Neutral Silylyne Complex of Molybdenum: Synthesis, Properties, and Access to Silaiminoacyl Complexes via [2+3] Cycloaddition.

A neutral silylyne complex of molybdenum was synthesized by the stepwise dehydrogenation method and its properties were compared with those of the tungsten analog. The complex takes a dimeric form as crystals but afford a monomer-dimer equilibrium in solution. The replacement of the central metal from W to Mo led to a monomer dominant (~98 %) solution at room temperature. The monomer-dimer dynamics was investigated based on thermodynamic parameters. The molybdenum silylyne complex underwent [2+2] cycloaddition with alkynes much faster than the tungsten analog. The reactions with organic azides led to the formation of the first example of silaiminoacyl complexes through [2+3] cycloaddition. The structures and bonding aspects of the products were clarified by multiple measurements and DFT calculations.

Identification and quantification of diphenhydramine, haloperidol, and its metabolite, reduced haloperidol in a saponified brain specimen that was immersed in the sea water for more than 10 years.

In forensic toxicology, blood and urine specimens are commonly used for detecting and quantifying drugs and their metabolites. When the cadaver is so damaged or decomposed such that the specimens mentioned above cannot be collected, it is necessary to perform drug analysis using alternative specimens such as hair, nails, oral fluids and meconium. Adipocere is resistant to further degradation; it is thus possible to be used as an alternative specimen to analyze drugs and their metabolites. Some researchers indeed have reported drug concentrations in saponified samples that were collected years after decedents' deaths. In this study, we subjected saponified brain, which remained under sea for over 10 years after death, to forensic toxicological analysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Using product ion scan analysis, we confirmed the presence of diphenhydramine, haloperidol, and reduced haloperidol, a metabolite of haloperidol. In addition, drugs and metabolite quantification were performed using the standard addition method. Correlation coefficients of the calibration curves were over 0.98. Analyte concentrations in the saponified brain were as follows: diphenhydramine was 1.84 ng/g, haloperidol was 1.30 ng/g, and reduced haloperidol was 3.02 ng/g. Our results suggest that it can be possible to quantify not only parent drugs but also their metabolites in saponified brain. These findings indicate that saponified tissues could be applied as alternative specimens for forensic toxicology, and could be useful as supporting information for victim identification.

A simple and rapid method for quantifying aconitines and their metabolites in whole blood by modified QuEChERS and liquid chromatography/tandem mass spectrometry (LC/MS/MS).

Aconitum contains highly toxic alkaloids such as aconitine, hypaconitine, jesaconitine, and mesaconitine. Since Aconitum ingestion causes fatal intoxication, it is important to analyze aconitines and their metabolites in the blood. In forensic toxicology, postmortem drug redistribution is known as one factor that would hamper accurate evaluation of concentrations. Therefore, it is recommended to collect multiple blood samples from various sites and compare the results to avoid miss identification of causative compounds for intoxication. In this study, we evaluated aconitines and their metabolites in postmortem blood specimens from ten sites by QuEChERS extraction and liquid chromatography-tandem mass spectrometry (LC/MS/MS). The recovery rates and matrix effects of analytes were approximately 74-80% and 94-100%, respectively. The correlation coefficients were over 0.99. The validation studies revealed that accuracies and precisions were around 97-2% (intraday) and 100-4% (interday), respectively. Finally, the concentrations of aconitine and jesaconitine were from 2.72 to 7.20 ng/mL and from 14.9 to 26.3 ng/mL, respectively. The concentrations of mesaconitine were from 0.32 to 0.88 ng/mL in four samples and detected in two. The concentrations were highest in the right atrium and lowest in the femoral vein. Our results suggest that aconitine and jesaconitne are accumulated in right atrium blood after death, and that right atrium specimen is suitable for measuring aconitine compounds in fatal intoxication cases.

