Effect of bovine lactoferrin on the immune responses of captive bottlenosed dolphins (Tursiops truncatus) being transported over long distances.

Bovine lactoferrin was administered orally, in feed, to six bottlenosed dolphins (Tursiops truncatus) before they were transported for approximately six hours; their stress responses were compared with those of five untreated dolphins. During the journey the dolphins had an increased plasma concentration of cortisol, and lymphopenia, eosinopenia and mild neutrophilia, indicating a stress response. The administration of lactoferrin did not affect the function of the dolphins' polymorphonuclear leucocytes, but affected their leucogram by maintaining the number of circulating eosinophils.

A new deoxyuridine-5'-triphosphatase in Yoshida sarcoma cells involved in deoxyuridine 5'-triphosphate metabolism.

A magnesium-independent deoxyuridine-5'-triphosphatase was found in Yoshida sarcoma cells but not in normal rat liver. The phosphatase is specific for deoxyuridine 5'-diphosphate and deoxyuridine triphosphate, and its Km for deoxyuridine triphosphate is 2.7 X 10(-7) M. The enzyme was not inhibited by fluoride and required no divalent cations. Thus it differs from known nucleotide phosphatases. Deoxyuridine monophosphokinase, which is detectable in a crude extract of normal rat liver, could not be detected in an extract of Yoshida sarcoma cells. However, with hydroxylapatite column chromatography of the extract, a deoxyuridine 5'-monophosphate kinase activity as high as that in normal rat liver was found in fractions separated from the phosphatase activity. Thus the absence of detectable deoxyuridine 5'-monophosphate kinase activity in the crude extract of Yoshida sarcoma cells is due to the presence of this nucleotide phosphatase.

Characterization of pyrimidine nucleoside monophosphokinase in normal and malignant tissues.

It was found that there are two kinds of pyrimidine nucleoside, monophosphokinase deoxythymidine 5'-monophosphate-deoxyuridine 5'-monophosphate (dTMP-dUMP) kinase and cytidine 5'-monophosphate-deoxycytidine 5'-monophosphate-uridine 5'-monophosphate-doexyuridine 5'-monophosphate (CMP-dCMP-UMP-dUMP) kinase, and their molecular weights were calculated to be 46,000 and 26,000, respectively, by gel filtration. dTMP-dUMP kinase phosphorylated dTMP with a Km of 3.1 X 10(-5)M and dUMP with a Km of 7.7 X 10(-4) M. dTMP phosphorylation catalyzed by dTMP-dUMP kinase was inhibited competively by dUMP with a Ki of 2.0 X 10(-3) M. Similarly, phosphorylation of dUMP by this enzyme was inhibited competively by dTMP with a Ki of 2.5 X 10 (-5) M. CMP-dCMP-UMP-dUMP kinase of Yoshida sarcoma phosphorylated dUMP with a Km of 3.1 X 10(-3) M and dCMP with a Km of 7.1 X 10 (-4) M, but it did not phosphorylate dTMP. Phosphorylation of dUMP BY CMP-dCMP-UMP-dUMP kinase was inhibited competitively by DCMP and dTMP with Ki's of 6.9 X 10(-4) and 3.0 X 10(-3) M, respectively, and phosphorylation of dCMP was inhibited completely by dUMP a Ki of 2.2 X 10(-3) M. Relative Vmax activity of this enzyme was 345 nmoles/mg protein with dCMP and 127 nmoles/mg protein with dUMP.

Conversion of dNTP to dNMP dependent on DNA synthesis in isolated Yoshida sarcoma nuclei.

Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 +/0- 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGMP hydrolysis catalyzed by N-nucleotidase. (d) GDP has no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one.

Formation of deoxyribonucleoside 5'-monophosphates dependent on DNA synthesis in nuclei isolated from regenerating rat liver.

