PKA implicated in the phosphorylation of Cx43 induced by stimulation with FSH in rat granulosa cells

Connexin 43 (Cx43)-mediated gap junctional communication in granulosa cells is crucial for germ line development and postnatal folliculogenesis. We previously showed that follicle-stimulating hormone (FSH) promoted phosphorylation of Cx43 in rat primary granulosa cells. We further identified Ser365, Ser368, Ser369, and Ser373 in the carboxy-terminal tail as the major sites of phosphorylation by FSH, and found that the phosphorylation of these residues was essential for channel activity. In this study, we investigated the protein kinase(s) responsible for FSH-induced phosphorylation. H89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, inhibited FSH-induced phosphorylation both in vivo and in vitro, whereas PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, had little effect on the phosphorylation level. Ca2+-dependent protein kinase (PKC) appeared to negatively regulate phosphorylation. Phosphopeptide mapping with or without H89 treatment indicated that PKA could be responsible for phosphorylation of the four serine residues. In addition, the purified catalytic subunit of PKA could phosphorylate the recombinant C-terminal region of Cx43, but not the variant in which all four serine residues were substituted with alanine. These results suggest that FSH positively regulates Cx43-mediated channel formation and activity through phosphorylation of specific sites by PKA.     

Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene

Phytoplasmas are plant pathogenic bacteria that infect more than 700 plant species. Because phytoplasma-resistant cultivars are not available for the vast majority of crops, the most common practice to prevent phytoplasma diseases is to remove infected plants. Therefore, developing a rapid, accurate diagnostic method to detect a phytoplasma infection is important. Here, we developed a phytoplasma detection assay based on loop-mediated isothermal amplification (LAMP) by targeting the groEL gene and 16S rDNA. We designed 19 primer sets for the LAMP assay and evaluated their amplification efficiency, sensitivity, and spectra to select the most suitable primer sets to detect Candidatus Phytoplasma asteris. As a result, DNA was efficiently amplified by one of the primer sets targeting the groEL gene, and LAMP assay sensitivity with this primer set was 10-fold higher than that of the polymerase chain reaction. Moreover, the groEL gene was successfully amplified from several strains of Ca. Phytoplasma asteris by this primer set, indicating that the groEL gene can be used as a LAMP assay target gene for a broad range of phytoplasma strains. Additionally, a simple DNA extraction method that omits the homogenizing and phenol extraction steps was combined with the LAMP assay to develop a simple, rapid, and convenient diagnostic method for detecting phytoplasma.     

Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE

Posttranslational modification of alpha-dystroglycan (α-DG) by the like-acetylglucosaminyltransferase (LARGE) is required for it to function as an extracellular matrix (ECM) receptor. Mutations in the LARGE gene have been identified in congenital muscular dystrophy patients with brain abnormalities. However, the precise function of LARGE remains unclear. Here we found that LARGE could act as a bifunctional glycosyltransferase, with both xylosyltransferase and glucuronyltransferase activities, which produced repeating units of [-3-xylose-α1,3-glucuronic acid-β1-]. This modification allowed α-DG to bind laminin-G domain-containing ECM ligands.     

Effects of extracellular lactate on production of reactive oxygen species by equine polymorphonuclear leukocytes in vitro

Objective-To evaluate effects of extracellular lactate on viability, shape change, lactate metabolism, and reactive oxygen species (ROS) production in equine polymorphonuclear leukocytes (PMNs). Sample-PMNs isolated from equine venous blood samples. Procedures-PMNs were incubated with 0 to 300mM lactate for 30 minutes before each experiment. Viability was assessed via trypan blue exclusion. Shape change was assessed via flow cytometry and light microscopy. Relative quantification of monocarboxylic acid transporter and lactate dehydrogenase lactate dehydrogenase (LDH) isotype mRNAs was performed with a real-time PCR assay. Effects of lactate at a pH of 7.4 to 6.0 on ROS production in response to phorbol 12-myristate 13-acetate, opsonized zymosan, or N-formyl-methionyl-leucyl-phenylalanine was assessed by luminol-dependent chemiluminescence. Results-Lactate had no effect on viability of PMNs but did alter their size and density. Monocarboxylic acid transporter 1 and lactate dehydrogenase B mRNA values were not altered. Monocarboxylic acid transporter 4 and lactate dehydrogenase A mRNA values were significantly decreased. Lactate incubation of cells significantly decreased PMN-derived luminol-dependent chemiluminescence and induced different sensitivities to stimulants (phorbol 12-myristate 13-acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine). The response ratio to N-formyl-methionyl-leucyl-phenylalanine revealed that PMNs were primed by incubation with up to 50mM lactate, significantly increasing the production of ROS. Incubation with lactate and acidic pH caused a synergistic effect on ROS production. Conclusions and Clinical Relevance-Extracellular lactate potentially has a direct effect on the capacity to produce ROS by equine PMNs, which may be associated with alterations in innate immune functions within a short period after high-intensity exercise.     

