Time-dependent changes in protein phosphorylation patterns in rat brain synaptosomes caused by deltamethrin

Effects of deltamethrin, a powerful pyrethroid insecticide, on the protein phosphorylation and dephosphorylation processes during depolarization in rat brain synaptosomes were studied by using [³²P]phosphoric acid as a starting radiotracer and high external concentration of potassium ions or veratridine (10^?-5 M) as depolarizing agents. At the onset of depolarization there was a quick rise in phosphorylation in various synaptic proteins for about 15–30 s followed by a gradual decline in levels of phosphorylation. The effect of deltamethrin (10^?-7 M) on this system was found to be dependent on the length of preincubation of the synaptosome with the pesticide prior to depolarization. At an early stage (0–3 min preincubation period) it caused a modest suppression of protein phosphorylation activities. When the period of deltamethrin preincubation was extended to 5–20 min, however, it caused a significant increase in protein phosphorylation throughout the depolarization period. At the later stage of the action of deltamethrin (e.g. preincubation period of 30–40 min), deltamethrin-treated synaptosomes no longer responded to the depolarization signal to raise the level of phosphorylation on many proteins. These results indicate that deltamethrin's actions on the synaptic process are complex. Depending on the length of exposure, its effects on protein phosphorylation responses in intact synaptosomes could be either stimulatory or inhibitory. To study the cause of deltamethrin-induced synaptic block at the later stage, effects of deltamethrin on protein kinases were studied by using lysed synaptic membranes with [gamma-³²P]ATP. Deltamethrin was shown to inhibit calcium–calmodulin-dependent protein phosphorylation activities at 10^?-7 M when given directly to the enzyme source 10 min prior to the addition of [³²P]ATP. Such an observation helps to explain the inhibitory action of deltamethrin on protein phosphorylation which occurs at the late stage of its action (i.e. preincubation time > 20 min).     

Molecular heterogeneity in clinical isolates of Malassezia pachydermatis from dogs

Molecular investigation of 16 strains, conventionally identified to be Malassezia pachydermatis, isolated from dogs in Japan was carried out by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD band patterns of 13 clinical isolates were identical to that of standard strain of M. pachydermatis (CBS-1879). The other three clinical isolates were different from the standard strain of M. pachydermatis in RAPD patterns, and two of the three isolates were identical. About 620 bp genomic DNA fragments of the CHS2 gene were amplified from the same 16 clinical isolates of M. pachydermatis by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of the 16 clinical isolates revealed that the 13 strains were genetically very close to the standard strain of M. pachydermatis and the other two isolates were genetically close to the standard strain of M. furfur rather than M. pachydermatis. The remaining one isolate was phylogenetically distinct from all the seven Malassezia species reported so far.     

Ultrastructural Changes in Calvarial Osteoblast Cytoskeleton after Prostaglandin E2 Administration in Rats

Recent studies on the cytoskeleton of osteoblasts have been made mainly using cultured cells. However, the morphology of cultured cells may be altered during subculture. Therefore, cytoskeletal changes of calvarial osteoblasts were investigated in situ by electron microscopy using the detergent perfusion method to preserve cell morphology as well as selectively observing the cytoskeleton in the presence of a high concentration of prostaglandin E2 (PGE2) in the calvarial periphery in rats. Rats were perfused with a mixture of Triton X-100 and glutaraldehyde, then the cytoskeleton was observed by transmission electron microscopy. In osteoblasts of the control group, thick bundles of microfilaments ran parallel to the long axis of the cells immediately below the cell membrane adjacent to the osteoid layer. In contrast, in the osteoblasts of the PGE2-administered group, the external morphology was changed to an asteroid or cubic shape, and thick bundles of microfilaments immediately below the cell membrane adjacent to the osteoid were not observed, although microfilament fibres, with a diameter of 5-6 nm, were observed in the cytoplasm.     

Efficacy of enamel matrix protein applied to spontaneous periodontal disease in two dogs

Enamel matrix protein (EMP) was applied for regeneration of periodontal tissue in 2 dogs with spontaneous periodontal disease. Case 1 had bony resorption around the root and root apex of the maxillary fourth premolars. Case 2 had vertical resorption of bone between the mandibular first and second molars. A flap was formed in the buccal gingiva, and EMP was applied onto the surface of the exposed root. One or 4 months postoperatively, increased bone level and clinical attachment were recognized. EMP was therefore suggested to be effective to induce regeneration of periodontal tissues in the cases with periodontal disease.     

Clinical evaluation of growth hormone secretion in cattle using insulin tolerance test

Growth hormone secretion was evaluated in cattle. Clinically healthy bovine growth hormone (bGH) concentrations were 10.7 +/- 1.6 ng/ml in Holstein and 7.8 +/- 3.9 ng/ml in Japanese black cattle. The bGH concentration alternated at three-hour intervals, and tended to be higher at midnight and lower in the morning and before feeding. Insulin tolerance test (ITT) at an insulin dosage of 0.25 U/kg showed a significant increase of bGH concentration to 331 +/- 153% at 60 to 90 min after injection. In ITT applied to five under-growth calves of Japanese black cattle, the basal bGH concentrations were lower and peak values after insulin injection were shown to be significantly low. The ITT is useful for the clinical examination of bGH secretion in cattle.     

Synthesis and insecticidal activity of benzoheterocyclic analogues of N'-benzoyl-N-(tert-butyl)benzohydrazide: Part 3. Modification of N-tert-butylhydrazine moiety

Nineteen analogues were synthesized by modifying the tert-butylhydrazine moieties of N'-tert-butyl-N'-(3,5-dimethylbenzoyl)-5-methyl-2,3-dihydro-1,4-benzodioxine-6-carbohydrazide and N'-tert-butyl-N'-(3,5-dimethylbenzoyl)-5-methylchromane-6-carbohydrazide (chromafenozide), and the synthesized analogues were evaluated for their insecticidal activity against Spodoptera litura F. While all of the synthesized analogues had insecticidal activity inferior to those of the lead compounds, several of the analogues nonetheless showed high insecticidal activity. Chromafenozide has shown very high selectivity toward lepidopteran species.     

Experimental and clinical studies on plasma leptin in obese dogs

Leptin is a protein synthesized and secreted primarily by adipocytes, and the circulating leptin concentration is elevated in obese humans and rodents. Recently, we have established a sandwich enzyme-linked immunosorbent assay for canine leptin. In the present study, plasma leptin concentrations were measured in experimentally developed obese beagles and in clinically obese dogs. When 5 male beagles were given a high-energy diet for 3 months, all of them became obese and the plasma leptin concentration significantly increased from 2.4+/-1.2 to 4.9+/-0.9 ng/ml, positively correlating with body fat content estimated by the deuterium oxide dilution method (r=0.87). The leptin concentrations of plasma samples collected from 59 dogs in veterinary practices were compared with their body condition scores (BCS). The plasma leptin concentrations of obese dogs were 9.7+/-0.7 and 12.3+/-1.5 ng/ml at BCS=4 and BCS=5, respectively, which were significantly higher than those of optimal (BCS=3) dogs (2.7+/-0.3 ng/ml). There was no significant effect of sex and breed. A weak positive correlation (r=0.37) was found between the plasma leptin concentration and age, probably due to the lesser content of visceral fat in puppies younger than 1 year old. These results indicate that plasma leptin is a good index of adiposity in dogs regardless of breed, age and sex, and may be useful for quantitative assessment of obesity in small animal practice.