Age-related DNA methylation analysis for forensic age estimation using post-mortem blood samples from Japanese individuals.

As one of external visible characteristics (EVCs) in forensic phenotyping, age estimation is essential to providing additional information about a sample donor. With the development of epigenetics, age-related DNA methylation may be used as a reliable predictor of age estimation. With the aim of building a feasible age estimation model for Japanese individuals, 53 CpG sites distributed between 11 candidate genes were selected from previous studies. The DNA methylation level of each target CpG site was identified and measured on a massive parallel platform (synthesis by sequencing, Illumina, California, United States) from 60 forensic blood samples during the initial training phase. Multiple linear regression and quantile regression analyses were later performed to build linear and quantile age estimation models, respectively. Four CpG sites on four genes- ASPA, ELOVL2, ITGA2B, and PDE4C -, were found to be highly correlated with chronological age in DNA samples from Japanese individuals (|R| > 0.75). Subsequently, an independent validation dataset (n = 30) was used to verify and evaluate the performance of the two models. Comparison of mean absolute deviation (MAD) with other indicators showed that both models provide accurate age predictions (MAD: linear = 6.493 years; quantile = 6.243 years). The quantile model, however, can provide the changeable prediction intervals that grow wider with increasing age, and this tendency is consistent with the natural aging process in humans. Hence, the quantile model is recommended in this study.

Carcinoma ex pleomorphic adenoma of the buccal region is composed of salivary duct carcinoma and squamous cell carcinoma components.

A 73-year-old female presented with an asymptomatic mass in the left buccal region that she had first noticed 4 years earlier. The tumor, which was located in the buccal space, was clinically diagnosed as a salivary gland tumor and treated by excision. Histopathological examination revealed a capsule of connective tissue consisting of three different histopathological neoplastic areas in a large, fibrous, hyalinizing stromal background. The neoplastic lesion contained two malignant and one benign element, with histological characteristics consistent with squamous cell carcinoma (SCC), salivary duct carcinoma (SDC) and pleomorphic adenoma (PA). The SCC nests showed no continuity with the buccal mucosa. Both the SCC and SDC nests were surrounded by non-atypical myoepithelial cells, suggesting that both components may have developed from transformation of metaplastic luminal epithelial cells of PA. The tumor was diagnosed as a non-invasive carcinoma (SCC and SDC) ex pleomorphic adenoma (Ca-ex-PA). There was no evidence of recurrence 16 months after operation.

Cystadenocarcinoma (papillary cystadenocarcinoma) of the submandibular gland.

Cystadenocarcinoma (papillary cystadenocarcinoma) of the salivary gland is a rare malignant neoplasm. Major locations of this neoplasm are the parotid gland, the sublingual gland, and minor salivary glands, while occurrence in the submandibular gland is extremely rare. As far as we know, only one report, written in the French language, has been published concerning cystadenocarcinoma of the submandibular gland, but no report is available in the English language. In this report, we describe a case of cystadenocarcinoma arising from the submandibular gland of a 54-year-old male patient. The neoplasm is a low-grade carcinoma, and pre-operative examination may not show typical characteristics of malignant neoplasms. Therefore, as was true in this case, the differential diagnosis from benign lesions is sometimes difficult. This is the first report on cystadenocarcinoma of the submandibular gland in the English language and the first to show a computed tomography (CT) scan and magnetic resonance imaging (MRI) of this neoplasm in the submandibular gland.

Renal tubular effects of endothelin-B receptor signaling: its role in cardiovascular homeostasis and extracellular volume regulation.

The role of the endothelin-B receptor in vascular homeostasis is controversial because the receptor has both pressor and depressor effects in vivo. One potential depressor mechanism of endothelin-B activation is through the promotion of natriuresis and diuresis in the renal tubule. Recent studies demonstrate that rodents genetically deficient for the endothelin-B exhibit sodium-dependent hypertension due to an absence of tonic inhibition of the epithelial sodium channel in the distal nephron. These studies suggest that the predominant role of endothelin-B receptors in the basal physiologic state may be to regulate renal sodium excretion relative to the level of oral salt intake.