Calreticulin expression in neoplastic versus normal dog mammary glands: a cDNA subtraction-based study

This is the first report describing the expression of canine calreticulin (cCRT) in canine mammary gland tumour (MGT). Using cDNA subtraction method, it is found that mRNAs of CRT, cathepsin A, ovostatin, and lactotransferrin were differentially expressed in mammary adenocarcinoma as compared to hyperplasia, both of which were obtained from the dog. Furthermore, the mRNA expression levels of CRT and cathepsin A were significantly higher in canine MGT samples than in nontumour samples. In contrast, immunohistochemical studies have indicated that the expression of cCRT protein found to be detected in most of mammary gland tissues and was not correlated to the types of canine MGTs. Furthermore, cCRT was molecularly cloned, and the amino acid sequence of cCRT was found to be very similar to those of other species. Further studies are required to elucidate additional roles of cCRT in canine MGT.     

A novel method for the reinforcement of harp soundboard

The effects of a carbon fiber-reinforced plastic (CFRP) overlay on the wooden soundboard of a harp were compared to those of conventional veneer reinforcement with respect to the vibrational properties and bending strength. CFRP reinforcement has a minimal effect on the vibrational properties of the soundboard in its width direction, whereas conventional veneer reinforcement significantly reduces the acoustic conversion efficiency of the soundboard. The CFRP-reinforced soundboard also has sufficient bending strength in its longitudinal direction. These results indicate that CFRP is a promising material for the reinforcement of the wooden soundboards of harps to minimize the reduction of the sound amplitude.     

Vibrational properties of harp soundboard with respect to its multi-layered structure

The vibrational properties of a harp soundboard were investigated with respect to its multi-layered structure. The surfaces of harp soundboards are usually reinforced with veneer; however, this reduces the specific dynamic Young’s modulus (E′/ρ) and significantly increases the internal friction (Q ^?1) of soundboards. Since smaller E′/ρ and greater Q ^?1 values impart a smaller acoustic conversion efficiency, the attachment of veneer is predicted to reduce the amplitude of the sound produced, as suggested by harp makers. The vibrational properties of veneer-reinforced wood are elucidated using a multi-layered model comprising base wood, a glue layer, veneer and a varnish layer. The results of calculations suggest that a thinner veneer attached with minimal glue would increase the sound amplitude.     

Bacterial Pleuritis with Thickened Mesothelial Hyperplasia in a Young Beagle Dog

A five-month-old male beagle dog suddenly became moribund. Bloody fluid accumulated in the thoracic and abdominal cavities, and soft yellow flecks were floating in the thoracic fluid. The mediastinum and pericardium became dark reddish with villous thickening. Other parietal and pulmonary pleurae were rough, and the organs adhered to each other. Histologically, most mediastinal pleura formed papillary projections covered by a single layer of mesothelial cells. Many macrophages and neutrophils infiltrated the submesothelial connective tissue. At the mediastinum adjacent to the pericardium, cuboidal mesothelial cells proliferated solidly and formed a thick surface stratum. The flecks consisted of gram-negative filamentous or small bacillary (coccoid) bacteria. In the right posterior lobe of the lung, neutrophilic infiltration and a large encapsulated abscess including a bacterial colony were present. We diagnosed this case as “bacterial pleuritis with thickened mesothelial hyperplasia”. The cause of the pleuritis might be a chronic pleural infection spread via the lung abscess.     

Optimal forest rotation periods: integrating timber production and carbon sequestration benefits in Pinus tabulaeformis plantations on the Loess Plateau,P.R. China

Determining the optimal rotation period was a crucial component of forest sustainable management strategies, especially under climate change. This paper had two objectives: (1) to determine the economic benefits and optimal rotation periods for timber production when coupled to carbon sequestration, as predicted by time series prediction models for Pinus tabulaeformis plantations in China; and (2) to evaluate how different carbon prices and interest rates affected optimal rotation periods using the forest land expectation value. The results suggested that time series prediction models were valuable for estimating timber volumes and carbon sequestrations based on surveys of different-aged stands. Importantly, since integrating carbon sequestrations into timber production benefits did not increase optimal rotation periods, this should promote P. tabulaeformis plantation management. In the sensitivity analysis, a higher carbon price increased the profitability of carbon sequestration and timber production, but not optimal rotation periods, though they were reduced under higher interest rates. In conclusion, incorporating both timber production and carbon sequestration benefits would sharply increase forest-based revenues, while realizing the carbon sequestration potential of P. tabulaeformis plantations. This approach was clearly useful to the development of reforestation/afforestation projects trying to mitigate climate change and also provided a theoretical basis for sustainable forest management.     

Barriers to natural regeneration in temperate forests across the USA

New Forests - For millennia, natural disturbance regimes, including anthropogenic fire and hunting practices, have led to forest regeneration patterns that created a diversity of forest lands...