Soft-hydrothermal processing of red cedar bedding reduces its induction of cytochrome P450 in mouse liver.

Red cedar-derived bedding materials cause changes in cytochrome P450-dependent microsomal enzyme systems in laboratory animals. We examined the effect of essential oil of red cedar (EORC), as well as the effect of bedding from which it had been removed, on the hepatic expression cytochrome P450s in mice. EORC was obtained from liquid extracts of red cedar bedding by a soft-hydrothermal process and was administered orally to mice. Between days 1 and 2 after administration, hepatic P450s were significantly induced as follows: CYP3As, 7.1x; CYP1As, 1.6x; CYP2E1, 1.5x; CYP2Cs, 1.6x. A housing study of mice indicated that red cedar bedding increased the levels of these P450s in mouse liver, whereas mice housed in cedar bedding from which EORC had been removed (ST-cedar bedding) showed significantly lower levels of P450s, especially CYP3As, CYP1As and CYP2E1. Soft-hydrothermal processing partially removed many components of EORC. In particular, several volatile sesquiterpenes, naphthalene-derived aromatics and 4,4-dimethyl-13alpha-androst-5-ene were decreased in the ST-cedar bedding, suggesting that these may be responsible for P450 induction. This study demonstrated that the removal of these volatile compounds by soft-hydrothermal processing can decrease the hepatic P450-inducing effect of red cedar bedding.

Use of soft hydrothermal processing to improve and recycle bedding for laboratory animals.

Cage bedding for laboratory rodents can influence animal wellbeing and thus the experimental data. In addition, a large amount of used bedding containing excrement is discharged as medical waste from life science institutes and breeding companies. We developed a ground-breaking system to improve fresh bedding and recycle used bedding by applying a soft hydrothermal process with high-temperature and high-pressure dry steam. The system removes both harmful organic components and aromatic hydrocarbons that can affect animals' metabolism. The purpose of the present study was to evaluate the chemical and physical properties of the improved fresh bedding and the recycled used bedding treated by the system. The results showed that 68-99% of the predominant aromatic hydrocarbons were removed from fresh bedding treated at 0.35 MPa and 140 degrees C for 120 min ('improved bedding'). In addition, 59.4-99.0% of predominant harmful organic compounds derived from excrement were removed from used bedding treated at 0.45 MPa and 150 degrees C for 60 min ('recycled bedding'). The soft hydrothermal treatment increased the number of acidic functional groups on the bedding surface and gave it the high adsorptive efficiency of ammonia gas. Harmful substances such as microorganisms, heavy metals and pesticides decreased below the detection limit. The results clearly showed that the improved and recycled bedding is safer for laboratory rodents and has the potential to ameliorate conditions in primary and secondary enclosures (e.g. cages and animal rooms) used for maintaining laboratory animals. This process may be one of the most advanced techniques in providing an alternative to softwood and other bedding, economizing through the recycling of used bedding and reducing bedding waste from animal facilities.

Effect of intrasplenic transplantation of immortalized human hepatocytes in the treatment of acetaminophen-induced acute liver failure SCID mice.

OBJECTIVE: We have established immortalized human hepatocytes by transduction of HPV16 E6/E7 and hTERT (HHE6E7T-1). The cells retained the characteristics of differentiated hepatocytes, but the functional characteristics such as albumin secretion, ureogenesis, and glyconeogenesis decreased gradually as the passages progressed beyond 200 population doublings (PDs). In this report, we transplanted minimally differentiated HHE6E7T-1 cells into the spleens of acute liver failure severe combined immunodeficiency (SCID) mice to examine the potential of the cells to redifferentiate in vivo. MATERIALS AND METHODS: Acute liver failure was induced in SCID mice by intraperitoneal injection of 400 mg/kg acetaminophen. Two hours later, HHE6E7T-1 cells at 200 PDs were transplanted: group 1 (n = 10) 50 microL phosphate-buffered saline (PBS); group 2 (n = 9), lysate of 1 x 10(6) HHE6E7T-1 cells at 200 PDs resuspended in 50 microL PBS; and group 3 (n = 8), 1 x 10(6) HHE6E7T-1 cells. Survival rates at 7 days after transplantation were compared. Blood glucose levels, plasma ammonia levels, and spleen histology were examined at 24 hours after transplantation. RESULTS: Survivals in each group were: 30% for group 1, 33% for group 2, and 100% for group 3. The survival of group 3 was significantly higher than groups 1 or 2 (P < .01). Plasma ammonia levels in group 3 (200 +/- 34 microg/dL) were significantly lower than those in group 1 (325 +/- 92 microg/dL; P < .05). Blood glucose levels in group 3 (110 +/- 20 mg/dL) were significantly higher than those in group 1 (83 +/- 14 mg/dL; P < .05). Upon histologic examination of spleen, the clusters of HHE6E7T-1 cells were clearly identified. CONCLUSIONS: The immortalized human hepatocytes, HHE6E7T-1 at 200 PDs, improved the survival of acute liver failure mice through possible redifferentiation in vivo.

