Purification and characterization of feline ghrelin and its possible role

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser3, has been isolated from rat and human stomach as an endogenous ligand for the growth hormone secretagogue receptor. Here, we purified feline ghrelin and examined its possible physiological role in cats. The major active form of feline ghrelin is a 28-amino acid peptide octanoylated (C8:0) at Ser3; except for one amino acid residue replacement, this structure is identical to those of rat and human ghrelins. However, much structural divergence in peptide length and fatty acid modification was observed in feline ghrelin: peptides consisting of 27 or 26 amino acids lacking Gln14 and/or Arg28 were found, and the third serine residue was modified by octanoic acid (C8:0), decanoic acid (10:0), or unsaturated fatty acids (C8:1, C10:1 and C10:2). In agreement with the structural divergence, two kinds of cDNA with different lengths were isolated. Administration of synthetic rat ghrelin increased plasma growth hormone levels in cats, with a potency similar to that in rat or human. Plasma levels of ghrelin in cats increased approximately 2.5-fold after fasting. The present study indicates the existence of structural divergence in feline ghrelin and suggests that, as in other animals, ghrelin may play important roles in GH release and feeding in cats.     

Characterization of an endopolygalacturonase Gene cppg1 from Phytopathogenic Fungus Chondrostereum purpureum

Chondrostereum purpureum, a phytopathogenic fungus, produces endopolygalacturonase (endoPG) which has been suggested to have a causal role in the silver-leaf symptom of apple trees. In this paper, we detected C. purpureurn-derived endoPG at the infection sites using ELISA with a polyclonal antibody against endoPG I. A gene encoding endoPG I and its homolog were also isolated from the C. purpureum genome. The endoPG I gene was designated as cppg1. The cppg1 gene is the first fungal endoPG gene reported in the Basidiomycetes. Received 31 May 2000/ Accepted in revised form 13 September 2000     

Hepatocyte growth factor transduces different intracellular signals in aortic and umbilical venous endothelial cells

Endothelial cells are important for maintenance of vascular integrity by producing a variety of bioactive molecules such as nitric oxide (NO). Recent evidence has suggested that there are some differences in characteristics between endothelial cells from different origins. Here we examined responses of two typical endothelial cells to hepatocyte growth factor (HGF), which induces endothelium-dependent relaxation of microvessels. Stimulation of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) with HGF increased endothelial NO synthase activity, accompanied with an increase of activity-related site-specific phosphorylation of protein kinase B/Akt. However, HGF stimulated phosphorylation of p38 mitogen-activated protein kinase (MAPK) only in HUVEC, but not in BAEC, while it induced phosphorylation of p44/p42 MAPK in both cells. These results suggest that HGF transduces different intracellular signals between aortic and umbilical venous endothelial cells, and that the differences might represent divergent endothelial responses to growth factors, especially those that activate receptor-tyrosine kinases.     

Isolation of Salmonella from diarrheic feces of pigs

A survey of Salmonella was carried out in fecal samples of 887 pigs with diarrhea collected from 235 pig farms between April 1996 and March 2001. Salmonella was isolated from 84 feces (9.5%) of 887 pigs and from 45 (19.1%) of 235 farms. The higher prevalence was found in weaned pigs (12.4%) and fattening pigs (17.3%) than in sows (4.2%) and suckling pigs (4.5%). Isolation rates of S. Typhimurium were higher from weaned and fattening pigs than from the others. Therefore, risk of horizontal infection of S. Typhimurium will increase, if no adequate health managements are practiced when weaned and fattening pigs have diarrhea.     

Isolation and serological survey of Salmonella in pigs in Japan

A total of 267 fecal and serum samples collected from individual pigs reared on a Salmonella-positive farm were subjected to bacteriological and serological examinations of Salmonella. Salmonella was isolated from 47 pigs (17.6%) and prevalence of antibody to lipopolysaccharide (LPS) of S. Typhimurium, which was partly common to S. O4, 12: d: -, was observed in 90 pigs (33.7%). Salmonella was isolated from 26 (28.9%) of 90 antibody-positive pigs and 21 (11.9%) of 177 antibody-negative pigs. Twenty-one of 36 pigs (58.3%) positive for S. O4, 12: d: -, five of 10 pigs (50.0%) positive for S. Havana, and none for S. Anatum had antibodies. Thus, seropositive rates were higher than isolation-positive rates, and antibody prevalence was associated with serovars of the isolates. Then, we analyzed antibody prevalence among pigs on Japanese pig farms. The antibodies to LPS of S. Typhimurium were found in 195 of 1,498 pigs (13.0%) and in at least one serum sample on 35 of 52 farms (67.3%). Our results indicate that Salmonella does not seem to be so prevalent in pigs though it is widely prevalent among pig farms.     

