Sales of veterinary antimicrobial agents for therapeutic use in food-producing animal species in Japan between 2005 and 2010.

The use of veterinary antimicrobial agents in animals can result in the emergence and selection of resistant bacteria in food-producing animals. This study elucidated the use of veterinary antimicrobial agents in Japan in terms of milligrams of active ingredient sold per kilogram of biomass between 2005 and 2010. Data on sales of antimicrobial agents and on the biomass of the target animal species were compiled from statistics published bythe Japanese Ministry of Agriculture, Forestry and Fisheries. The quantities of antimicrobials used varied between animal species: the highest usage was observed in pigs (392 to 423 mg/ kg), followed by beef cattle (45 to 67 mg/kg), broiler chickens (44 to 63 mg/kg) and dairy cattle (33 to 49 mg/kg). For the animal species combined, usage of third- and fourth-generation cefalosporins, fluoroquinolones and macrolides ranged from 0.10 to 0.14 mg/kg biomass, 1.1 to 1.3 mg/kg biomass and 7.8 to 10.6 mg/kg biomass, respectively.

Influenza A virus encephalopathy with symmetrical thalamic lesions.

UNLABELLED: During an epidemic of influenza A infection in Japan, a 7-year-old boy was admitted to our hospital because of high fever, convulsions, coma, and liver dysfunction on the 2nd day of a cold-like illness. His serum CPK was markedly elevated, but there was no hyperammonaemia or hypoglycaemia. His CSF showed an increased protein level, but the cell count and glucose level were normal. CT and MRI of the brain showed symmetrical thalamic lesions, and he was diagnosed with acute necrotizing encephalopathy in childhood. He had a significant increased in antibodies to influenza A H1N1 in serum and CSF, but the CSF was negative for influenza virus using virus isolation and a polymerase chain reaction assay. CONCLUSION: Antibody production without detectable levels of influenza virus in cerebrospinal fluid suggests that virus infection occurred, but the virus did not replicate in sufficient numbers in his central nervous system. The thalamic lesion, the hallmark of acute necrotizing encephalopathy in childhood, may be initiated by a local virus infection and develop with subsequent local changes such as breakdown of the blood-brain barrier and the extravasation of blood.

Two novel missense mutations in calcium-sensing receptor gene associated with neonatal severe hyperparathyroidism.

Familial hypocalciuric hypercalcemia (FHH) is characterized by lifelong asymptomatic hypercalcemia without PTH hypersecretion and is inherited as an autosomal dominant trait with near 100% penetrance. In contrast, neonatal severe hyperparathyroidism (NSHPT) is a life-threatening disorder characterized by marked hypercalcemia and PTH hypersecretion. FHH/NSHPT results from inactivating mutations of the human calcium-sensing receptor (Casr) gene on chromosome 3q13.3-24. Nearly 30 different mutations of the Casr gene associated with FHH/NSHPT have been reported previously. In this report, genetic analysis of 1 Japanese NSHPT family revealed 2 novel mutations at codon 185 (CGA-->TGA/Arg-->Ter) in exon 4 of the Casr gene and at codon 670 (GGG-->GAG/Gly-->Glu) in exon 7. The Arg185Ter change was shown to occur in the proband's unaffected father and paternal grandmother as well as in the proband. The other mutation in exon 7 was shown in the proband's unaffected mother of Philippine origin as well as in the proband. This family is the first case of manifestation of more than 1 mutation in a proband's chromosomes; 1 mutation was obtained from the unaffected father, and the other was from the unaffected mother. Our observations have given us important keys to help elucidate the structure-function relationships of the Casr.

Preoperative simultaneous cisplatin- or carboplatin-based chemotherapy and radiotherapy for squamous cell carcinoma of the oral cavity.

BACKGROUND: Encouraging results have been reported with cisplatin- or carboplatin-based chemotherapy regimens and simultaneous irradiation treatment in advanced and unresectable head and neck head and neck cancer. We have therefore examined the effectiveness of such therapy on tumor control, survival, and toxicity in patients with advanced oral squamous cell carcinoma. METHODS: Forty-one patients with squamous cell carcinoma of the oral cavity (including soft palate) were treated preoperatively with cisplatin or carboplatin, and 5-fluorouracil or peplomycin in combination with simultaneous irradiation to a target volume of 40Gy, and 2-6 weeks later, curative surgery was performed. RESULTS: Thirty-eight patients (91.7%) had Stage III or IV disease, and three patients had Stage II lesions. The preoperative clinical responses of the primary tumor were: 25 patients (61.0%) achieved a complete response (CR), 15 (36.6%) a partial response (PR), only 1 patient (2.4%) had stable disease or no change (NC). The overall response rate was 97.6%. Histological effects according to the grading system of Shimosato and coworkers [Jpn J Clin Oncol 1:19-35, 1971] were seen in 38/41 (92.7%). Of clinical CR patients, 73.9% were also histologic negative for tumor. Side effects of this therapy were relatively low and reversible. With a median follow-up of 52.8 months (range 17-92 months), 5-year cumulative survival rates were 81.5% for all patients, 100% for Stage II, 88.6% for Stage III, and 76.4% for Stage IV patients, respectively. There was no significant postoperative morbidity. CONCLUSIONS: This preoperative chemoradiotherapy regimen was highly active, well tolerated, and appeared to have a survival benefit even for advanced carcinomas of the oral cavity.

