truncated cTnT (cTnT-ND), suggesting a role of calcium overloading. Ouabain treatment of primary cultures of mouse cardiomyocytes in hypokalemic media, another calcium overloading condition, also produced cTnT-ND. Exploring the molecular mechanisms, we found that cTnT phosphorylation was primarily in the NH(2)-terminal region and the level of cTnT phosphorylation did not change under the calcium overloading conditions. However, alternatively spliced cTnT variants differing in the NH(2)-terminal primary structure produced significantly different levels of cTnT-ND in vivo in transgenic mouse hearts. The results suggest that stress conditions involving calcium overloading may convey an increased sensitivity of cTnT to the restrictive μ-calpain proteolysis, in which structure of the NH(2)-terminal variable region may play a determining role.     

Microenvironment mesenchymal cells protect ovarian cancer cell lines from apoptosis by inhibiting XIAP inactivation

Epithelial ovarian carcinoma is characterized by high frequency of recurrence (70% of patients) and carboplatin resistance acquisition. Carcinoma-associated mesenchymal stem cells (CA-MSC) have been shown to induce ovarian cancer chemoresistance through trogocytosis. Here we examined CA-MSC properties to protect ovarian cancer cells from carboplatin-induced apoptosis. Apoptosis was determined by Propidium Iodide and Annexin-V-FITC labelling and poly-ADP-ribose polymerase cleavage analysis. We showed a significant increase of inhibitory concentration 50 and a 30% decrease of carboplatin-induced apoptosis in ovarian cancer cells incubated in the presence of CA-MSC-conditioned medium (CM). A molecular analysis of apoptosis signalling pathway in response to carboplatin revealed that the presence of CA-MSC CM induced a 30% decrease of effector caspases-3 and -7 activation and proteolysis activity. CA-MSC secretions promoted Akt and X-linked inhibitor of apoptosis protein (XIAP; caspase inhibitor from inhibitor of apoptosis protein (IAP) family) phosphorylation. XIAP depletion by siRNA strategy permitted to restore apoptosis in ovarian cancer cells stimulated by CA-MSC CM. The factors secreted by CA-MSC are able to confer chemoresistance to carboplatin in ovarian cancer cells through the inhibition of effector caspases activation and apoptosis blockade. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway and the phosphorylation of its downstream target XIAP underlined the implication of this signalling pathway in ovarian cancer chemoresistance. This study reveals the potentialities of targeting XIAP in ovarian cancer therapy.     

The HDAC inhibitor,MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells

Epidermal growth factor receptor (EGFR), which promotes cell survival and division, is found at abnormally high levels on the surface of many cancer cell types, including many cases of non-small cell lung cancer. Erlotinib (Tarceva), an oral small-molecule tyrosine kinase inhibitor, is a so-called targeted drug that inhibits the tyrosine kinase domain of EGFR, and thus targets cancer cells with some specificity while doing less damage to normal cells. However, erlotinib resistance can occur, reducing the efficacy of this treatment. To develop more effective therapeutic interventions by overcoming this resistance problem, we combined the histone deacetylase inhibitor, MPT0E028, with erlotinib in an effort to increase their antitumor effects in erlotinib-resistant lung adenocarcinoma cells. This combined treatment yielded significant growth inhibition, induced the expression of apoptotic proteins (PARP, γH2AX, and caspase-3), increased the levels of acetylated histone H3, and showed synergistic effects in vitro and in vivo. These effects were independent of the mutation status of the genes encoding EGFR or K-Ras. MPT0E028 synergistically blocked key regulators of the EGFR/HER2 signaling pathways, attenuating multiple compensatory pathways (e.g., AKT, extracellular signal-regulated kinase, and c-MET). Our results indicate that this combination therapy might be a promising strategy for facilitating the effects of erlotinib monotherapy by activating various networks. Taken together, our data provide compelling evidence that MPT0E028 has the potential to improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents.     

Improved fatigue resistance in Gsα-deficient and aging mouse skeletal muscles due to adaptive increases in slow fibers

Genetically modified mice with deficiency of the G protein α-subunit (G(s)α) in skeletal muscle showed metabolic abnormality with reduced glucose tolerance, low muscle mass, and low contractile force, along with a fast-to-slow-fiber-type switch (Chen M, Feng HZ, Gupta D, Kelleher J, Dickerson KE, Wang J, Hunt D, Jou W, Gavrilova O, Jin JP, Weinstein LS. Am J Physiol Cell Physiol 296: C930-C940, 2009). Here we investigated a hypothesis that the switching to more slow fibers is an adaptive response with specific benefit. The results showed that, corresponding to the switch of myosin isoforms, the thin-filament regulatory proteins troponin T and troponin I both switched to their slow isoforms in the atrophic soleus muscle of 3-mo-old G(s)α-deficient mice. This fiber-type switch involving coordinated changes of both thick- and thin-myofilament proteins progressed in the G(s)α-deficient soleus muscles of 18- to 24-mo-old mice, as reflected by the expression of solely slow isoforms of myosin and troponin. Compared with age-matched controls, G(s)α-deficient soleus muscles with higher proportion of slow fibers exhibited slower contractile and relaxation kinetics and lower developed force, but significantly increased resistance to fatigue, followed by a better recovery. G(s)α-deficient soleus muscles of neonatal and 3-wk-old mice did not show the increase in slow fibers. Therefore, the fast-to-slow-fiber-type switch in G(s)α deficiency at older ages was likely an adaptive response. The benefit of higher fatigue resistance in adaption to metabolic deficiency and aging provides a mechanism to sustain skeletal muscle function in diabetic patients and elderly individuals.     