Neurovirulence of H7N7 influenza A virus: brain stem encephalitis accompanied with aspiration pneumonia in mice.

A mouse-adapted influenza A virus, A/equine/London/1416/73-MA (H7N7) caused viral pneumonia, ganglionitis and encephalitis after intranasal inoculation in mice. Virological and pathological data suggested that this virus spreads to the brain by both hematogenous and transneuronal routes, and produces encephalitic lesions similar to those seen in mice infected with H5 highly pathogenic avian influenza A viruses by intranasal infection. Some mice infected with this strain were affected by aspiration pneumonia, which may be caused by neurogenic dysfunction of the pharyngeal/laryngeal reflex due to brain stem encephalitis.

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in ovine from São Paulo State, Brazil.

Serum samples from 597 sheep from São Paulo State, in the southeastern region of Brazil, were tested to determine the prevalence of antibodies directed against Toxoplasma gondii (> or = 1:64) and Neospora caninum (> or = 1:50) using the indirect fluorescent antibody test (IFAT). The animals were divided into three groups based on their age: < or = 1 year, 1-4 years, and > or = 4 years. Antibodies to T. gondii were observed in 34.7% of the samples with titers ranging from 64 to 16,384 and IgG antibodies directed against N. caninum were observed in 9.2%, with titers ranging from 50 to 3200. Only 3.5% of the sheep were positive for both agents. All farms had at least one positive animal for T. gondii, and 26 of the 30 farms had at least one positive animal for N. caninum. An association between seroprevalence and age was observed for T. gondii (P = 0.001), but not to N. caninum (P = 0.343). It was not possible to associate seroprevalence to T. gondii and the presence of domestic or feral cats, since in all farms there was at least one positive sheep. There was no association between seropositivity to N. caninum and the presence of domestic (P = 1.000) and feral dogs (P = 0.550).

MCG measurement in the environment of active magnetic shield.

MCG (Magnetocardiography) measurement by a SQUID gradiometer was attempted with only active magnetic shielding (active shielding). A three-axis-canceling-coil active shielding system, where three 16-10-16 turns-coil sets were put in the orthogonal directions, produces a homogeneous magnetic field in a considerable volume surrounding the center. Fluxgate sensors were used as the reference sensors of the system. The system can reduce environmental magnetic noise at low frequencies of less than a few Hz, at 50 Hz and at 150 Hz. Reducing such disturbances stabilizes biomagnetic measurement conditions for SQUIDs in the absence of magnetically shielded rooms (MSR). After filtering and averaging the measured MCG data by a first-order SQUID gradiometer with only the active shielding during the daytime, the QRS complex and T wave was clearly presented.

Bayesian classification of myocardial excitation abnormality using magnetocardiogram maps for mass screening.

We propose a novel classification method based on the Bayes rule to utilize the magnetocardiogram (MCG) in noninvasive mass screening. The cardiac excitation is directly tracked by maps of the MCG field generated by myocardial excitation current through the excited wave front. To adopt the characteristics of the excited wave fronts as a parameter for the Bayes theorem, we developed a parameterization procedure that consists of a two-dimensional wavelet approximation and a cluster analysis of magnetic field maps. With the parameter determined by this procedure, the probability of a subject to belong to a disease group or to the normal group is estimated by the Bayes theorem. The subject is classified into the group of the highest probability. We applied the proposed method to ST-T period of MCG data of 6 old myocardial infarction (OMI) patients and 15 normal controls. The method showed sensitivity of 83%; specificity, 100%; positive predictive value, 100%; and negative predictive value, 94% in the classification of OMI patients and normal controls. The processing time is less than 5 seconds per one subject. It suggests a possible application of the proposed method in mass screening of abnormal MCG patterns.

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.