A simple method for the determination of glyphosate, glufosinate and their metabolites in biological specimen by liquid chromatography/tandem mass spectrometry: an application for forensic toxicology.

Glyphosate (GLYP) and glufosinate (GLUF) are phosphorus-containing amino acid type herbicides that are used worldwide. With their rising consumptions, fatal intoxication cases due to these herbicides, whether accidental or intentional, cannot be ignored. Both compounds are difficult to detect, and their pretreatment for instrumental analysis are complicated and time-consuming. Our aim was to develop a simple and rapid quantification method for the two herbicides and their metabolites with liquid chromatography/tandem mass spectrometry (LC/MS/MS). We also compared 2-amino-4-phosphonobutyric acid and DL-2-amino-5-phosphonopentanoic acid as alternative internal standards (IS) to GLYP13C2 15N. Herbicide-containing specimens were highly diluted, evaporated to dryness, and derivatized with acetate/acetic anhydride and trimethyl orthoacetate for 30 min. at 120°C. Our optimized LC conditions successfully separated the target analytes, with acceptable linearities (R 2>0.98) and matrix effects (65%-140%). Accuracy and precision ranged from 80.2 % to 111 %, and from 1.3 % to 13 % at the higher concentration, respectively.The concentration of the herbicides and their metabolites were investigated in a postmortem case of suspected herbicide poisoning cases, in which we detected GLYP and its metabolites. Using one of the three ISs, the GLYP concentrations ranged from 3.1 to 3.5 mg/mL, and 3.3 to 4.5 mg/mL in plasma and urine, respectively; GLYP metabolite concentrations in plasma and urine were 18 to 20 µg/mL and 44 to 54 µg/mL. We thus succeeded in developing a rapid method without extraction for measuring GLYP and GLUF along with their metabolites, and demonstrated its practical applicability.

Reactions of a Silylyne Complex with Aldehydes: Formation of W-Si-O-C Four-Membered Metallacycles and Their Metathesis-Like Fragmentation.

A tungsten silylyne complex having a W≡Si triple bond reacted with two molecules of aldehydes at room temperature to give W-Si-O-C four-membered metallacycles by [2+2] cycloaddition and subsequent formyl hydrogen transfer from one aldehyde molecule to another. Upon heating to 70 °C, the four-membered metallacycles underwent metathesis-like fragmentation cleanly to afford carbyne complexes and "silanoic esters," in a manner similar to that of metallacyclobutadiene, an intermediate of alkyne metathesis reactions, and dimerization of the latter products gave 1,3-cyclodisiloxanes. The "silanoic ester" was also trapped by pivalaldehyde to give a [2+2] cycloaddition product in high yield.

Pericardial fluid is suitable as an alternative specimen for the measurement of ß-hydroxybutyrate within 96 h after death.

We examined postmortem ß-hydroxybutyrate (BHB) levels in the body fluids obtained from 253 forensic autopsy cases whose causes of death were determined. Postmortem changes of BHB levels according to postmortem intervals (PMI) in various body fluids (plasma, urine, vitreous humor, and pericardial fluids) were investigated to determine appropriate alternative specimens as plasma samples. Our study has indicated the following points: 1) the BHB levels in plasma specimens from three sampling sites showed no significant differences, 2) postmortem changes of BHB levels in plasma and pericardial fluids could be negligible within 96 h PMI, while urine and vitreous humor BHB levels showed postmortem changes, and 3) pericardial fluid would thus be most suitable as an alternative to plasma in postmortem BHB level. We have also proposed that BHB levels could be applicable for the diagnosis of metabolic disorders in forensic autopsy.

STR Genotyping from a Dry-Cleaned Skirt in a Sexual Assault Case.

