Role of ω-Conotoxin GVIA-Sensitive Ca2+ Entry in Angiotensin II-Stimulated [3H]Phorbol 12, 13-Dibutyrate Binding in Bovine Adrenal Medullary Cells

Abstract: The relative contributions of Ca²⁺ influx and intracellular Ca²⁺ mobilization were examined for angiotensin II-stimulated [³H]phorbol 12, 13-dibutyrate binding, which reflects the level of activated protein kinase C in bovine chromaffin cells. Angiotensin II receptors activate phospholipase C in chromaffin cells, leading to a shortlived mobilization of intracellular Ca²⁺. Angiotensin II-stimulated [³H]phorbol 12, 13-dibutyrate binding was largely blocked in Ca²⁺-free buffer and by pretreatment with the Ca²⁺-channel blocker ω-conotoxin GVIA. The [³H]phorbol 12, 13-dibutyrate binding response to [Sar¹]angiotensin II also appeared to be voltage sensitive, as no additivity was observed with the response to the depolarizing agent 4-aminopyridine (3 m M ). Threshold sensitivities of the extra-and intracellular Ca²⁺-mobilizing pathways to angiotensin II were similar, and all examined effects of angiotensin II in these cells were apparently mediated by losartan-sensitive (AT₁-Iike) receptors. The dependence of angiotensin II-stimulated [³H]phorbol 12, 13-dibutyrate binding on extracellular Ca²⁺ entry, in contrast to stimulation by other phospholipase C-linked receptor agonists (bradykinin and methacholine), suggests that angiotensin II preferentially stimulates protein kinase C translocation to the plasma membrane, rather than to internal membranes, in bovine adrenal medullary cells.     

Production of pectin methylesterase from Erwinia chrysanthemi B374 in Bacillus subtilis

Summary The gene coding for pectin methylesterase (PME) of Erwinia chrysanthemi B374 (pme) was cloned by a polymerase chain reaction. The pme gene was expressed in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the -amylase gene from B. amyloliquefaciens. The cultivation of B. subtilis cells carrying the cloned pme resulted in efficient secretion of PME into the culture medium based on enzymatic and sodium dodecyl sulphate-polyacrylamide gel electrophoresis characterizations. The NH₂-terminal sequence analysis of the secreted PME revealed two different NH₂-termini. Heterologous processing was probably due to a second putative signal peptidase cleavage site at the joint region between the PME and -amylase signal peptide. Offprint requests to: R. Heikinheimo     

Vectoring gypsy moth nuclear polyhedrosis virus byApanteles melanoscelus [Hym.: Braconidae]

Gypsy mothLymantria dispar L. larvae were exposed toApanteles melanoscelus (Ratzeburg) females contaminated with nuclear polyhedrosis virus. Three methods of contamination (ovipositor, total body surface, and exposure to infected hosts) and two exposure periods (2 and 24 hours) were tested. A significantly greater incidence of larval mortality caused by virus occurred among larvae exposed to contaminated than among larvae exposed to uncontaminated parasites for 2 and 24 hours. No significant differences occurred in larval mortality caused by virus for the 3 methods of contamination for the 2- and 24-hour tests or in parasite emergence from larvae parasitized by contaminated or uncontaminated parasites.     

A feasibility study of altered spatial distribution of losses induced by eddy currents in body composition analysis

Background  

Tomographic imaging has revealed that the body mass index does not give a reliable state of overall fitness. However, high measurement costs make the tomographic imaging unsuitable for large scale studies or repeated individual use. This paper reports an experimental investigation of a new electromagnetic method and its feasibility for assessing body composition. The method is called body electrical loss analysis (BELA).     

Central boreal mire plant communities along soil nutrient potential and water content gradients

Peatlands have traditionally been exploited in forestry and agriculture over the boreal region, yet they also provide substantial source of fuel production. The large-scale exploitation of peatlands has raised a concern about the diversity of mire plant communities. We studied composition of mire plant communities along soil nutrient potential and water content gradients, to recognize the areas with the high plant diversity. Soil electrical conductivity (EC_b) was measured to characterise soil nutrient regimes and soil dielectric permittivity (DP) the soil (volumetric) water regimes. A total of 115 mire sites were studied in the central boreal region of south-western Finnish Lapland. We found that Ward’s hierarchical cluster analysis produced eight stable EC_b and DP clusters with discrete vegetation compositions. On the basis of a locally weighted regression analysis (Loess), Carex dioica L., Comarum palustre L., Equisetum fluviatile L., Menyanthes trifoliata L., and Scorpidium scorpioides (Hedw.) Limpr. were found as indicator species for nutrient-rich regimes as designated by high soil EC_b. The soil EC_b is a diagnostic measure of plant diversity as EC_b?>?7 mSm^?1 resulted in a considerable increase in species richness. Our classification method, based on electrical measurements, provides a simple way to classify mires and focus detailed research to areas with potentially high conservation value.     