Quantification of TECTA and DFNA5 expression in the developing mouse cochlea.

TECTA and DFNA5 are the mouse orthologues of the human deafness-associated genes TECTA and DFNA5. To determine how expression of these genes is regulated during development, relative mRNA abundance was examined in mice by non-radioactive RT-PCR. TECTA mRNA was detected on embryonic day 15 (E15), increased to its highest level on postnatal day 3 (P3) and then dramatically decreased by P15. Low levels persisted (adulthood, P45 to 67) with mean mRNA abundance after P15 less than 25% of P3 levels. DFNA5 mRNA expression was constant throughout these time points. These results imply that TECTA is transcribed at a particularly high level during tectorial membrane morphogenesis. In contrast, DFNA5 is present in both the developing and mature cochlea.

Rats expressing human cytosolic copper-zinc superoxide dismutase transgenes with amyotrophic lateral sclerosis: associated mutations develop motor neuron disease.

Some cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding cytosolic, copper-zinc superoxide dismutase (SOD1). We report here that rats that express a human SOD1 transgene with two different ALS-associated mutations (G93A and H46R) develop striking motor neuron degeneration and paralysis. As in the human disease and transgenic ALS mice, pathological analysis demonstrates selective loss of motor neurons in the spinal cords of these transgenic rats. In spinal cord tissues, this is accompanied by activation of apoptotic genes known to be activated by mutant SOD1 protein in vitro and in vivo. These animals provide additional support for the proposition that motor neuron death in SOD1-related ALS reflects one or more acquired, neurotoxic properties of the mutant SOD1 protein. The larger size of this rat model as compared with the ALS mice will facilitate studies involving manipulations of spinal fluid (implantation of intrathecal catheters for chronic therapeutic studies; CSF sampling) and spinal cord (e.g., direct administration of viral- and cell-mediated therapies).

Seroprevalence of Neospora caninum in female water buffaloes (Bubalus bubalis) from the southeastern region of Brazil.

Antibodies to Neospora caninum were assayed in sera of 222 female water buffaloes from Ribeira Valley of São Paulo State, Brazil, using an indirect fluorescent antibody test (IFAT) and Neospora agglutination test (NAT). IFAT antibodies were found in 64% of buffaloes with titers of 1:25 (42 buffaloes), 1:50 (53 buffaloes), 1:100 (31 buffaloes), 1:200 (10 buffaloes), 1:400 (3 buffaloes), or > or =1:800 (3 buffaloes). NAT antibodies were found in 53% of buffaloes; in titers of 1:40 in 52 buffaloes, 1:80 in 27 buffaloes, 1:160 in 21 buffaloes, and > or =1:320 in 17 buffaloes. Results indicate a high prevalence of N. caninum exposure in water buffaloes in Brazil and warrant an investigation of the role of N. caninum as an abortifacient in water buffaloes.

[Peritoneal sclerosis in children under treatment with peritoneal dialysis].

Peritoneal dialysis(PD) is an established method of dialysis for children at the end stage of renal failure. Among several problems associated with PD, peritoneal sclerosis is one of the most serious complications. We examined four children on PD who were diagnosed as having peritoneal sclerosis in 1997 and 1998. Three of these cases were switched to hemodialysis(HD) and one is now undergoing a switch from PD to HD. Although all of the four patients had a history of bacterial peritonitis, they had been maintained in a fairly good condition on PD. The mean duration of PD was 9 years(4-14 years). All cases were anuric and had been maintained on PD without any problems of peritoneal function. From these observations, we should consider peritoneal sclerosis even in patients with a good clinical condition during the long-term period of PD. We recommend serial peritoneal biopsies, at least at the time of peritoneal catheter changes, to check for peritoneal sclerosis. The patients who are diagnosed as having sclerotic thickening of the peritoneal membrane should be switched from PD to HD to prevent the occurrence of life-threatening peritoneal sclerosis.

Green fluorescent protein-transgenic rat: a tool for organ transplantation research.

The purpose of this study is to evaluate green fluorescent protein (GFP) transgenic rats for use as a tool for organ transplantation research. The GFP gene construct was designed to express ubiquitously. By flow cytometry, the cells obtained from the bone marrow, spleen, and peripheral blood of the GFP transgenic rats consisted of 77, 91, and 75% GFP-positive cells, respectively. To examine cell migration of GFP-positive cells after organ transplantation, pancreas graft with or without spleen transplantation, heart graft with or without lung transplantation, auxiliary liver and small bowel transplantation were also performed from GFP transgenic rat to LEW (RT1(1)) rats under a 2-week course of 0.64 mg/kg tacrolimus administration. GFP-positive donor cells were detected in the fully allogenic LEW rats after organ transplantation. These results showed that GFP transgenic rat is a useful tool for organ transplantation research such as cell migration study after organ transplantation without donor cell staining.

