Molecular analysis of cytosolic and mitochondrial malate dehydrogenases isolated from domestic cats (Felis catus).

Malate dehydrogenase (MDH) plays crucial roles in energy and cellular metabolism. In this study, we describe the identification and characterization of cytosolic MDH (MDH1) and mitochondrial MDH (MDH2) in liver of domestic cat (Felis catus). To clone the feline full-length MDH genes, we performed rapid amplification of cDNA ends. The MDH1 gene encoded a protein of 334 amino acids and the MDH2 gene encoded a protein of 338 amino acids, containing a 24-amino acid mitochondrial target sequence. The feline MDH1 and MDH2 proteins shared, respectively, 98.8-93.7 and 96.7-94.4% homology with dog, giant panda, horse, cow, pig, human, mouse, and rat. The feline MDHs had a highly conserved active motif, which contained important residues for catalysis and coenzyme binding. The putatively acetylated lysine residues that regulate MDH activity were also conserved at K118, K121, and K298 in MDH1, and K185, K301, K307, and K314 in MDH2. Both MDH1 and MDH2 mRNAs were ubiquitously expressed, but these expression levels varied in a tissue-specific manner. Both MDH genes were expressed at considerably high levels in heart and skeletal muscle, but at low levels in lung and spleen.

Pollen tube attraction by the synergid cell.

In flowering plants, guidance of the pollen tube to the embryo sac (the haploid female gametophyte) is critical for successful fertilization. The target embryo sac may attract the pollen tube as the final step of guidance in the pistil. We show by laser cell ablation that two synergid cells adjacent to the egg cell attract the pollen tube. A single synergid cell was sufficient to generate an attraction signal, and two cells enhanced it. After fertilization, the embryo sac no longer attracts the pollen tube, despite the persistence of one synergid cell. This cessation of attraction might be involved in blocking polyspermy.

IgG-mediated histamine release from canine mastocytoma-derived cells.

BACKGROUND: Recent data suggest that normal tissue mast cells can express functional receptors for IgG under certain conditions. However, little is known about IgG receptor expression and functional consequences in mast cell neoplasms. METHODS: In this study, neoplastic mast cells were obtained from a dog with cutaneous mastocytoma (CM-MC) and from a dog with visceral mastocytoma (VI-MC). Both cell populations were characterized morphologically and functionally. RESULTS: Most cells proliferated constantly in suspension without particular supplements. Doubling times of CM-MC and VI-MC were 52.2 and 27.5 h, respectively. Both cell types were sensitive to formalin fixation, did not contain heparin and were tryptase and chymase positive. Electron microscopy showed fine granules with electron-dense content in both cell populations. The total histamine content of CM-MC and VI-MC was 0.25 and 0.10 pg/cell, respectively. Calcium ionophore A23187 and substance P induced dose-dependent histamine release, whereas compound 48/80 had no effect. Most significantly, both cell types, when sensitized with monomeric dog IgG, released histamine upon stimulation by anti-dog IgG. CONCLUSIONS: Dog mastocytoma-derived cells may be useful to study the regulation of neoplastic mast cell growth and differentiation, as well as IgG receptor-mediated activation in neoplastic mast cells. Further research is required to clarify the pathophysiological significance of constitutive expression of IgG receptors in neoplastic (canine) mast cells.

Site-specific single amino acid changes to Lys or Arg in the central region of the movement protein of a hybrid bromovirus are required for adaptation to a nonhost.

A hybrid Cowpea chlorotic mottle virus (CCMV) [CCMV(B3a)] in which the CCMV 3a movement protein gene is replaced by the 3a (B3a) gene of Brome mosaic virus cannot infect cowpea systemically. Previously, analysis of RNA3 cDNA clones constructed from cowpea-adapted mutants derived from CCMV(B3a) revealed that a single codon change in the B3a gene allowed CCMV(B3a) to infect cowpea systemically. In this study, to extend the analysis of the CCMV(B3a) adaptation mechanism, we directly sequenced B3a gene RT-PCR products prepared from 28 cowpea plants in which cowpea-adapted mutants appeared, and found seven patterns of a codon change localized at five specific positions in the central region (Ser(118), Glu(132), Glu(138), Gln(178), and Ser(180)). All of the patterns involved an amino acid change to Lys or Arg. Mutational analysis of the B3a gene demonstrated that a single codon change resulting in either Lys or Arg at any of the five positions was sufficient for the adaptation of CCMV(B3a) to cowpea. In contrast, CCMV(B3a) variants with a codon change resulting in Lys or Arg at three other positions (137, 155, and 161) in the B3a gene not only showed lack of systemic infection of cowpea but also showed weakened initial cell-to-cell movement in the inoculated leaves and diminished B3a accumulation in protoplasts. These results suggest that adaptive changes in the B3a gene are site-specifically selected in cowpea plants.