Hydrolysis of deoxyribonucleoside 5'-triphosphate, resulting in deoxyribonucleoside 5'-monophosphate formation dependent on DNA synthesis, was observed in nuclei isolated from regenerating rat liver. The intensity of the hydrolysis in nuclei varied at different times after partial hepatectomy, showing its maximum at 48 h. The rates of DNA synthesis altered corresponding to the intensities of hydrolysis. Proportionality between decrease in DNA synthesis and decrease in dNMP production was also observed in nuclei treated with various inhibitors of DNA synthesis. The formation of dNMP was detected with the four DNA substrates, indicating no involvement of specific dNTPase . Although regenerating nuclei contained a nonspecific dNTPase activity that can cause release of dNMP , this activity was independent of DNA synthesis and not inhibited by inhibitors of DNA synthesis. These results indicated that regenerating liver nuclei had two different activities for dNMP production; one is DNA synthesis-dependent, and the other is a non-specific dNTPase activity. This paper has focused on the former activity.

Hepatoprotective action of adenovirus-transferred HNF-3gamma gene in acute liver injury caused by CCl(4).

Hepatocyte nuclear factor-3gamma (HNF-3gamma) is an important regulator of liver-specific genes and the expression of this factor is reduced in the liver injured by carbon tetrachloride (CCl(4)). Wistar rats were infected with a recombinant adenovirus carrying the cDNA for HNF-3gamma (AxCAHNF3gamma) via the tail vein and were treated with CCl(4) by intraperitoneal injection. Liver damage, such as swelling of the hepatocytes and increases in serum marker enzymes were markedly alleviated by AxCAHNF3gamma infection. Interestingly, hepatocyte growth factor (HGF) was strongly induced in the AxCAHNF3gamma-infected liver. Likewise, HNF-1alpha and HNF-1beta levels were increased, but HNF-3alpha and HNF-3beta levels were depressed in the liver. Our results suggest that the transduced HNF-3gamma gene leads to a hepatoprotective effect via the induction of HGF by the combined actions of liver-enriched transcription factors.

A novel mutation of CHRNA4 responsible for autosomal dominant nocturnal frontal lobe epilepsy.

OBJECTIVE: To identify the mutation responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in a nonwhite family. BACKGROUND: ADNFLE is newly recognized as an entity of idiopathic partial epilepsy. Recently, two different mutations of the neuronal nicotinic acetylcholine receptor alpha4 subunit (CHRNA4) gene were identified in a white family as a cause of ADNFLE. METHODS: Four affected and three unaffected individuals in three generations of a Japanese family with ADNFLE, and 100 unrelated healthy Japanese volunteers were studied. Clinical features and EEG findings in affected individuals were consistent with those of ADNFLE reported in white families with ADNFLE. Mutations within the CHRNA4 gene were screened for using single-strand conformation polymorphism analysis (SSCA) and were determined by direct sequencing. The mutation identified was sought in volunteers by the amplification refractory mutation system. RESULTS: A C-to-T exchange (C755T) was found in exon 5 of the CHRNA4 gene on one allele of affected individuals. C755T segregated in affected individuals and was not found in 200 alleles obtained from the volunteers. C755T replaced serine 252 (Ser252) in the second membrane-spanning domain (M2) of CHRNA4 with a leucine. Ser252 is conserved characteristically in the alpha-subunit of acetylcholine receptor and is considered to play an important role in channel function. CONCLUSION: C755T is a novel missense mutation of the CHRNA4 gene causing autosomal dominant nocturnal frontal lobe epilepsy in this Japanese family.

Studies on nuclear acid deoxyribonucleases and proofreading exonuclease isolated from rat liver nuclei.

Three kinds of deoxyribonucleases (peaks A, B, and C) were separated from purified rat liver nuclei on DEAE-cellulose column chromatography, and their characteristics were partially studied. Peaks A and B had endonuclease activities under acidic conditions with low substrate specificity and did not require divalent metal ions. Peak C had an exonuclease activity under alkaline conditions with substrate specificity for denatured DNA or single stranded homopolymer and required divalent cations. Peak C degraded 3'-terminally mismatched substrate much faster than 3'-terminally matched substrate.

Cellular factors for stimulation of nucleosomal template activity for in vitro DNA synthesis.