Advanced glycation end-products in diabetic nephropathy.

Advanced glycation end-products (AGEs) are the pigmented and fluorescent adduct formed by a non-enzymatic reaction between sugar and protein. Since AGEs are generated in high glucose milieu, then induce the structural and functional alteration of matnx proteins, and have biological effects on various kinds of cells including mesangial cells, AGEs have been implicated in tissue damage of diabetic nephropathy. To elucidate the factor(s) that determine the AGEs level in diabetic nephropathy, we quantitated the plasma pentosidine level of different status of diabetic nephropathy by HPLC assay. The plasma pentosidine level in diabetic nephropathy was found to be determined by factors such as renal function control of glucose and the patient's age; of these, renal function was the most critical factor. For a better understanding of the pathological role of AGEs in diabetic nephropathy, we then examined renal tissues of diabetic nephropathy immunohistochemically using antibodies specific for AGE proteins. Immunohistochemistry revealed the positive immunostaining for AGEs in the expanded mesangial area of diffuse diabetic glomerulosclerosis. The degree of staining was stronger than that in patients of IgA nephropathy with a similar degree of mesangial expansion. The nodular lesions, characteristic of diabetic nephropathy, were also stained positive for AGEs. These findings suggests a potential link of AGEs accumulation, which may be determined by renal function, control of glucose and age, to renal tissue damage in diabetic nephropathy.

An HL-60 variant cell line defective in cholesterol synthesis.

Human promyelocytic leukemia cell line, HL-60, is known to proliferate exponentially in a serum-free synthetic medium supplemented with insulin and transferrin (BREITMAN, T.R. et al. (1980). Exp. Cell. Res., 126: 494-498). Among four HL-60 cell lines tested in this medium (serum-free medium), however, a cell line, HL-60/Biken ceased to proliferate after two days culture and most of the cells died within a week. Addition of purified serum lipoprotein (LDL or HDL) to the serum-free medium almost completely restored the proliferating activity of the cells. Total lipids extracted from the lipoproteins could replace the lipoproteins in promoting cell proliferation. Among various lipid components of the lipoproteins, only cholesterol showed a high stimulatory effect on cell proliferation, whereas other lipids tested were ineffective, except for sphingomyelin, cerebroside, and phosphatidic acid which showed limited stimulatory effects. As for the intermediates of cholesterol biosynthesis, desmosterol was also effective, whereas lanosterol was rather inhibitory. Chromatographic analyses of the lipids synthesized by HL-60/Biken cells and wild type cells (HL-60/RCB) cultured in serum-free medium, clearly demonstrated that, in HL-60/Biken cells, cholesterol synthesis was almost completely blocked and lanosterol was accumulated 10-fold that in wild type HL-60/RCB cells. All of these results indicate that HL-60/Biken is a variant cell line defective in cholesterol synthesis in the process synthesizing desmosterol from lanosterol. The variant HL-60 cells showed a marked resistance to cell differentiation along both monocytic and granulocytic pathways when compared with wild type HL-60 cells. The cell line may be useful for the study of the role of cholesterol in cell differentiation.

Apoptosis induced by adenosine in human leukemia HL-60 cells.

Among several nucleosides and nucleotides, which showed strong inhibition of growth of HL-60 cells, only adenosine (Ado) specifically induced typical apoptotic death of the cells, accompanying double-strand cleavage of DNA into nucleosomal size fragments, and subsequent apoptotic body formation. A marked enhancement of endogenous poly(ADP-ribosyl)ation activity in the cell was detected at a relatively early stage of cell death, whereas other nucleosides and nucleotides tested were ineffective on poly(ADP-ribosyl)ation activity, suggesting that the enzyme activation is closely related to apoptosis. The observed Ado effect was not mediated by Ado receptors, in contrast to the Ado-induced apoptotic death of thymocytes, judging from the facts that all of the receptor agonists tested did not substitute for Ado and that a receptor antagonist did not inhibit the effect of Ado. Ado transport into the cell seemed to be essential for the induction of apoptosis, since an inhibitor of Ado transport (dipyridamole) strongly suppressed apoptosis. Cytochalasin B blocked Ado-induced apoptotic body formation without affecting activation of endogenous poly(ADP-ribosyl)ation activity in the cell. Thus, the process of apoptosis in HL-60 cells induced by Ado seems to be separated into at least two steps, an initial step of DNA degradation and a following morphological change. While the adenine moiety of Ado was essential for its apoptosis-inducing activity, the sugar was replaceable, and various analogs with modified sugar were inducers of apoptosis, although they were less efficient than Ado.

