Purification and characterization of S-modulin, a calcium-dependent regulator on cGMP phosphodiesterase in frog rod photoreceptors.

S-modulin is a 26 kDa protein that regulates light sensitivity of cGMP phosphodiesterase in a Ca(2+)-dependent manner in frog rod outer segments (ROSs). In the present study, we purified S-modulin by taking advantage of a hydrophobic interaction between Phenyl Sepharose and S-modulin at high Ca2+ concentrations. The yield was greater than 90%. 45Ca(2+)-binding experiment showed that S-modulin is a Ca(2+)-binding protein. At high Ca2+ concentrations, S-modulin binds to ROS membranes. The binding target of the Ca2+/S-modulin complex is possibly a ROS membrane lipid(s), but it was difficult to identify. The binding was observed mainly at greater than 1 microM Ca2+. The amino acid sequence deduced from proteolytic fragments of S-modulin was approximately 80% and 60% identical to those of recovering and visinin, respectively.     

Use of the Bacillus subtilis subtilisin signal peptide for efficient secretion of TEM beta-lactamase during growth.

We report the development of an efficient Bacillus subtilis secretory system, with the secreted product stably maintained in the medium for 100 h. The system is based on characterization of the subtilisin signal peptidase cleavage site and promoters, catabolite repression of sporulation, presence of a vegetative secreting mechanism, and availability of a protease-deficient strain.     

Solubilization and purification of Artemia salina (Na,K)-activated ATPase and NH2-terminal amino acid sequence of its larger subunit

Membrane bound (Na,K)-ATPase partially purified from the nauplius larva of the brine shrimp, Artemia salina, was solubilized with the non-ionic detergent C12E8 in the presence of KCl. The addition of KCl was essential for protecting the enzyme against inactivation. With solubilization the enzyme could then be purified to apparent homogeneity. Electron microscopic observation of the purified enzyme revealed a homogeneous population of particles with a diameter of approximately 4 nm. The larger (alpha) subunit of the enzyme formed double bands on sodium dodecyl sulfate-polyacrylamide gels. NH2-terminal sequence analysis of the alpha subunit revealed the possible presence of two isoforms of (Na,K)-ATPase. At the third position a small but distinct amount of lysine was found in addition to glycine, suggesting that the two forms are different from each other at least at the third residue. The NH2-terminal sequence determined is as follows. NH2-Ala-Lys-Gly (Lys)-Lys-Gln-Lys-Lys-Gly-Lys-Asp-Leu-Asn-Glu-Leu-Lys-Lys-Glu-Leu-Asp-Il e-Asp -Phe-His-Lys-Ile-Pro- The sequence is abundant in hydrophilic amino acids, especially lysine, and is quite different from those of vertebrate enzymes reported so far.     

The Effects of Interleukin‐6 Signal Blockade on Immune System,Reproductive and Skeletal Development in Juvenile Mice

Interleukin‐6 (IL‐6) is involved in the pathogenesis of multiple disorders, including juvenile autoimmune diseases. IL‐6 participates in a broad spectrum of physiological events, and the IL‐6 receptor (IL‐6R) is widely distributed across multiple organs. The interrelationship of development phases in juveniles together with organs involved in IL‐6 signaling called for evaluations of anti–IL‐6R antibody induced effects in a juvenile mouse model to assess the safety of such an approach in human juvenile arthritis. Here we show that naive mice in which IL‐6 signals have been transiently blocked during the juvenile period develop normally. The fatal immunogenic reactions recorded earlier by repeated administration of the chosen rat anti‐mouse IL‐6R antibody, MR16‐1, to mice were avoided successfully by application of a high loading dose followed by lower maintenance doses, with the support of modeling data. The high loading‐dose regimen enabled us to conduct assessments without any major interference due to immunogenicity. Transient and complete inhibition of IL‐6 signals from postnatal days 22 to 79 in mice exhibited no biologically important changes in sexual maturation or development of immune and skeletal systems. Although tendencies toward reductions of peripheral blood T‐cell counts were observed, normal levels of antigen‐specific IgG/IgM antibody productions indicating sufficient immunological functions were confirmed. Our results demonstrate that blockage of IL‐6R by the neutralizing antibody does not affect juvenile development. This may be in part due to the generation or existence of compensatory pathways in the whole body system.     