Nucleosomes isolated from Yoshida sarcoma chromatin by micrococcal nuclease treatment were relatively inactive as templates for in vitro DNA synthesis. However, the template activity increased by trypsin digestion of nucleosomes or addition of heparin to the reaction mixture. This indicates that the nucleosomal template activity is masked. A crude extract of Yoshida sarcoma cells stimulated the nucleosomal template activity. The stimulatory factor was separated into three peaks by DEAE cellulose column chromatography. The same three peaks were observed in normal rat liver extract with much lower activities, but enhanced in regenerating liver. The factors seem to stimulate DNA synthesis by activating DNA template in nucleosomes without degrading histones or changing the primary structure of nucleosomal DNA.

Effects of gastrointestinal and related hormones on glycogenolysis and gluconeogenesis in cultured liver cells.

When isolated rat liver cells were incubated for 15 min in the presence of vasoactive intestinal peptide, secretin, gastrin, caerulein or glucagon at concentrations ranging from 0.2 microgram to 2 microgram per ml, glycogenolysis was stimulated. Among the gastrointestinal hormones or peptides tested, vasoactive intestinal peptide had the highest stimulatory activity. However, somatostatin was inhibitory for liver glycogenolysis. Combination experiments showed that somatostatin also inhibited the stimulatory effects of vasoactive intestinal peptide and secretin, but not that of glucagon, while glucagon and vasoactive intestinal peptide, or glucagon and secretin showed additive effects on glycogenolysis, but secretin and vasoactive intestinal peptide did not. The results suffest that the receptor site of glucagon is different from those of secretin and vasoactive intestinal peptide. Slight but significant stimulation of gluconeogenesis was also observed by vasoactive intestinal peptide and secretin. The evidence presented in this paper indicates that the so-called enterohepatic axis, in which a part of serum glucose levels is regulated directly by gastrointestinal hormones, exists and that the axis is inhibited by somatostatin.

Hyperplastic innervation of vasoactive intestinal peptide in human gallbladder with cholelithiasis.

The vasoactive intestinal peptide (VIP) immunoreactive nerve fibres in the gallbladder from 14 human patients with cholelithiasis was examined by immunohistochemical method. In the chronic cholecystitis, hyperplastic VIP immunoreactive nerves were observed around the hypertrophied muscle bundles, Rokitansky Aschoff Sinus and in the mucosal layer. However, in the acute cholecystitis and gangrenous cholecystitis, reduction or disappearance of VIP nerve fibres was observed. These reductions or disappearances of VIP immunoreactive nerves may secondly result from severe tissue damage. These results suggest that hyperplastic VIP nerves cause gallbladder relaxation, stasis and mucosal fluid unbalance, which may closely correlate to gallstone formation.

Production of nitric oxide from endothelial cells by 31-amino-acid-length endothelin-1, a novel vasoconstrictive product by human chymase.

Human chymase selectively converts big endothelin (ET)-1 to 31-amino-acid-length ET-1 [ET-1(1-31)]. In this study we examined effect of ET-1(1-31) on endothelial function. ET-1(1-31) evoked contraction in a concentration-dependent manner at > 10(-8) M, which was about 10 times weaker than that of conventional ET-1 [ET-1(1-21)]. BQ485, an ETA receptor antagonist, completely abolished ET-1(1-31)-induced contraction, but BQ788, an ETB receptor antagonist, slightly enhanced it, suggesting that ET-1(1-31) relaxes artery via endothelium. On endothelial cells, ET-1(1-21) and ET-1(1-31) increased [Ca2+]i and produced NO, both of which were significantly inhibited by BQ788 and not by BQ485. These results indicate that ET-1(1-31) increased [Ca2+]i and produced NO in endothelial cells through ETB receptor similarly with ET-1(1-21), although slight difference in effect on smooth muscle cells.

Scanning electron microscopic observations of nerves in the guinea-pig gallbladder after an acetylcholinesterase histochemistry.