Cloning and expression of a gene encoding N-glycosyltransferase (ngt) from Saccarothrix aerocolonigenes ATCC39243.

In the course of our bioconversion studies on the derivatives of an indolocarbazole, J-104303, Saccharothrix aerocolonigenes ATCC39243 was found to convert J-104303, which was added into the culture medium, to its glycosylated derivative, J-109384. In order to clone the gene having the ability to convert J-104303 to J-109384, a library of Saccharothrix aerocolonigenes ATCC39243 DNA fragments was constructed using Streptomyces lividans TK21 and pIJ702 as host strain and vector, respectively. By examining more than 5,000 transformants, one was found to convert J-104303 to J-109384. Sequence analysis of the inserted DNA fragment revealed an open reading frame with 1,245 base pairs, named ngt. The transformant containing this ngt gene was also found to introduce a D-glucose moiety into 6-N-methylarcyriaflavin C. Furthermore, when ngt was introduced into Streptomyces mobaraensis BA13793, a producer of J-104303, the resulting transformant produced J-109384 directly.

Salt-sensitive hypertension in endothelin-B receptor-deficient rats.

The role of the endothelin-B receptor (ET(B)) in vascular homeostasis is controversial because the receptor has both pressor and depressor effects in vivo. Spotting lethal (sl) rats carry a naturally occurring deletion in the ET(B) gene that completely abrogates functional receptor expression. Rats homozygous for this mutation die shortly after birth due to congenital distal intestinal aganglionosis. Genetic rescue of ET(B)(sl/sl) rats from this developmental defect using a dopamine--hydroxylase (DBH)-ET(B) transgene results in ET(B)-deficient adult rats. On a sodium-deficient diet, DBH-ET(B);ET(B)(sl/sl) and DBH-ET(B);ET(B)(+/+) rats both exhibit a normal arterial blood pressure, but on a high-sodium diet, the former are severely hypertensive. We find no difference in plasma renin activity or plasma aldosterone concentration between salt-fed wild-type, DBH-ET(B);ET(B)(+/+) or DBH-ET(B);ET(B)(sl/sl) rats, and acute responses to intravenous L-NAME and indomethacin are similar between DBH-ET(B);ET(B)(sl/sl) and DBH-ET(B);ET(B)(+/+) rats. Irrespective of diet, DBH-ET(B);ET(B)(sl/sl) rats exhibit increased circulating ET-1, and, on a high-sodium diet, they show increased but incomplete hypotensive responses to acute treatment an ET(A)-antagonist. Normal pressure is restored in salt-fed DBH-ET(B);ET(B)(sl/sl) rats when the epithelial sodium channel is blocked with amiloride. We conclude that DBH-ET(B);ET(B)(sl/sl) rats are a novel single-locus genetic model of severe salt-sensitive hypertension. Our results suggest that DBH-ET(B);ET(B)(sl/sl) rats are hypertensive because they lack the normal tonic inhibition of the renal epithelial sodium channel.

Elevation of blood pressure by genetic and pharmacological disruption of the ETB receptor in mice.

Exogenously administered endothelin (ET) elicits both pressor and depressor responses through the ETA and/or the ETB receptor on vascular smooth muscle cells and ETB on endothelial cells. To test whether ETB has pressor or depressor effects under basal physiological conditions, we determined arterial blood pressure (BP) in ETB-deficient mice obtained by crossing inbred mice heterozygous for targeted disruption of the ETB gene with mice homozygous for the piebald (s) mutation of the ETB gene (ETBs/s). F1 ETB-/s and ETB+/s progeny share an identical genetic background but have ETB levels that are approximately (1)/(8) and (5)/(8), respectively, of wild-type mice (ETB+/+). BP in ETB-/s mice was significantly higher, by approximately 20 mmHg, than that in ETB+/s or ETB+/+ mice. Immunoreactive ET-1 concentration in plasma as well as respiratory parameters was not different between ETB-/s and ETB+/s mice. A selective ETB antagonist, BQ-788, increased BP in ETB+/s and ETB+/+ but not in ETB-/s mice. Pretreatment with indomethacin, but not with NG-monomethyl-L-arginine, can attenuate the observed pressor response to BQ-788. The selective ETA antagonist BQ-123 did not ameliorate the increased BP in ETB-/s mice. Moreover, BP in mice heterozygous for targeted disruption of the ETA gene was not different from that in wild-type controls. These results suggest that endogenous ET elicits a depressor effect through ETB under basal conditions, in part through tonic production of prostaglandins, and not through secondary mechanisms involving respiratory control or clearance of circulating ET.