The time-course of the effects of contralateral sound on the level of distortion product otoacoustic emissions.

The effects of the addition of contralateral noise on the level of distortion product otoacoustic emissions (DPOAEs) were examined. In the present study, the DPOAEs were recorded for a relatively long period (2 minutes), and the time-course of the effects of contralateral sound on the level of DPOAEs were considered. In general, the addition of the contralateral noise resulted in suppression of the level of DPOAEs. The time-course of this suppression appeared to depend on the level of the contralateral noise. When the level of the contralateral noise was low, the suppression of the level of DPOAEs seemed to be largely unchanged for at least 2 minutes. In contrast, when a relatively high level of contralateral noise was used, the suppression of the level of DPOAEs decreased with time.

Effects of perineural capsaicin treatment on cardiopulmonary reflexes elicited by laryngeal instillations of capsaicin and distilled water in sevoflurane-anesthetized dogs.

The aim of this study was to determine the effect of perineural capsaicin (CAPS) treatment on cardiopulmonary reflexes elicited by topical laryngeal instillation of CAPS and distilled water (DW) in sevoflurane-anesthetized dogs. Cardiopulmonary reflexes elicited by CAPS (10 microg/ml, 10 ml) were attenuated by perineural CAPS treatment to the superior laryngeal nerves (SLNs) (P<0.05), whereas those by DW (10 ml) remained unaffected (P>0.05). The reflex responses to DW that remained even after the perineural CAPS treatment were eliminated by laryngeal anesthesia with lidocaine. These results suggest that cardiopulmonary reflexes from the laryngeal mucosa elicited by CAPS instillation can be blocked by perineural CAPS treatment to the SLNs, which may result from inhibition of the laryngeal CAPS-sensitive C-fiber afferents.

Effects of perineural capsaicin treatment on compound action potentials of superior laryngeal nerve afferents in sevoflurane-anesthetized dogs.

Effects of perineural capsaicin (CAPS) treatment on compound action potentials of the superior laryngeal nerve (SLN) afferents were studied in 6 sevoflurane-anesthetized dogs. Perineural CAPS (100 microg/ml) to the bilateral SLNs reduced (P<0.01) the peak and integral amplitudes of the C-wave of the compound action potential. By contrast, the perineural CAPS had no effect on the A-wave component (P>0.05). Removal of the perineural CAPS recovered the C-wave to pretreatment level. The perineural CAPS treatment selectively blocks C-wave compound action potentials of the SLN afferents, providing a useful tool for studies of laryngeal C-fibers in respiratory physiology.

High efficiency selection of full-length cDNA by improved biotinylated cap trapper.

We report here an improved protocol for the preparation of full-length cDNA libraries that improves the previously reported method (Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics, 137, 327-336), that allows long cDNAs to be cloned more efficiently. One potential disadvantage of the original biotinylated CAP trapper protocol is the exposure of mRNA to chemical and enzymatic attacks during the biotinylation of the cap structure, before the first-strand cDNA synthesis (and selection of full-length cDNA by biotinylated cap). Here, we show that the biotinylation of the cap structure is very specific and effective even if biotinylation is performed on the mRNA/cDNA hybrid produced by the first-strand cDNA synthesis reaction. Consequently, mRNA remains protected from chemical and enzymatic degradation during the overnight biotinylation step, thus making it possible to select full-length cDNAs of longer average size. We herein report the efficiency and specificity of the new version of the protocol for cap structure biotinylation and capture of full-length cDNA.

High-efficiency full-length cDNA cloning by biotinylated CAP trapper.

We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 x 10(7)/10 micrograms starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.

[Two cases of pseudomyxoma peritonei that responded to intraperitoneal administrations of a combined CDDP, 5-FU and MMC therapy].