Dielectric properties of human skin at an acupuncture point in the 50-75 GHz frequency range: a pilot study

The reason for using acupuncture points as exposure sites in some applications of millimeter wave therapy has been unclear. Acupuncture points have been suspected to exhibit particular direct current (DC), low‐frequency electrical and optical properties compared to surrounding skin. To assess if such a biophysical correlation could exist at millimeter wave frequencies used in the therapy, we investigated the dielectric properties of an acupuncture point on the forearm skin within the 50–75 GHz range. These properties were compared with those of a neighboring ipsilateral control area and a corresponding contralateral control area. The complex reflection coefficient at the end of an open‐ended rectangular waveguide loaded with a Teflon plug was measured with a vector network analyzer. A suitable model of the aperture admittance was used to calculate the dielectric properties of the skin at the measured spots. Statistical analyses were conducted with an ANOVA to compare the three sites. From these analyses, the dielectric properties of the acupuncture site were found to be somewhat different from those of surrounding non‐acupuncture sites from 50 to about 61 GHz, in the case of the real part of the complex permittivity. Bioelectromagnetics 32:360–366, 2011. © 2011 Wiley‐Liss, Inc.     

High throughput analysis of grape genetic diversity as a tool for germplasm collection management

Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI?=?0.839 and 0.865, respectively) than V. vinifera cultivars (GDI?=?0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI?=?0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management.     

A new B-chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B-chain structure.

The solution structure of a new B-chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D-NMR and molecular modeling. This structure is compared with that of the oxidized B-chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res.46, 424-433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B-chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B-chain. 2D-NMR experiments confirmed, among multiple conformations, that the B-chain mutant presents defined secondary structures such as a alpha-helix between residues B9 and B19, and a beta-turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B-chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B-chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B-chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B-chains.     

Quantitative relationship between plasma potassium levels and QT interval in beagle dogs

The aim of the current study was to establish the quantitative relationship between plasma potassium concentrations and the QT interval of the electrocardiogram in dogs. Furosemide, a potent diuretic, was given at increasing doses (5-60 mg/kg) to five male and five female beagle dogs. Electrocardiogram (ECG) was recorded three times each day, simultaneous to blood sampling for measurement of plasma potassium. Furosemide treatment produced a clear hypokalaemia, which was associated with an increase in QT and corrected QT intervals (QTc) duration. On average, the slopes of the negative linear correlation between potassium plasma levels and QT or QTc were steeper in females than in males. These results show that a decrease in potassium plasma level may explain a concomitant increase in QT duration in a toxicity study in dogs, in particular if potassium values are decreased below 3.3 mmol/L. Correction of QT interval for K+ plasma level has, therefore, been established separately for males and females. A global formula correcting QT for K+ and heart rate simultaneously was established. Hypokalaemia was also associated with changes in the morphology of the T wave recorded in CV5RL, in particular, with a flattening and/or a notching of the wave (appearance of a second peak), biphasic aspect or inversion of polarity. These changes are probably related to an increased heterogeneity of repolarization between different populations of cardiomyocytes. In conclusion, hypokalaemia is quantitatively associated with an increase in QT and QTc duration in dogs. The relationship is apparently stronger for females than for males. A formula may be used to correct QT for potassium plasma level.     

Nitrogen uptake and metabolism in Populus x canescens as affected by salinity

External salinization can affect different steps of nitrogen (N) metabolism (ion uptake, N assimilation, and amino acid and protein synthesis) depending on the inorganic N source. Here, we assessed the net uptake of N supplied as nitrate or ammonium and N assimilation (combining metabolite analyses with molecular biological approaches) in grey poplar (Populus x canescens) plants grown under saline (75 mM NaCl) and control conditions. The specific (micromol N g(-1) dry weight fine roots h(-1)) and total plant (micromol N per plant h(-1)) N net uptake rates, total plant N content, total plant biomass and total leaf protein concentration were reduced under saline conditions when plants were supplied with ammonium. In both nutritional groups, salt treatment caused pronounced accumulation of soluble N compounds in the leaves. The mRNAs of genes coding for enzymes catalyzing rate-limiting steps of both proline synthesis and degradation (delta-1-pyrroline-5-carboxylate synthase and proline dehydrogenase) as well as for NADH-dependent glutamate synthase were accumulated under saline conditions. Whereas under control conditions the plant N status seemed to be superior when ammonium was supplied, the N balance of ammonium-fed plants was more severely affected by salt stress than that of plants supplied with nitrate. Possible metabolic implications of stress-related accumulation of particular amino acids are discussed.