Endothelin in the central control of cardiovascular and respiratory functions.

1. Exogenously administered endothelin (ET) modulates the activity of cardiovascular and respiratory neurons in the central nervous system (CNS) and, thus, affects arterial blood pressure (ABP) and ventilation. However, a physiological role(s) for endogenous ET in the CNS has not been elucidated. To address this question, we examined ABP and ventilation in mutant mice deficient in ET-1, ETA and ETB receptors and endothelin-converting enzyme-1, which were made by gene targeting. 2. Respiratory frequency and volume was measured in mice by whole body plethysmography when animals breathed normal room air and hypoxic and hypercapnic gas mixtures. A few days after respiratory measurements, a catheter was implanted into the femoral artery under halothane anaesthesia. On the following day, the ABP of awake mice was measured through the indwelling catheter and heart rate was calculated from the ABP signal. After 2 h ABP measurement, arterial blood was collected through the catheter and pH and the partial pressures of O2 and CO2 were measured by a blood gas analyser. 3. Compared with corresponding controls, the mean (+/- SEM) ABP in ET-1+/- and ETB-deficient mice was significantly higher (118 +/- 2 vs 106 +/- 3 mmHg for ET-1+/- (n = 22) and ET-1+/+ (n = 17) mice, respectively; 127 +/- 3 vs 109 +/- 4 mmHg for ETB-/s (n = 9) and ETB+/s (n = 9) mice, respectively; P < 0.05 for both). In ET-1+/- mice, PCO2 tended to be higher and PO2 was significantly lower than corresponding values in ET-1+/+ mice. Under resting conditions, there was no significant difference in respiratory parameters between mutants and their corresponding controls. However, reflex increases of ventilation to hypoxia and hypercapnia were significantly attenuated in ET-1+/-, ET-1-/- and ETA-/- mice. 4. In another series of experiments in ET-1+/- mice, we found that sympathetic nerve activity (SNA) was augmented and reflex excitation of phrenic nerve activity (PNA) in response to hypoxia and hypercapnia was blunted. Attenuation of the reflex PNA response to hypercapnia was also observed in the medulla-spinal cord preparation from ET-1-/- mice. 5. Elevation of ABP in ETB-deficient mice was most likely due to a peripheral mechanism, because SNA and respiratory reflexes were not different from those in control animals. 6. We conclude that endogenous ET-1 plays an important role in the central neural control of circulation and respiration and that ETA receptors mediate this mechanism.

Basaloid-squamous cell carcinoma of the oral mucosa: report of two cases and study of the proliferative activity.

Two cases of basaloid-squamous cell carcinoma (BSC) of the oral mucosa are described. The first case occurred at the floor of the mouth in a 58-year-old man, and the second case occurred at the mandibular gingiva in a 79-year-old woman. The laboratory data of the first case showed a positive response to hepatitis C virus antibody. In the first case, the tumor mass measured 4 x 4 cm in size, and was located at the lingual side of the median mandible beside the sublingual gland. In the second case, the tumor mass measured 25 x 15 mm in size, and was located in the alveolar mucosa of the right mandible. Histologically, both tumors showed a neoplastic epithelium arranged in a solid pattern with evidence of peripheral palisading, central necrosis, and some squamous differentiation. The proliferative activities of the BSC were compared with conventional squamous cell carcinomas (SCC) in the oral floor and gingiva, respectively, by employing a sensitive argyrophilic nuclear organizer region (AgNOR) staining method. The number of AgNOR per nucleus of the BSC was higher than that of any other SCC cases. The results support the opinion that BSC of the oral mucosa has a worse prognosis than conventional SCC.

Basaloid-squamous cell carcinoma of the floor of the mouth: characterization of a cell line.