In this sexual assault case, the standard preliminary semen examinations could not confirm physically or biochemically whether the accused's semen had stained the victim's skirt because the skirt had been dry-cleaned for stain removal and had been worn for more than a year after the assault. Fortunately, however, a photograph taken just after the assault was found in the court records that showed white stains on the checkered skirt. The locations of the stains were estimated based on the checkered pattern of the fabric, and microscopic examination using Baecchi's staining revealed the presence of spermatozoa. Further analysis indicated the male DNA profile generated from the sperm cells was consistent with the suspect's DNA using three multiplex STR typing systems for a total of 21 autosomal and 17 Y chromosomal short tandem repeats (STRs). Ultimately, the result of the DNA profile played a very useful role as additional evidence.

Species specificities among primates probed with commercially available fluorescence-based multiplex PCR typing kits.

To assess species specificities among primates of signals from short tandem repeat (STR) loci included in two commercially available kits, mainly the AmpFlSTR Identifiler kit and additionally the GenePrint PowerPlex 16 system, we analyzed 69 DNA samples from 22 nonhuman primate species representing apes, Old World Monkeys (OWMs), New World Monkeys (NWMs), and prosimians. Each prosimian species and the NWM cotton-top tamarin apparently lacked all STR loci probed. Only one peak, the amelogenin-X peak, was evident in samples from all other NWMs, except the owl monkey. In contrast, several loci, including the amelogenin-X peak, was evident in samples from each OWM species. Notably, for each ape sample, the amelogenin peaks were concordant with morphological gender of the individual. Among the primates, especially in apes, the numbers of alleles for STR loci were increasing according to their phylogenetic order: prosimians

Cloning and transcriptional expression of mouse mannosyltransferase IV/V cDNA, which is involved in the synthesis of lipid-linked oligosaccharides.

We cloned the mouse mannosyltransferase IV/V gene (mALG11) from FM3A cells by a bioinformatic approach. The ORF contained 1476 bp encoding 492 amino acids. The cloned mALG11 complemented the growth defect of the Saccharomyces cerevisiae ALG11Δ mutant. In addition, we detected a variant cDNA by alternate splicing that had an additional four-nucleotide ATGC insertion at base 276 of the ORF. Consequently the variant cDNA encoded a truncated protein with 92 amino acids, lacking the glycosyltransferase group-1 domain. The variant cDNA occurs in many mouse strains according to EST database searches. Moreover, we detected it in FM3A cDNA, but we did not detect any such variants in the human EST database or in HeLa cDNA, although human ALG11 (hALG11) genomic DNA has the same sequence around the intron-exon boundaries as those of mALG11 genomic DNA. Hence, we concluded that there is different transcriptional control mechanism between mALG11 and hALG11.

Effects of extracranial-intracranial bypass for patients with hemorrhagic moyamoya disease: results of the Japan Adult Moyamoya Trial.

BACKGROUND AND PURPOSE: About one half of those who develop adult-onset moyamoya disease experience intracranial hemorrhage. Despite the extremely high frequency of rebleeding attacks and poor prognosis, measures to prevent rebleeding have not been established. The purpose of this study is to determine whether extracranial-intracranial bypass can reduce incidence of rebleeding and improve patient prognosis. METHODS: This study was a multicentered, prospective, randomized, controlled trial conducted by 22 institutes in Japan. Adult patients with moyamoya disease who had experienced intracranial hemorrhage within the preceding year were given either conservative care or bilateral extracranial-intracranial direct bypass and were observed for 5 years. Primary and secondary end points were defined as all adverse events and rebleeding attacks, respectively. RESULTS: Eighty patients were enrolled (surgical, 42; nonsurgical, 38). Adverse events causing significant morbidity were observed in 6 patients in the surgical group (14.3%) and 13 patients in the nonsurgical group (34.2%). Kaplan-Meier survival analysis revealed significant differences between the 2 groups (3.2%/y versus 8.2%/y; P=0.048). The hazard ratio of the surgical group calculated by Cox regression analysis was 0.391 (95% confidence interval, 0.148-1.029). Rebleeding attacks were observed in 5 patients in the surgical group (11.9%) and 12 in the nonsurgical group (31.6%), significantly different in the Kaplan-Meier survival analysis (2.7%/y versus 7.6%/y; P=0.042). The hazard ratio of the surgical group was 0.355 (95% confidence interval, 0.125-1.009). CONCLUSIONS: Although statistically marginal, Kaplan-Meier analysis revealed the significant difference between surgical and nonsurgical group, suggesting the preventive effect of direct bypass against rebleeding. CLINICAL TRIAL REGISTRATION URL: http://www.umin.ac.jp/ctr/index.htm. Unique identifier: C000000166.