A rationally designed synthetic mimic of the discontinuous CD4-binding site of HIV-1 gp120

Synthetic mimetics of the CD4-binding site of HIV-1 gp120 are promising candidates for HIV-1 entry inhibition, as well as immunogen candidates for the elicitation of virus-neutralizing antibodies. On the basis of the crystal structure of gp120 in complex with CD4, we have used a recently introduced strategy for the generation of structurally diverse scaffolds to design and synthesize a scaffolded peptide, in which three fragments, making up the sequentially discontinuous binding site of gp120 for CD4, are presented in a nonlinear and discontinuous fashion through a molecular scoffold, which restrains conformational flexibility. The affinities of this molecule to CD4, as well as to the broadly neutralizing antibody mAb b12, whose epitope overlaps the CD4-binding site of gp120, were determined in competitive binding assays.     

Plasmatic exosomes from prostate cancer patients show increased carbonic anhydrase IX expression and activity and low pH

Abstract

Acidity, hypoxia and increased release of exosomes are severe phenotypes of tumours. The regulation of pH in tumours involves the interaction of several proteins, including the carbonic anhydrases which catalyze the formation of bicarbonate and protons from carbon dioxide and water. Among CA isoforms, CA IX is over-expressed in a large number of solid tumours, conferring to cancer cells a survival advantage in hypoxic and acidic microenvironment, but there isn’t evidence that CA IX expression could have a real clinical impact. Therefore, in this study for the first time the expression and activity of CA IX have been investigated in the plasmatic exosomes obtained from patients with prostate carcinoma (PCa). For this purpose, the study was performed through different methodological approaches, such as NTA, western blot analysis, enzyme activity assay, Nanoscale flow cytometry, ELISA, confocal microscopy. The results showed that PCa exosomes significantly overexpressed CA IX levels and related activity as compared to healthy donors. Furthermore, CA IX expression and activity were correlated to the exosome intraluminal pH, demonstrating for the first time that PCa exosomes are acidic. Our data suggest the possible use of the exosomal CA IX expression and activity as a biomarker of cancer progression in PCa.     

Accumulation of altered aspartyl residues in erythrocyte membrane proteins from patients with sporadic amyotrophic lateral sclerosis

Spontaneous protein deamidation of labile asparagines (Asn), generating abnormal l-isoaspartyl residues (IsoAsp), is associated with cell aging and enhanced by an oxidative microenvironment. The presence of isopeptide bonds impairs protein structure/function. To minimize the damage, IsoAsp can be “repaired” by the protein l-isoaspartyl/d-aspartyl O-methyltransferase (PIMT) and S-adenosylmethionine (AdoMet) is the methyl donor of this reaction. PIMT is a repair enzyme that initiates the conversion of l-isoAsp (or d-Asp) residues to l-Asp residues. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease principally affecting motor neurons. The condition of oxidative stress reported in familial and sporadic forms of ALS prompted us to investigate Asn deamidation in ALS tissue. Erythrocytes (RBCs) were selected as a model system since they are unable to replace damaged proteins and protein methylesterification is virtually the only AdoMet-consuming reaction operating in these cells. Our data show that, in vitro assay, abnormal IsoAsp residues were significantly higher in ALS patients erythrocyte membrane proteins with an increased methyl accepting capability relative to controls (p < 0.05). Moreover, we observed a reduction in AdoMet levels, while AdoHcy concentration was comparable to that detected in the control, resulting in a lower [AdoMet]/[AdoHcy] ratio. Then, the accumulation of altered aspartyl residues in ALS patients is probably related to a reduced efficiency of the S-adenosylmethionine (AdoMet)-dependent repair system causing increased protein instability at Asn sites. The increase of abnormal residues represents a new protein alteration that may be present not only in red blood cells but also in other cell types of patients suffering from ALS.     

Salivary flow rate and risk of malnutrition – a study among dentate,community‐dwelling older people

doi: 10.1111/j.1741‐2358.2012.00679.x Salivary flow rate and risk of malnutrition – a study among dentate, community‐dwelling older people Objective: To analyse the relation between unstimulated and stimulated salivary secretion and the risk of malnutrition among home‐dwelling elderly people. Background: Saliva has an important role in eating. Despite this, there are only a few studies on the role of salivary secretion in the development of malnutrition among elderly people. Materials and methods: The study population consisted of 157 subjects aged 75 or older. This was a part of GeMS study carried out in Kuopio, in eastern Finland. The data used in this study were collected by means of interviews and geriatric and oral clinical examinations. The risk of malnutrition was measured using the Mini Nutritional Assessment Short‐Form. Logistic regression models were used to estimate odds ratios (OR) and their 95% Confidence Intervals (CI). Results: Subjects with a low unstimulated salivary flow rate (<0.1 ml/min) or stimulated salivary flow rate (<1.0 ml/min) had no statistically significant increase in risk of malnutrition, OR: 1.3, CI: 0.5–3.9, OR: 1.5, CI: 0.5–4.2, respectively, when compared with those with a normal unstimulated and stimulated salivary flow rate. Conclusion: Our results do not support the concept that low salivary secretion is an important risk factor for malnutrition among community‐dwelling elders.