Pseudomyxoma peritonei, which is the seeding of the peritoneum by mucin-secreting metastatic deposits and the filling of the peritoneal cavity by these secretions, is a malignancy that even after excision of the metastatic areas has a poor prognosis, since no effective therapy has yet been established. Herein, we report two cases of pseudomyxoma peritonei that responded to a combined postsurgical therapy consisting of CDDP, 5-FU and MMC. Case 1 involved a 56-year-old woman who underwent an appendectomy, a bilateral ovariectomy, and an omentectomy, due to pseudomyxoma peritonei. Further, a subcutaneous implant-type reservoir was sited intraperitoneally for postoperative chemotherapy. At 30 days after surgery, therapy commenced, consisting of 100 mg of CDDP and 20 mg of MMC administered intraperitoneally and 1,000 mg of 5-FU continuously infused by intravenous drip. This combined therapy was given for 5 days and repeated 8 times. As a result, the serum CEA value, which had elevated postoperatively, fell within the respective normal range, and at 4 years postoperatively, this patient remains alive. Case 2 concerns a 59-year-old woman who underwent an appendectomy, a right ovariectomy, and an omentectomy due to pseudomyxoma peritonei, and was postoperatively given 50 mg intraperitoneal administration of CDDP. She received 2 courses of postoperative chemotherapy similar to that given case 1 but with the addition of a 500 mg 5-FU intraperitoneally. At 8 months postoperatively, she is alive and disease-free. Given the above results, intraperitoneal administrations of this combined CDDP-5-FU-MMC therapy may be effective for patients with pseudomyxoma peritonei.

[In vivo neurochemical effects of electroconvulsive shock on the forced swimming rat].

The effects of electroconvulsive shock (ECS) on monoamine metabolism in the forced swimming rat were studied using on-line microdialysis. In the present experiment, we measured forced swimming-induced immobility time and interstitial concentrations of dopamine (DA), its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and serotonin (5-HT), its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the anterior hypothalamus. Mean immobility time was short in the ECS-treated group compared with shamECS group but the difference was not statistically significant. DA and 5-HT tended to increase following a single ECS. Interstitial concentration of 5-HIAA increased significantly. Forced swimming test caused an increase in both DA and 5-HT turnover in the hypothalamus. In contrast, pretreatment with ECS tends to suppress these forced swimming-induced increases in DA and 5-HT turnover. These data suggest that ECS may regulate excess activation in central dopaminergic and serotonergic system following forced swimming stress.

Effects of pulsing electromagnetic fields on the ligament healing in rabbits.

Effects of pulsing electromagnetic fields (PEMFs) on ligament healing were investigated using 80 rabbits. All animals received square resection (4 mm x 4 mm) of both patellar ligaments in full thickness at their center. They were divided into 4 groups of 20 rabbits each and stimulated with different electromagnetic intensity of 0 (control), 2, 10, and 50 gauss (G) for 6 hr daily. Pulse frequency and pulse width were 10 Hz and 25 microseconds, respectively. After PEMFs stimulations for 1, 2, 3 and 4 weeks, 5 animals of each group were euthanized and the regenerated tissue at the defective portion was investigated histologically and biomechanically. Histologically, the tissue stimulated by PEMFs showed an earlier increase in capillaries and fibroblasts and more matured, prominent longitudinal orientation of collagen fibers than those of control groups. Among the rabbits stimulated electromagnetically, those stimulated at 50 G revealed the earliest ligament healing. Tensile strength of regenerated ligament tissues of any PEMFs groups increased significantly at 1 and 2 weeks after operation, however, at 3 and 4 weeks after operation, there were no significant differences between groups. Among these values, those of 50 G group were the highest consistently during most of the experimental period. From the above results, PEMFs enhanced the earlier stage of ligament healings and 50 G gauss seemed to be the most effective among the 3 field intensities used. This promoting effect may potentiate the earlier recovery of the function after the ligament injury.

Pathological studies on local tissue reactions in guinea pigs and rats caused by four different adjuvants.

