Establishment of a canine lens epithelial cell line

Some immortalized lens epithelial cell lines have been established and are useful for molecular analysis. The establishment of additional cell lines must, however, enable a variety of in-vitro examinations. The objective of this study was to establish a new canine lens epithelial cell line by isolating CLC-1 cells from the lens tissue of a dog with cataracts. In CLC-1 cells, transforming growth factor beta (TGF-β) treatment significantly decreased gene expression of an epithelial marker and elevated that of mesenchymal markers; these characteristics are similar to those of a human lens epithelial cell line. Interestingly, CLC-1 cells exhibited lower expression of an epithelial marker and higher expression of mesenchymal markers than an anterior lens capsule. These results suggest that CLC-1 cells were derived from a cell population that was committed to epithelial-mesenchymal transition in cataract lens tissue. In conclusion, CLC-1 cells could be useful for analyzing molecular pathogenesis in canine cataracts.     

Anaesthetic and cardiopulmonary effects of balanced anaesthesia with medetomidine-midazolam and butorphanol in dogs

The anaesthetic and cardiopulmonary effects of combinations of medetomidine (Me), midazolam (Mi) and butorphanol (Bu) were evaluated in dogs. The characterization of anaesthetic effects was assessed using a scoring system. The combinations tested were 20 or 40 micrograms/kg Me and 0.5 mg/kg Mi (20Me-Mi or 40Me-Mi) followed by either an intravenous injection of physiological saline solution (PSS) or Bu (0.1 or 0.3 mg/kg). The mixture of Me and Mi was injected intramuscularly, followed 15 min later by an intravenous injection of Bu or PSS in all six groups. The combined Me-Mi induced deep sedation but not profound anaesthesia. The effect of the subsequent Bu administration was observed in the scores related to its analgesic effect. There were no significant differences between the two doses of Bu, following either 20Me-Mi or 40Me-Mi in the duration of anaesthesia, heart and respiratory rates, rectal temperature, and anaesthetic and analgesic scores except for palpebral reflex, and interdigital web clamping scores. Therefore, we concluded that the addition of 0.1 mg/kg Bu to Me-Mi elicits adequate anaesthesia with adequate analgesic effect, and side-effects such as bradycardia, hypertension, and slight respiratory acidosis in some dogs.     

Effects of antioxidants on induction of apoptosis in bursal cells of Fabricius during in vitro cultivation

After physically disrupting cell contacts, apoptosis of bursal cells of Fabricius was induced during in vitro cultivation. The percentage of apoptotic cells increased with incubation time and approximately 70% cells represented apoptosis after 6 hr of incubation. The induction of apoptosis was significantly inhibited by treatment of the cells with ascorbic acid (vitamin C), but not with trolox, a vitamin E analog. An intense DNA ladder pattern was shown at 6 hr post-isolation, which is a biochemical hallmark of apoptosis. Treatment of the cells with ascorbic acid inhibited the DNA fragmentation, but trolox did not. To monitor the intracellular production of reactive oxygen species (ROSs), the intensity of fluorescence emitted from DCFH-DA was measured. The intensity of fluorescence from cells incubated for 0.5-2 hr was approximately 2-fold higher than that from cells at 0 hr. The relative intensity of fluorescence decreased immediately after the addition of ascorbic acid to the cells. The intensity from the cells treated with ascorbic acid was 20-30% of that from the control cells at each incubation time. For trolox, the intensity was 50-70% of that from the control cells at each 1 to 2 hr incubation time. When ROSs-induced lipid peroxidation was assessed using cis-parinaric acid (PnA) as a monitor molecule, lipid peroxidation was found to occur in the control cells after isolation of the bursal cells. Treatment of the cells with trolox reduced lipid peroxidation, but treatment with ascorbic acid enhanced peroxidation.     

Purification of swine carbonic anhydrase isoenzyme III and measurement of its levels in tissues and plasma

The changes in the levels of carbonic anhydrase isozyme III (CA‐III) in swine plasma and urine have not been previously determined or reported. CA‐III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA‐III from swine muscle tissues and determined CA‐III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA‐III in the tissues of female swine (age, 3 months) and plasma of young swine (age, 1–5 months) and adult female pigs (age, 2–3 years) were determined using the ELISA system for swine CA‐III. The mean (± SD) levels of CA‐III in the skeletal muscles were 3.8 ± 3.2 mg/g (wet tissue), and in the plasma, 230 ± 193 ng/ml at 1 month, 189 ± 208 ng/ml at 2 months, 141 ± 148 ng/ml at 3 months, 78 ± 142 ng/ml at 4 months and 53 ± 99 ng/ml at 5 months. The mean level of CA‐III in the plasma samples from 2‐ to 3‐year‐old pigs was 18 ± 60 ng/ml. CA‐III in the plasma samples was found to decrease from 1 month until 3 years of age (p < 0.01). We performed far‐western blotting to clarify the cause of the observed decrease in CA‐III in plasma. Our results demonstrated that CA‐III is bound to the transferrin and albumin. In addition, we determined that the levels of CA‐III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.     

Effect of pH-shifting on the gel forming characteristics of salt-ground meat from walleye pollack

ABSTRACT:   In order to elucidate the mechanism of the changes in gel forming characteristics of fish meat by pH-lowering, the gelation-temperature curve and the gelation-moisture content curve were examined using the acidified walleye pollack surimi or neutralized one after acidification. In the gelation-temperature curve, the gel strength was highest at 30°C and lowest at approximately 50–60°C, irrespective of pH shifting. The gel strength at 30°C and 80°C decreased with the decrease in pH value. The neutralization of acidified surimi improved the gel strength, but it was considerably lower than the original gel strength. The gel strength at 50–60°C was not affected by pH lowering. The gel strength at 80°C could not be revived to the original by pH readjustment, either in the presence or in the absence of EDTA. These results suggest that irreversible changes of meat protein take place under the low pH, and the oxidation ability of sulfhydryl (SH) groups of protein molecule is not affected by pH-shifting.     

A morphological study off a thyroid carcinoma in a medaka, Oryzias latipes (Temminck & Schlegel)

A large mass protruding beyond the left operculum was found in an adult male medaka, Oryzias latipes (Temminck & Schlegel). Histologically, the mass was mainly composed of thyroid follicles measuring up to 292 μm in diameter. The follicular epithelium consisted of cuboidal or squamous cells. The follicles lacked colloidal material in the lumen. The thyroid tissue infiltrated into the kidney. Electron microscopically, the tumour cells were characterized by dilation and vesiculation of rough endoplasmic reticulum, which extended throughout the cytoplasm, and an irregular outline of the nucleus. Mitotic figures and partly degenerate cells were frequently observed. Based on the above features, the tumour was diagnosed as a follicular carcinoma of the thyroid, the first reported case in the medaka.