Establishment of a Genetic Transformation System for the Marine Pennate Diatom Fistulifera sp. Strain JPCC DA0580—A High Triglyceride Producer

A genetic transformation system for the marine pennate diatom, Fistulifera sp. JPCC DA0580, was established using microparticle bombardment methods. Strain JPCC DA0580 has been recently identified as the highest triglyceride (60 % w/w) producer from a culture collection of 1,393 strains of marine microalgae, and it is expected to be a feasible source of biodiesel fuel. The transformation conditions for strain JPCC DA0580 were optimised using the green fluorescent protein gene (gfp) and the gene encoding neomycin phosphotransferase II (nptII). The most efficient rate of transformation was attained when tungsten particles (0.6 μm in diameter) were used for microparticle bombardment. The effect of endogenous and exogenous promoters on the expression of nptII was examined. Endogenous promoters were more efficient for obtaining transformants compared with exogenous promoters. Southern hybridisation analysis suggested that nptII integrated into the nuclear genome. This genetic manipulation technique should allow us to understand the mechanisms of high triglyceride accumulation in this strain, thereby contributing to improving BDF production.     

Autophagy in normal tissues of camel (Camelus dromedarius) with focus on immunoexpression of LC3 and LC3B

Autophagy is a highly regulated intracellular pathway for degradation and recycling of cytoplasmic protein aggregates and entire organelles. The autophagic pathway is stimulated by nutrient starvation, which prompted us to study the desert camel. Various organs of the camel undergo ecological and physiological stress due to food and water deprivation, dehydration and long exposure to solar radiation. We investigated the immunohistochemical expression of specific biomarkers of autophagy under normal conditions as a baseline for later work on stressed individuals. The autophagy-specific biomarkers, microtubule-associated protein1 light chain 3 (LC3), and its cleaved variant, LC3B, were strongly expressed in the cytosol of all tissues examined. The cytosolic immunoreactivity of LC3 was relatively weak, diffuse and vacuolar, while that of LC3B was stronger, punctate and at lower levels. LC3 appears to be associated with the autophagosomal membranes, either free or lysosome-bounded. LC3B is specific for the autophagosome-lysosome complexes and their degraded, granular contents. Autophagy was strongly expressed in CNS neurons and intestinal neural elements, which suggests a protective function for the nervous system. Autophagic markers also were seen in deformed immune-competent cells with fragmented nuclei in lymph nodes, spleen and gut-associated lymphoid tissue (GALT), which suggests a “suicidal” activity of eliminating unneeded cells. Autophagy, as measured by LC3 and LC3B expression, may participate in a general regulatory mechanism in tissues of the desert camel.     

Increased number and altered phenotype of lymphatic vessels in peripheral lung compartments of patients with COPD

Background

De novo lymphatic vessel formation has recently been observed in lungs of patients with moderate chronic obstructive pulmonary disease (COPD). However, the distribution of lymphatic vessel changes among the anatomical compartments of diseased lungs is unknown. Furthermore, information regarding the nature of lymphatic vessel alterations across different stages of COPD is missing. This study performs a detailed morphometric characterization of lymphatic vessels in major peripheral lung compartments of patients with different severities of COPD and investigates the lymphatic expression of molecules involved in immune cell trafficking.

Methods

Peripheral lung resection samples obtained from patients with mild (GOLD stage I), moderate-severe (GOLD stage II-III), and very severe (GOLD stage IV) COPD were investigated for podoplanin-immunopositive lymphatic vessels in distinct peripheral lung compartments: bronchioles, pulmonary blood vessels and alveolar walls. Control subjects with normal lung function were divided into never smokers and smokers. Lymphatics were analysed by multiple morphological parameters, as well as for their expression of CCL21 and the chemokine scavenger receptor D6.

Results

The number of lymphatics increased by 133% in the alveolar parenchyma in patients with advanced COPD compared with never-smoking controls (p < 0.05). In patchy fibrotic lesions the number of alveolar lymphatics increased 20-fold from non-fibrotic parenchyma in the same COPD patients. The absolute number of lymphatics per bronchiole and artery was increased in advanced COPD, but numbers were not different after normalization to tissue area. Increased numbers of CCL21- and D6-positive lymphatics were observed in the alveolar parenchyma in advanced COPD compared with controls (p < 0.01). Lymphatic vessels also displayed increased mean levels of immunoreactivity for CCL21 in the wall of bronchioles (p < 0.01) and bronchiole-associated arteries (p < 0.05), as well as the alveolar parenchyma (p < 0.001) in patients with advanced COPD compared with never-smoking controls. A similar increase in lymphatic D6 immunoreactivity was observed in bronchioles (p < 0.05) and alveolar parenchyma (p < 0.01).

Conclusions

This study shows that severe stages of COPD is associated with increased numbers of alveolar lymphatic vessels and a change in lymphatic vessel phenotype in major peripheral lung compartments. This novel histopathological feature is suggested to have important implications for distal lung immune cell traffic in advanced COPD.