Since it was first described in 1986, basaloid-squamous cell carcinoma (BSC) has been considered a distinct variant of squamous cell carcinoma that occurs in a variety of anatomic sites, including the head and neck region. We report the characterization of the first cell line established from a basaloid-squamous cell carcinoma of the floor of the mouth. The cell line exhibited a highly invasive capacity, indicating that BSC has very aggressive behavior. This cell line may be a useful model for elucidation of the biological characteristics of BSC.

[A case of probable Creutzfeldt-Jakob disease with a point mutation of prion protein gene codon 180 and atypical MRI findings].

A case of probable Creutzfeldt-Jakob disease (CJD) is reported. A 70-year-old Japanese woman was admitted with a complaint of amnesia. She initially developed Klüver-Bucy syndrome, hypergraphia, and later showed myoclonus and startle response. She developed akinetic mutism within nine months from the onset. Prion protein gene codon 180 point mutation (Met/Ile) was detected and we diagnosed her as CJD. Serial MRI studies revealed abnormal T2 elongation and thickening limited to the cerebral cortices, which started at bilateral temporal lobes and later extended to frontal, parietal, and occipital lobes with relative sparing of hippocampi and central gyri. Serial EEG did not show periodic synchronous discharge (PSD). Three cases of CJD with a point mutation of codon 180 were reported in the past. There are several common features in the past cases and the present case, i.e. 1) negative familial history, 2) late onset, 3) T2 high intensity at cerebral cortices on MRI, and 4) negative PSD. These may be characteristic features of CJD with a point mutation of codon 180.

Physiological significance of plasma sulfoconjugated dopamine in patients with hypertension--clinical and experimental studies.

Sulfoconjugated catecholamines, especially dopamine sulfate, have recently attracted much attention because of the possibility of their conversion to active free dopamine by tissue arylsulfatase. In the present study, we have measured the plasma levels of free and sulfoconjugated dopamine in patients with hypertension and have investigated the physiological significance of sulfoconjugation. Results showed that the plasma level of dopamine sulfate in patients with essential hypertension was higher than the level in control subjects, and was highest in patients with renal hypertension. However, the plasma level of free dopamine showed no significant difference between patients with hypertension and normal subjects. Moreover, after normalization of blood pressure in hypertensive patients with medication, the plasma levels of conjugated dopamine decreased to almost the control value. In the experimental study, dopamine sulfate inhibited angiotensin II-induced aldosterone release from bovine adrenal cortical cells to a similar extent as produced by free dopamine. From these results, we have concluded that plasma sulfoconjugated dopamine may regulate free dopamine in the plasma of patients with hypertension, and it may have some physiological effects on blood pressure regulation.

Diethyldithiocarbamate, a superoxide dismutase inhibitor, reduces indomethacin-induced gastric lesions in rats.

We examined the effect of diethyldithiocarbamate (DDC), the superoxide dismutase (SOD) inhibitor, on the development of gastric lesions induced by indomethacin in rats. Indomethacin (25 mg/kg) was given subcutaneously, and gastric acid secretion, motility, lipid peroxidation, vascular permeability, and myeloperoxidase as well as gastric lesions were measured. Indomethacin produced high-amplitude contractions of the stomach and caused hemorrhagic lesions in the corpus mucosa with significant increase in neutrophil-related processes such as myeloperoxidase activity, vascular permeability and lipid peroxidation. These changes caused by indomethacin were all significantly inhibited by prior administration of atropine (3 mg/kg s.c.). Pretreatment of the animals with DDC (75-1,000 mg/kg s.c.) prevented these lesions induced by indomethacin in the corpus mucosa in a dose-dependent manner (> 100 mg/kg), though at high doses (> 750 mg/kg) some damage was found in both the antrum and duodenum. DDC showed a significant inhibition against the gastric mucosal SOD activity (> 400 mg/kg), yet potently suppressed the increase of lipid peroxidation, vascular permeability, and myeloperoxidase activity caused by indomethacin. DDC dose-dependently (> 75 mg/kg) inhibited the enhancement of gastric motility caused by indomethacin and showed a weak antisecretory effect at high doses (> 750 mg/kg). These results showed that DDC reduced indomethacin-induced gastric lesions by suppressing gastric motility, despite inhibiting SOD activity. This study also indicates the prime importance of gastric hypercontraction in the pathogenesis of this lesion model and suggests that other events including the neutrophil-related processes may be secondary to gastric hypercontraction caused by indomethacin.