Three-dimensional structures of the nerves of the guinea-pig gallbladder, after histochemical demonstration of the acetylcholinesterase activity and HCl hydrolysis-collagenase digestion, were examined by scanning electron microscope. HCl-collagenase digestion facilitated easy identification of silver- and gold-intensified acetylcholinesterase-positive nerve fibers at a high accelerating voltage (25 kV), due to their strong reflection image. Ganglia were either triangular or ovoidal in shape. Dense para- and peri-vascular nerve fibers occurred around the cystic artery. There were a few intramuscular nerve fibers with varicosity-like structures among smooth muscle bundles. Dense branched and tapering nerve fibers with varicosities in the lamina propria mucosae were closely attached to epithelial cells. The acetylcholinesterase-positive fibers in the lamina propria and peri- and para-vascular nerves, and fewer positive fibers in the smooth muscle layer probably represent cholinergic nerves involved in the concentration of biliary compounds and lesser in the motor function of the smooth muscle.

Clinical, biochemical and ultrastructural study on the pathogenesis of hyperornithinemia-hyperammonemia-homocitrullinuria syndrome.

A 10-year-old boy with the hyperornithinemia, hyperammonemia and homocitrullinuria (HHH) syndrome is described. With dietary restriction of protein intake and supplementary administration of L-ornithine and L-arginine, the high concentration of ammonia decreased and the clinical signs of truncal ataxia and lethargy improved. A deficiency of ornithine transport into liver mitochondria was demonstrated biochemically, and glycogen granules and smooth surface endoplasmic reticulum were increased, but mitochondria showed normal construction ultrastructurally. Cranial computed tomography (CT) showed diffuse white matter low density and cerebellar vermis atrophy. The impairment of ornithine transport and energy production in the central nervous system may be related to the cranial CT findings and neurological signs.

Effect of protein kinase C on glucose-mediated insulin secretion in HIT-T15 cells.

OBJECTIVE: To clarify the regulation of protein kinase C on glucose-mediated insulin secretion. MAIN OUTCOME MEASURES: We examined the effect of protein kinase C on the cytosolic free Ca(2+) concentration ([Ca(2+)]i) and the activity of Ca(2+)-activated K(+) channels (K(Ca)-channel) in the insulinoma cell line, HIT-T15. RESULTS: Glucose at a concentration of 10 mmol/L increased the secretion of insulin. This increase was partly inhibited by 1 nmol/L staurosporine, a protein kinase C inhibitor. Staurosporine (1 nmol/L) also attenuated the glucose-induced elevations in [Ca(2+)]i. On the contrary, glibenclamide (100 nmol/L) specifically blocked ATP-sensitive K(+) channels, and increased both [Ca(2+)]i and insulin secretion, but staurosporine had no effect on them. Patch clamp studies showed that 10 mmol/L glucose almost completely blocked K(Ca) channel activity, an effect that was reversed by 1 nmol/L staurosporine. Phorbol 12-myristate 13-acetate (1 mmol/L), a protein kinase C activator, also decreased K(Ca) channel activity. CONCLUSIONS: These results indicate that the activation of protein kinase C is involved in the glucose-induced release of insulin by modulating K(+) channel function in HIT-T15 cells.

[Mechanism of efficacy of erythromycin on diffuse panbronchiolitis--effect of erythromycin on cytokine mRNA expression in human whole blood model].

Recently, "low-dose and long-term" erythromycin (EM) has been reported to be effective in treatment of diffuse panbronchiolitis (DPB), but its mechanism is still obscure. We studied the effect of EM on cytokine mRNA expression by using LPS-stimulated human whole blood as an experimental vivo model. IL-8 mRNA was expressed in biphasic fashion with peak expression at 6 hours and 20 hours from the start of LPS stimulation. When whole blood was pretreated with EM (2 micrograms/ml) for 1 hours. IL-8 mRNA expression was depressed at 20 hours (p < 0.025) from the start of LPS (1 microgram/ml) stimulation. However, when pretreated for 12 hours, it was not depressed. EM (2 micrograms/ml) also depressed IL-1 beta (p < 0.025) and TNF alpha (p < 0.05) mRNA expressions at 6 hours from the start of LPS stimulation. From the above results, it was suggested that the direct inhibition of IL-1 beta and TNF alpha production by EM resulted in subsequent depression of production of IL-8 that is a potent chemotactic factor for neutrophil, and consequently, EM acts to protect the bronchiole tissues of DPB patients from destruction by proteolytic enzymes released from neutrophils. This assumption seems to be supported by our previous observation that when patients with DPB were treated with EM a marked decrease in number of neutrophil in bronchoalveolar lavage fluid (BALF) was accompanied by clinical and radiographic improvement.