Transsphenoidal microsurgical results of female patients with prolactinomas.

OBJECTIVE: We investigated surgical cure rate and surgical complications of patients with macroprolactinomas who desired pregnancy to evaluate the efficacy of transsphenoidal surgery. METHODS: Surgical cure rate was investigated in 138 female patients who were under 40 years old. RESULTS: We found a significant correlation between serum prolactin levels and adenoma volume (r=0.004; p<0.0001), adenoma volume and age (r=-0.213; p<0.03), and proliferative index of the adenoma and age (r=-0.15; p<0.007). Seventy-seven out of 81 patients with enclosed macroadenoma were considered cured, and therefore the overall surgical cure rate was 95%. However, during long-term follow-up, recurrence of adenomas with hyperprolactinemia was seen in 5 out of 81 patients (6%), and the long-term cure rate in patients with enclosed macroadenomas was 89%. Adenomas that did not invade the cavernous sinus showed a significantly higher surgical curability and lower serum prolactin levels, and a smaller size than those adenomas that invaded the cavernous sinus. CONCLUSIONS: The long-term surgical cure rate was found to be 89% and this success rate far surpasses the complication rate of 39% during pregnancy by dopamine agonist therapy. Thus, transsphenoidal surgery should be considered as a first-line treatment for female patients who desire pregnancy.

The natural course of unruptured cerebral aneurysms in a Japanese cohort.

BACKGROUND: The natural history of unruptured cerebral aneurysms has not been clearly defined. METHODS: From January 2001 through April 2004, we enrolled patients with newly identified, unruptured cerebral aneurysms in Japan. Information on the rupture of aneurysms, deaths, and the results of periodic follow-up examinations were recorded. We included 5720 patients 20 years of age or older (mean age, 62.5 years; 68% women) who had saccular aneurysms that were 3 mm or more in the largest dimension and who initially presented with no more than a slight disability. RESULTS: Of the 6697 aneurysms studied, 91% were discovered incidentally. Most aneurysms were in the middle cerebral arteries (36%) and the internal carotid arteries (34%). The mean (±SD) size of the aneurysms was 5.7±3.6 mm. During a follow-up period that included 11,660 aneurysm-years, ruptures were documented in 111 patients, with an annual rate of rupture of 0.95% (95% confidence interval [CI], 0.79 to 1.15). The risk of rupture increased with increasing size of the aneurysm. With aneurysms that were 3 to 4 mm in size as the reference, the hazard ratios for size categories were as follows: 5 to 6 mm, 1.13 (95% CI, 0.58 to 2.22); 7 to 9 mm, 3.35 (95% CI, 1.87 to 6.00); 10 to 24 mm, 9.09 (95% CI, 5.25 to 15.74); and 25 mm or larger, 76.26 (95% CI, 32.76 to 177.54). As compared with aneurysms in the middle cerebral arteries, those in the posterior and anterior communicating arteries were more likely to rupture (hazard ratio, 1.90 [95% CI, 1.12 to 3.21] and 2.02 [95% CI, 1.13 to 3.58], respectively). Aneurysms with a daughter sac (an irregular protrusion of the wall of the aneurysm) were also more likely to rupture (hazard ratio, 1.63; 95% CI, 1.08 to 2.48). CONCLUSIONS: This study showed that the natural course of unruptured cerebral aneurysms varies according to the size, location, and shape of the aneurysm. (Funded by the Ministry of Health, Labor, and Welfare in Japan and others; UCAS Japan UMIN-CTR number, C000000418.).