Genetic alteration of penicillin non-susceptible Streptococcus pneumoniae observed throughout recurrence of acute otitis media detected by amplified fragment length polymorphism analysis.

The prevalence of penicillin non-susceptible Streptococcus pneumoniae (PNSSP) is increasing among isolates from acute otitis media (AOM). Repeated episodes of antibiotic exposure are a well-known risk factor for the isolation of PNSSP although otitis-prone or recurrent AOM cases frequently require repeated courses of antibiotic treatment. In order to evaluate the chronological alteration of S. pneumoniae during recurrences of AOM, strains of S. pneumoniae were isolated from 11 patients, each of whom had experienced 2-4 episodes of AOM, were examined. Every bacterial specimen obtained from a single episode of recurrent AOM was examined by PCR-based penicillin-binding protein (PBP) assay, serotyping, and amplified fragment length polymorphism (AFLP), then compared to other samples from the same case. Two cases (18.2%) showed strain diversity during repeated antibiotic treatments by serotyping or PBP-assay. By AFLP analysis, 6 cases (54.5%) demonstrated heterogeneous strains during recurrent AOM. Clonal survivors of previous episodes of AOM were not always the cause of subsequent episodes of AOM, even in otitis-prone cases.

Effect of D-glucose on nitric oxide release from glomerular endothelial cells.

BACKGROUND: Altered glomerular production of nitric oxide (NO) may be involved in hyperfiltration in early diabetic nephropathy. However little is known as to the role of glomerular endothelial cells (GECs) in diabetic hyperfiltration and their ability to release NO in response to hyperglycemia. METHODS: Using an established cell line, we directly monitored NO release from GECs in response to various concentrations of D-glucose, D-mannitol, and L-arginine, an NO synthase (NOS) agonist. L-Arginine-induced NO release was examined in the cells pretreated for different periods up to 24 h with 10 or 30 mM D-glucose. We also measured serially the accumulation of nitrite, the stable metabolite of NO, produced by the cells incubated for up to 24 h under 10 or 30 mM D-glucose conditions in the presence or absence of the NOS inhibitor, L-NAME. RESULTS: Direct measurement of NO demonstrated that D-glucose, but not D-mannitol, stimulation resulted in a rapid and dose-dependent increase in NO release by the cells. However, L-arginine-induced NO release was attenuated significantly in the cells preincubated for more than 12 h with 30 mM D-glucose compared to 10 mM D-glucose. The L-NAME-inhibitable production of nitrite in the media was significantly increased 1.5--2.0-fold until 6 h after incubation with 30 mM D-glucose compared to 10 mM D-glucose. CONCLUSIONS: We conclude that D-glucose, but not D-mannitol, produces a rapid and dose-dependent increase in NO release, whereas exposure to high D-glucose for more than 12 h may blunt NOS activity and/or NO stability in the GECs. These observations may therefore be important for glomerular endothelial dysfunction induced by hyperglycemia that is still tentative and may have a role in diabetic nephropathy.

Immunohistochemical localization of somatostatin receptor type 2A in rat and human tissues.

In this study, we elucidated the cellular localization of somatostatin receptor (SSTR) by immunohistochemistry using an antibody specific for SSTR type 2A (SSTR2A) in various organs of rat and human. SSTR2A expression was basically similar in rat and human, except in the pancreas and adrenal cortex. In the pituitary gland, the posterior lobe and the majority of growth hormone cells and some ACTH and TSH cells expressed SSTR2A. In rat adrenal gland, the zona glomerulosa strongly expressed SSTR2A, whereas zone-specific immunoreactivity was not observed in human. The adrenal medulla moderately expressed SSTR2A in both rat and human. SSTR2A immunoreactivity was observed in islet cells and some ductal cells in human pancreas, and also in acinar cells of rat pancreas. In gastrointestinal (GI) tract, the majority of crypt cells and nerve plexuses strongly expressed SSTR2A. The number of SSTR2A positive cells was much more than that of chromogranin A positive endocrine cells. In the kidney, the glomerular capillaries and collecting tubules, but not proximal tubules, showed immunoreactivity. SSTR2A immunoreactivity was observed not only in endocrine cells but also in non-endocrine cells.