We investigated pathological changes at the injection site in guinea pigs and rats for 16 weeks following a single intramuscular injection of one of the following oil adjuvant emulsions; oil adjuvant ISA-70, Freund's incomplete adjuvant, Freund's complete adjuvant, and aluminium phosphate gel. In the animals injected with ISA-70 emulsion prepared by manual shaking, grossly, there was partial thickening of subcutaneous tissue, discoloration of inter-muscular connective tissue, and swelling of the inguinal lymph nodes at 2 and 4 weeks post injection (PI). Histopathologically, ISA-70 injected sites revealed acute inflammatory changes at 72 hrs PI, and peak reactions consisting of macrophage accumulation around oil cysts and fibrosis were observed at 4 weeks PI. These changes were less severe and of shorter duration than those in the other three adjuvants. Guinea pigs and rats injected with materials containing inactivated Newcastle disease virus (NDV) antigen similarly showed an infiltration of plasma cells and lymphocytes in addition to the changes described above. ISA-70 containing NDV antigen induced similar hemagglutination-inhibition titer to that induced by Freund's incomplete adjuvant.

[A comparative study of cefepime for bacterial pneumonia].

The efficacy, safety and usefulness of cefepime (CFPM), a new cephem antibiotic, in bacterial pneumonia, were evaluated in a comparative study against ceftazidime (CAZ). Each drug was administered by intravenous drip infusion at a dose of 1.0 g (nominal potency) twice daily for 14 days, and the following results were obtained. 1. A total of 183 cases were enrolled in this study. Efficacy rates ("good" or better responses) as evaluated by the subcommittee were 90.3% (65/72) in the CFPM group and 94.0% (63/67) in the CAZ group, with no significant difference between the 2 groups. 2. Efficacy rates ("good" or better responses), as evaluated by attending physicians, (in the same bacterial pneumonia cases which were subjected to evaluation by the subcommittee) were 87.5% (63/72) in the CFPM group and 89.6% (60/67) in the CAZ group, with no significant difference between the 2 groups. 3. Bacteriologically, eradication rates were 96.9% (31/32) in the CFPM group and 96.7% (29/30) in the CAZ group with no significant difference between the 2 groups. 4. The incidence of side effects was 5.9% (5/85) in the CFPM group and 4.8% (4/84) in the CAZ group, with no significant difference between the 2 groups. No significant difference was also found between the 2 groups in the incidence of abnormal laboratory findings; 28.4% (23/81) of the case in the CFPM group and 34.1% (28/82) in the CAZ group. 5. As for overall usefulness of the drug in bacterial pneumonia cases, utility rates ("useful" or better evaluations) as evaluated by the subcommittee were 88.9% (64/72) in the CFPM group and 92.5% (62/67) in the CAZ group. The rates as evaluated by investigators (in cases judged as evaluable by the subcommittee) were 87.5% (63/72) and 85.1% (57/67), respectively. There were no significant differences between the 2 groups. These results indicated that CFPM is very useful for the treatment of bacterial pneumonia.

Multiplication of attenuated and virulent porcine parvoviruses in colostrum-deprived, neonatal pigs.

Colostrum-deprived, neonatal, 2 days old pigs were inoculated with the attenuated HT-/SK or the virulent 90HS strain of porcine parvovirus (PPV) by the oral or subcutaneous route and sacrificed 2, 4 or 6 days after inoculation. Then, comparison was made on viral multiplication in pigs between the two strains. Pigs inoculated with the HT-/SK strain showed no detectable viremia or HI antibody responses against PPV within 6 days after inoculation. Only in pigs inoculated by the subcutaneous route, a small amount of virus was recovered from the spleen, liver, or mesenteric lymph nodes. These viruses were distinguished from the parental virulent 90HS strain, as examined for rct maker in vitro. When pigs were inoculated with the virulent 90HS strain, viremia appeared in all of them 1 day after inoculation and continued for up to the sacrificed day. Moreover, a considerable amount of virus was also detected from all tissues, including brain, lung, liver, spleen, pancreas, small intestine, and lymph node tissues, in all pigs tested. HI antibodies were first detected 6 days after inoculation.

Effect of hypercholesterolemia on cholinephosphotransferase activity in rabbit and rat vessel walls.