Effect of loading frequency on fatigue life and dissipated energy of structural plywood under panel shear load

Wood-based panels are subjected to cyclic panel shear load caused by wind and seismic forces in such an application as the sheathing of bearing walls. The fatigue behavior of structural plywood under panel shear load with two different loading frequencies was examined. Pulsating panel shear load with a triangular waveform and loading frequency of 0.5 or 5 Hz was applied to the plywood specimens. Stress−strain hysteresis loops were measured throughout the fatigue tests. Fatigue life was highly dependent on loading frequency at more than 0.5 stress level. The deterioration of mechanical property and damage accumulation in plywood specimen was observed to be slower at higher loading frequency at more than 0.5 stress level. Analyses based on energy loss suggest that panel shear load with higher loading frequency causes less damage to the plywood specimen during one loading cycle at higher stress level, and that the fatigue damage accumulation causing failure might be dependent on stress level although it seems to be unaffected by loading frequency. Based on these results, a new fatigue failure model for plywood specimen was qualitatively developed by combining Weibull’s weakest link model and Daniels’ fiber bundle model.     

Fatigue of structural plywood under cyclic shear through thickness I: fatigue process and failure criterion based on strain energy

The fatigue behavior of plywood specimens under shear through thickness was examined on the basis of strain energy to obtain common empirical equations for the fatigue process and failure criterion under various loading conditions. Specimens were cut from commercial plywood panels of 9-mm thickness. Loading conditions were set as follows: a square waveform at a loading frequency of 0.5 Hz, a triangular waveform at 0.5 Hz, and a triangular waveform at 5.0 Hz. Peak stress applied was determined to be 0.5, 0.7, and 0.9 of static strength, that is, stress levels of 0.5, 0.7, and 0.9. The stress-strain relationships were measured throughout the fatigue test, and the strain energy was obtained at each loading cycle. Loading conditions apparently affected the relationship between stress level and fatigue life. On the other hand, the relationship between mean strain energy per cycle and fatigue life was found to be independent of loading conditions. Mean strain energy per cycle obtained as the fatigue limit was 5.85 kJ/m³ per cycle. Assuming that the accumulation of strain energy is a fatigue indicator, the fatigue process and failure criterion for the plywood specimens under the three loading conditions were commonly expressed by the relationship between cumulative strain energy and loading cycles.     

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) progress to date and future plans

The INHAND Proposal (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) has been operational since 2005. A Global Editorial Steering Committee (GESC) manages the overall objectives of the project and the development of harmonized terminology for each organ system is the responsibility of the Organ Working Groups (OWG), drawing upon experts from North America, Europe and Japan.Great progress has been made with 9 systems published to date – Respiratory, Hepatobiliary, Urinary, Central/Peripheral Nervous Systems, Male Reproductive and Mammary, Zymbals, Clitoral and Preputial Glands in Toxicologic Pathology and the Integument and Soft Tissue and Female Reproductive System in the Journal of Toxicologic Pathology as supplements and on a web site – www.goreni.org. INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in rodent toxicity and carcinogenicity studies. The guides provide representative photo-micrographs of morphologic changes, information regarding pathogenesis, and key references. During 2012, INHAND GESC representatives attended meetings with representatives of the FDA Center for Drug Evaluation and Research (CDER), Clinical Data Interchange Standards Consortium (CDISC), and the National Cancer Institute (NCI) Enterprise Vocabulary Services (EVS) to begin incorporation of INHAND terminology as preferred terminology for SEND (Standard for Exchange of Nonclinical Data) submissions to the FDA. The interest in utilizing the INHAND nomenclature, based on input from industry and government toxicologists as well as information technology specialists, suggests that there will be wide acceptance of this nomenclature. The purpose of this publication is to provide an update on the progress of INHAND.     

Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca²⁺ mobilization of the cells was evaluated using the Ca²⁺ indicator Fluo3 to monitor Ca²⁺ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca²⁺ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.