Adrenomedullin stimulates calcium efflux from adrenal chromaffin cells in culture: possible involvement of an Na+/Ca2+ exchange mechanism.

The effect of adrenomedullin, a hypotensive peptide, on Ca2+ efflux from cultured bovine adrenal chromaffin cells was examined. Adrenomedullin stimulated the efflux of 45Ca2+ from the cells in a concentration-dependent manner (10(-7)M - 3x10(-6)M). Adrenomedullin did not increase the intracellular free Ca2+ ([Ca2+]i) level and catecholamine secretion. The adrenomedullin-stimulated 45Ca2+ efflux was not inhibited by incubation with Ca2+-free medium, but was inhibited by incubation with Na+-free medium. These results indicate that adrenomedullin stimulates extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably through its stimulatory effect on membrane Na+/Ca2+ exchange.

Drug-dependent Ca2+ mobilization in organ-cultured rabbit ciliary processes.

This study was conducted to determine whether drug-dependent changes in cytosolic Ca2+ concentration take place in the ciliary nonpigment epithelial cells of rabbits under more physiological conditions. Iris-ciliary body from pigmented rabbits in organ-culture was loaded with a Ca(2+)-sensitive fluorescent dye, fura-2, and a video-imaging system with an image analyzer was employed. Using this method fluorescence from nonpigmented epithelial cells can be analyzed without interference from fluorescence from pigmented ciliary epithelial cells. Among the drugs studied, norepinephrine and carbachol induced Ca2+ transients in the nonpigmented epithelial cells of organ-cultured ciliary processes. Epinephrine, isoproterenol, dopamine, neuropeptide Y, and substance P at the concentration of 10(-6) to 10(-3) M failed to elicit a response. The cytosolic free Ca2+ concentration of the cells in the resting state, as determined by an in vitro calibration curve, was 166 nM. The peak free cytosolic Ca2+ concentration induced by norepinephrine was about 263 nM, and that induced by carbachol was more than 1,000 nM. The carbachol-induced response was larger in magnitude and longer in duration than that induced by norepinephrine. Not uncommonly, the carbachol-induced response lasted more than 15 min. The response was diminished in both peak height and duration by chelation of extracellular Ca2+. Atropine abolished the response showing the response being mediated by a muscarinic receptor.

The SH3 domain of Crk binds specifically to a conserved proline-rich motif in Eps15 and Eps15R.

The Crk protein belongs to the family of proteins consisting of mainly Src homology 2 and 3 (SH2 and SH3) domains. These proteins are thought to transduce signals from tyrosine kinases to downstream effectors. In order to understand the specificity and effector function of the SH3 domain of Crk, we screened an expression library for binding proteins. We isolated Eps15, a substrate of the epidermal growth factor receptor (EGFR) tyrosine kinase, and Eps15R, a novel protein with high sequence homology to the carboxyl-terminal domain of Eps15. Antibodies raised against a fragment of the Eps15R gene product immunoprecipitated a protein of 145 kDa. Eps15 and Eps15R bound specifically to the amino-terminal SH3 domain of Crk and coprecipitated equivalently with both c-Crk and v-Crk from cell lysates. The amino acid sequences of Eps15 and Eps15R featured several proline-rich regions as putative binding motifs for SH3 domains. In both Eps15 and Eps15R, we identified one proline-rich motif which accounts for their interaction with the Crk SH3 domain. Each binding motif contains the sequence P-X-L-P-X-K, an amino acid stretch that is highly conserved in all proteins known to interact specifically with the first SH3 domain of Crk. Furthermore, we found that immunoprecipitates of activated EGFR-kinase stably bound in vitro-translated Eps15 only in the presence of in vitro-translated v-Crk. Crk might therefore be involved in Eps15-mediated signal transduction through the EGFR.