3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, causes a rapid death of HL-60 cells cultured in serum-free medium.

HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10 micrograms/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serum-deprived growth condition.

Poly(ADP ribose) polymerase-defective mutant cell clone of mouse L1210 cells.

By a sequential mutation and selection utilizing N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen, we succeeded in separating a poly(ADP ribose) polymerase-defective mutant clone (Cl-3527) from a mouse L1210 cell clone (Cl-3). The enzyme activity per cell in Cl-3527 cells was only 8% of that in wild type L1210 (CCL 219) cells. Immunoblot analysis of the enzyme protein in crude extracts of the mutant and wild type cells revealed that the enzyme defect was manifested as the loss of a 113-kDa wild type enzyme band in Cl-3527. Further analysis of partially purified enzyme from Cl-3527 by immunoblotting revealed that the molecular size of the enzyme in Cl-3527 was 108 kDa and that the amount of the mutant enzyme protein was markedly decreased in Cl-3527. The mutant enzyme was much more heat-labile than the wild type enzyme but the Km for NAD+, requirements for Mg2+ and nicked DNA, and the inhibition by 3-aminobenzamide, a potent inhibitor of the enzyme, however, were not so different from those of wild type enzyme. The mutant cells showed prolonged doubling time, increased temperature-sensitivity, increased percentage of active enzyme on a treatment of cells at high temperature, and increased expression of plasma membrane NADase, compared to wild type cells. Introduction of wild type ADPR pol gene into Cl-3527 cells partially restored the ADPR pol activity and the heat-resistance.

Retinoic acid-specific induction of a protein kinase C isoform during differentiation of HL-60 cells.

Human promyelocytic leukemia cells (HL-60) were treated with several differentiation inducers, then the changes in the activity of cytosolic protein kinase C (PKC) isoforms were examined by hydroxylapatite chromatography and the species of the isoforms were determined immunologically. In three undifferentiated HL-60 cell lines examined, PKC alpha and beta isoforms were present, but PKC gamma isoform was not detected. When the cells were induced by dimethylsulfoxide, dibutyryl cAMP, or nicotinamide to differentiate into granulocytes, these two PKC isoforms each increased to about 2- to 3-fold. When retinoic acid was used as the inducer, in addition to PKC alpha and beta, a third PKC isoform appeared. This isoform was clearly distinct from rat PKC alpha, beta, and gamma, immunologically. This isoform showed a distinctly lower Ca(2+)-requirement (3 microM) than that of PKC alpha or beta (100 microM) and was more dependent on cardiolipin and phosphatidylethanolamine, compared with PKC alpha, beta, and gamma. These results suggest that while the increases in the activities of PKC alpha and beta isoforms are common in the differentiation program initiated by several inducers, including retinoic acid, the emergence of an unclassified PKC isoform is a retinoic acid-specific process.

Milk cream does not enhance 2,7-dimethylbenz[a]anthracene-induced mammary tumorigenesis.

We have previously reported that a diet enriched with butter showed an inhibitory effect on the development of mammary tumors in mice and rats. To solve the problem of whether the inhibitory effect of butter was caused by lipids of cow's milk, we have studied the effects of dried milk (WM), skim milk (SM) and milk cream (CR) on mammary tumorigenesis in rats. The lowest incidence of mammary tumors was observed in the CR group, although the difference from other groups was statistically not significant. However, the number of papillary carcinomas in the CR group was significantly lower than the WM group. The result indicates that milk lipids have no enhancing effect on mammary tumorigenesis.

Retinoic acid-induced differentiation-specific, C-kinase-dependent phosphorylation of cytosolic 44 and 32 kDa proteins in HL-60 cells.

The effect of various differentiation-inducers on the activity of Ca2+, phospholipid-dependent protein kinase (C-kinase) activity and endogenous protein phosphorylation by the kinase were examined in the extracts of HL-60 cells. Although all of the inducers, retinoic acid, dibutyryl cAMP, nicotinamide, dimethylsulfoxide, and 3-aminobenzamide increased the cytosolic C-kinase activity accompanied with the differentiation into mature myelocytes, only retinoic acid markedly enhanced Ca2+, phospholipid-dependent phosphorylation of 44 and 32 kDa proteins in the cytosol. These results suggest that the differentiation pathway induced by retinoic acid is different from the pathways induced by other inducers.