The activity of cholinephosphotransferase (EC 2.7.8.2, CPT) which catalyses de novo synthesis of phosphatidylcholine (PC) was studied in aortas of rabbits and rats, and in brain microvessels of rabbits with a cholesterol feeding-induced hypercholesterolemia. Cholesterol feeding produced a marked atheromatous change in rabbit aortas but not in rat aortas. The aortas of cholesterol-fed rabbits displayed a significantly higher CPT activity than the controls. On the other hand, the aortic CPT activity of cholesterol-fed rats was not different from that of control rats. The brain microvessels of cholesterol-fed rabbits having atheromatous aortic lesions did not show any lipid deposition, and CPT activity was similar to that of control rabbits. A tocopherol-deficient, high-cholesterol diet produced microscopical lipid deposits in rat aortas, and CPT activity of these aortas was significantly higher than that of aortas of rats on tocopherol-supplemented diets containing either a normal or high amount of cholesterol. The increase in CPT activity in atheromatous lesion might be closely related to lipid deposition in vessel walls and may be a cause of the increase in PC content in these lesions. Further studies are required to clarify the mechanism of activation of CPT activity in atheromatous conditions.

Effect of glycyrrhizin on stability of lysosomes in the rat arterial wall.

Studies were made on the effect of phospholipase A2 on the stability of arterial wall lysosomes, and the influence of glycyrrhizin on this effect. Lysosomal phospholipase A2 activity was found to be increased in the aorta of hypercholesterolemic rats. Treatment of the lysosomal fraction of the arterial wall with venom phospholipase A2 resulted in release of acid phosphatase, and addition of glycyrrhizin (0.1 mM) inhibited this release. Lysosomal phospholipase A2 in the arterial wall was also inhibited dose-dependently by glycyrrhizin. These results suggest that lysis of lysosomes was due to increase in phospholipase A2 activity, and that glycyrrhizin stabilized the lysosomes by inhibiting phospholipase A2 activity.

Effects of tocopherol deficiency on lipid metabolism in the arterial wall of rats on normal and high cholesterol diets.

The effects of dietary tocopherol deficiency on arterial wall enzymes involved in lipid synthesis and hydrolysis were studied in rats receiving normal diets and diets supplemented with 1% cholesterol. Arterial wall lipase and cholesterol esterase were associated with both the lysosome and microsome fractions, whereas acyl CoA synthetase, triglyceride synthesizing activity, cholesteryl ester synthesizing activity and cytidine diphosphatecholine-1,2-diacyl glycerol choline phosphotransferase (CPT) were found mainly in the microsomal fraction. When tocopherol was depleted from either the normal or high cholesterol diets, the following changes occurred in the arterial wall: (1) increase in thiobarbituric acid reactive substances; (2) decrease in lysosomal acid lipase and acid cholesteryl esterase; (3) decrease in the microsomal enzymes, acyl CoA synthetase, triglyceride synthesizing activity, cholesteryl ester synthesizing activity, neutral lipase and neutral cholesteryl esterase; and (4) increase in microsomal CPT. The results of these studies suggest that dietary tocopherol plays an important role in both lipid synthesis and degradation in the arterial wall, and the results may account for the accumulation of lipids and lipoperoxides in atherosclerotic lesions.

Opsonic activity and O-agglutinins against Escherichia coli in bovine colostrum.

Anticolibacillus antibody activity was examined in 16 samples of bovine colostrum by O-agglutination test with 6 serotypes of Escherichia coli, and its correlation with the manifestation of diarrhea was analyzed for newborn calves fed these colostrums. The 4 colostrums fed to newborn calves having diarrhea within a few days after birth had significantly lower agglutinin titers for all the serotypes tested than did the other colostrums. Other newborn calves fed on the remaining 12 colostrums did not manifest clinical signs of enteric disorders. The agglutinin spectrum against the E coli serotypes was similar between colostrum samples from the same stockfarm, but variable among the farms from which the colostrum had been collected. The agglutinin activity was sensitive to 2-mercaptoethanol and was found mainly in the macroglobulin fraction of gel filtration, indicating that the activity was due to antibodies of the immunoglobulin M type. This was evidenced directly by the agglutination test of purified immunoglobulins. Opsonic activity of colostrum and immunoglobulins purified therefrom was estimated by the rate of decrease in the number of viable E coli injected into mouse peritoneal cavity. The results indicated that the opsonic activity in colostrum was also attributed mainly to immunoglobulin M antibodies, although the contribution of immunoglobulin A and G antibodies was not ruled out. A part of the opsonic activity in colostrum seemed due to heat-labile component(s) as well.