Midaortic syndrome in childhood associated with a ruptured cerebral aneurysm: a case report.

BACKGROUND: We present a patient with a midaortic syndrome who presented with subarachnoid hemorrhage caused by rupture of an anterior communicating artery aneurysm. CASE DESCRIPTION: A 14-year-old boy with midaortic syndrome was admitted to our hospital because of subarachnoid hemorrhage due to rupture of an anterior communicating artery aneurysm. He also developed acute renal failure due to previously controlled hypotension. After blood dialysis, successful clipping of the aneurysm was performed. The postoperative course was complicated by malignant renovascular hypertension due to midaortic syndrome. Medical treatment failed to control his hypertension; left primary nephrectomy improved his condition. CONCLUSION: Although midaortic syndrome is rare, it may be significant as a cause of cerebral hemorrhage in childhood.

Prognostic factors and therapeutic strategy for anaplastic carcinoma of the thyroid.

Although anaplastic thyroid carcinoma (ATC) is one of the most aggressive malignancies, a few patients survive for a fairly long time after modern intensive treatment. We tried to identify prognostic factors of ATC to assist in deciding on the proper therapeutic strategy in individual patients. Of 47 patients with ATC (1976-1999), 3 patients with "incidental" ATC (largely differentiated thyroid carcinoma with a small region of ATC) were excluded because they had a favorable outcome. The 1-year survival rate of the remaining 44 patients with clinically distinct ATC was 16%. The presence of acute symptoms, large tumor (> 5 cm), distant metastasis, and leukocytosis (white blood cell count > or = 10,000/mm3) proved to be significant risk factors. Multivariate analysis by the Cox proportional hazard model showed that these four factors were independent factors for predicting death from ATC. We devised a novel prognostic index (PI) based on the number of these four unfavorable characteristics the patient possessed. Patients with a PI of < or =1 had a 62% survival rate at 6 months, whereas no patients with a PI of > or =3 survived longer than 6 months. All patients with a PI of 4 died from their disease within 3 months. Nine patients received multimodal treatment with a combination of surgery, external irradiation, and chemotherapy and had a long survival (mean 333 +/- 68 days; one patient is still alive and tumor-free), with a mean PI of 0.6. Our PI is useful as a means of selecting patients for aggressive therapy. When the PI is low, multimodal treatment should be attempted to obtain the best survival results; if it is high most patients are too seriously ill to tolerate intensive treatment and palliative therapy is recommended.

Real-time multisite observation of glutamate release in rat hippocampal slices.

A multichannel glutamate sensor was fabricated that consists of enzyme modified electrodes and has a high sensitivity and selectivity to glutamate. We placed a rat hippocampal slice on the sensor and monitored the current at four electrodes resulting from the stimulation with muscimol, a gamma-aminobutyric acid(A) (GABA(A)) receptor agonist. We obtained different glutamate concentration increases at the different positions, suppressed by bicuculline, a GABA(A) receptor antagonist. This demonstrated that the sensor can monitor the glutamate released via GABA(A) receptors pathways, and the difference in the concentrations may indicate differences in the distribution of GABA(A) receptor as well as diverse receptor functions. This multichannel sensor may be useful for non-invasive, real-time monitoring of glutamate distribution, which would make it a valuable tool for pharmacological analysis.

Suppression of thymic development by the dominant-negative form of Gads.

Gads, a hematopoietic-lineage-specific Grb2 family member, is involved in the signaling mediated by the TCR through its interactions with SLP-76 and LAT. Here, we generated transgenic mice expressing Grf40-dSH2, an SH2-deleted dominant-negative form of Gads, which is driven by the lck proximal promoter. The total number of thymocytes was profoundly reduced in the transgenic mice, whereas in the double-negative (CD4(-)CD8(-)) thymocyte subset, in particular the CD25(+)CD44(-) pre-T cell population, it was significantly increased. However, CD5 expression, which is mediated by pre-TCR stimulation, was significantly suppressed on the CD4(-)CD8(-) thymocytes of the transgenic mice. Furthermore, the SLP-76-dependent signaling was markedly suppressed as well. These data suggest that Gads plays an important role in the pre-TCR as well as TCR signaling in thymocytes.