[Antibiotics susceptibilities and other biological properties of methicillin-resistant Staphylococcus aureus: observation in the last 2 years in our university hospital].

We examined biological properties of strains of methicillin-resistant Staphylococcus aureus (MRSA) which were isolated in our ward in 1991 and 1992. A total of 47 MRSA strains were isolated in 1991 and 64 in 1992. The majority of these strains of MRSA were highly resistant to DMPPC, CEZ and IPM, and were intermediately resistant to MINO. All these strains were, however, sensitive to VCM. The number of coagulase type II strains increased from 22 (46.8%) to 51 (79.7%), and that of enterotoxin type A strains from 27 (57.4%) in 1991 to 47 (73.4%) in 1992. The number of strains which produce Toxic Shock Syndrome Toxin-1 (TSST-1) also increased from 19 (40.4%) to 45 (70.3%), and those strains that produce beta-lactamase decreased from 24 (51.1%) to 21 (32.8%). From the above results, we confirmed the recent change in types of the epidemic strains of MRSA. Namely, there was a marked increase in number of strains which produce type II coagulase type A enterotoxin and TSST-1. For the prevention of a patient to patient-, room to room- and ward to ward-spread, strict isolation was indicated both the infection patients, immunocompromised patients who were at high risk for the infection and the proved carriers. Treatment with VCM was started immediately if MRSA infection was thought plausible. These countermeasures seemed to succeed in reducing the incidence of the infection in our ward.

[Effect of macrolides on cytokine mRNA expression in human whole blood model].

Recently, low dose and long term use of Macrolides (Mls) has been reported to be effective in treatment of chronic lower respiratory tract infections, however its mechanism is still obscure. We evaluated the effect of Mls (EM, AZM, RKM) on cytokine mRNA expressions. We preincubated the whole blood with several concentrations of Mls and removed the Mls and then stimulated human whole blood with LPS as an experimental vivo model. In order to examine cytokine mRNA expressions, we used the RT-PCR method. Cytokine mRNA expressions were suppressed significantly (p < 0.05) by pretreatment with EM, AZM; moreover, the suppression was peaked at low concentrations (0.04 approximately 0.2 microgram/ml). Although, Cytokine mRNA expressions were not suppressed by pretreatment with RKM. These results suggest that EM, AZM have suppression on Cytokine mRNA expressions, and consequently, this suppression has a reasonable effect for DPB patients.

[PUVA therapy for cutaneous acute graft-versus-host disease--a case report].

We performed 8-methoxypsolaren and ultraviolet A (PUVA) therapy as a second line treatment for stage +3 cutaneous acute graft-versus-host disease (GVHD) in a 10-year-old girl who had undergone unrelated bone marrow transplantation. Although prednisolone as first line therapy was not effective, the cutaneous lesion began to improve after several times of PUVA treatment and completely disappeared following 11 times of irradiation without additive systemic immunosuppressive therapy. PUVA therapy was considered as a useful treatment for cutaneous acute GVHD.

[Addition of fentanyl to epidural lidocaine raises the toe temperature].

In order to investigate effects of addition of fentanyl epidurally on the onset of sympathectomy from epidural lidocaine, we have measured the toe temperature of 29 healthy patients undergoing elective lower extremity or lower abdominal surgeries. The latency of onset of the toe temperature was significantly shorter in patients receiving both epidural lidocaine and fentanyl compared with those receiving epidural lidocaine alone (258 +/- 135 vs 398 +/- 184 sec, P < 0.05 [mean +/- SD]). Osmolarity and pH of the epidural solutions were similar between the two groups. These results suggest, but do not indicate, that sympathectomy from epidural lidocaine is accelerated by the addition of fentanyl.