Physiological significance of plasma sulfoconjugated dopamine: experimental and clinical studies.

Sulfoconjugated catecholamines have been regarded simply as metabolites of free catecholamines. However, a conjugated form of the catecholamine, dopamine has recently attracted much attention because it is present at high levels in the plasma of humans and experimental animals. We carried out experimental and clinical studies to determine the physiological significance of this large amount of dopamine conjugate in the plasma. Clinical studies showed that the plasma level of dopamine sulfate decreased significantly during the acute phase of heart failure, whereas that of free dopamine increased. Moreover, the plasma level of conjugated dopamine in patients with essential hypertension was higher than that in control subjects, and being highest in patients with renal hypertension. In experimental studies, we examined the activity for deconjugating DA sulfate in homogenates of organs from dogs. The kidney and liver exhibited the highest activities, and in the heart, the activity was higher in the atrium than the ventricle. We also examined the effect of dopamine sulfate on isolated perfused rat heart. Dopamine sulfate was found to be converted to free dopamine, which was responsible for the positive inotropic action, in atrial tissue. Moreover, deconjugation of DA sulfate to the free form was accelerated by a high work lord on the heart. From these results, we conclude that the formation of dopamine sulfate plays a role in regulating the level of plasma free dopamine and that plasma dopamine sulfate may be a storage or reserve form of dopamine. Free (or active) dopamine may be formed through a deconjugation reaction when necessary.

Endogenous ET-1 contributes to liver injury induced by galactosamine and endotoxin in isolated perfused rat liver.

Injury to hepatocytes most likely occurs via disturbances in the microcirculation. The role of vasoconstriction due to the effect of endogenous endothelin-1 (ET-1) in the development of galactosamine (GalN)- and lipopolysaccharide (LPS)-induced liver injury was investigated. Using the multiple indicator dilution technique, we measured the volume of the hepatic sinusoids and the apparent Disse space as indicators of overall hepatic microcirculation. Serum purine nucleoside phosphorylase activity as a marker of damage to nonparenchymal cells increased and the volume of the sinusoids and the Disse space decreased prior to hepatocyte damage in rats treated intraperitoneally with GalN and LPS. Moreover, the amount of ET-1 release was elevated. When livers from untreated rats were perfused with ET-1 in a recirculating system, hepatocyte damage was observed similar to experiments with GalN and LPS. A monoclonal anti-endothelin antibody, AwETN40, diminished the extent of liver injury caused by GalN and LPS in isolated perfused rat liver. The present study suggests that vasoconstriction is an early event in GalN- and LPS-induced liver injury and that the development of hepatocyte damage is mediated via microcirculatory disturbances due to endogenous ET-1.

Effects of nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on duodenal alkaline secretory and ulcerogenic responses induced by mepirizole in rats.

The inhibition of nitric oxide (NO) production by NO synthase inhibitors stimulates HCO3- secretion in the rat duodenal mucosa. Therefore, we examined the effects of NG-nitro-L-arginine methyl ester (L-NAME, the NO synthase inhibitor) and nitroprusside (the exogenous NO donor) on the duodenal HCO3- and ulcerogenic responses in anesthetized rats. Animals were administered mepirizole (200 mg/kg, subcutaneously) for induction of duodenal ulcers, and gastric acid and duodenal HCO3- secretions were measured with or without pretreatment with L-NAME (5 mg/kg, intravenously) or nitroprusside (4 mg/kg, intravenously). Mepirizole increased acid secretion, decreased the acid-induced duodenal HCO3- secretion, and induced hemorrhagic lesions in the proximal duodenum. The inhibition of NO production by L-NAME potentiated the acid secretory response, increased the duodenal HCO3- secretion, and prevented the duodenal lesions, and these changes were all antagonized by simultaneous administration of L-arginine (200 mg/kg, intravenously) but not D-arginine. On the other hand, nitroprusside slightly reduced the acid response but further decreased the HCO3- output, resulting in aggravation of duodenal lesions induced by mepirizole. These data suggest that the inhibition of endogenous NO production by the NO synthase inhibitor L-NAME increases duodenal HCO3- secretion and protects the duodenal mucosa against acid injury.