Diagnosis of fulminant type 1 diabetes mellitus in an autopsy case with postmortem changes.

A Japanese female in her thirties was found dead in her apartment; the postmortem interval was estimated to be approximately 3 days. Several postmortem changes were evident. Acute gastroenteritis had been diagnosed 3 days earlier. On autopsy, no specific findings other than fatty liver were observed. On hematoxylin eosin staining, mild fibrosis, and invasion of neutrophils in the pancreatic parenchyma, severe fatty liver, and extensive vacuolation of renal tubular cells were observed. Biochemical analyses revealed extremely high ß-hydroxybutyrate concentrations in body fluids with moderate elevation of hemoglobin A1c. Toxicological analyses of organ and urine samples were negative. We concluded that severe ketoacidosis had occurred in the deceased. Subsequently, selective destruction of pancreatic ß-cells was demonstrated. Considered together, results indicated that the cause of death was fulminant type 1 diabetes mellitus. This report illustrates the fact that a combination of biochemical and immunohistochemical investigations can be useful for diagnosing this condition in cases with evident postmortem changes.

Prevalence and clinicoepidemiological features of moyamoya disease in Japan: findings from a nationwide epidemiological survey.

BACKGROUND AND PURPOSE: The objectives of the present study were to estimate an annual number of patients with moyamoya disease in Japan and to describe the clinicoepidemiological features of the disease. METHODS: The study consisted of 2 questionnaire surveys, which were distributed to randomly selected departments of neurosurgery, internal medicine, neurology, cerebrovascular medicine, and pediatrics in hospitals throughout Japan. The first survey inquired about the number of the patients treated in 2003, and the second requested additional detailed clinicoepidemiological information about each patient identified in the first survey. RESULTS: In 2003, the total number of patients treated in Japan was estimated at 7700 (95% confidence interval, 6300 to 9300). Sex ratio (women to men) of the patients was 1.8. For men, the peak of moyamoya disease was observed in patients aged 10 to 14 years and for women aged 20 to 24 years. Annual rate of newly diagnosed cases in 2003 was 0.54 per 100,000 population. Family history of moyamoya disease was found in 12.1% of the patients. The majority (77.9%) were treated as outpatients. CONCLUSIONS: Although the clinicoepidemiological features of the patients in the present study were almost similar to those obtained in previous ones, the estimated prevalence of moyamoya disease in Japan has almost doubled during the recent decade (3900 in 1994 and 7700 in 2003). The increase could partly be explained by the increase in newly diagnosed cases (0.35 in 1994 and 0.54 in 2003 per 100,000 population).

Radiological findings, clinical course, and outcome in asymptomatic moyamoya disease: results of multicenter survey in Japan.

BACKGROUND AND PURPOSE: Although the development of a noninvasive MR examination has increased the opportunity to identify asymptomatic patients with moyamoya disease who have experienced no stroke episodes, their clinical features are still unclear. This was the first multicenter, nation-wide survey focused on asymptomatic moyamoya disease in Japan and was designed to clarify their clinical features. METHODS: A clinical database of asymptomatic patients with moyamoya disease was collected from 12 participating hospitals in Japan between 2003 and 2006. In total, 40 patients were enrolled in this historical prospective cohort study. Of these, 6 underwent surgical revascularization, including superficial temporal artery to middle cerebral artery anastomosis and/or pial synangiosis. Their demographic and radiological findings as well as outcome were evaluated. RESULTS: On initial evaluation, cerebral infarction and disturbed cerebral hemodynamics were detected in approximately 20% and 40% of the involved hemispheres, respectively. Angiographical stage was more advanced in more elderly patients. Of 34 nonsurgically treated patients, 7 experienced transient ischemic attack (n=3), ischemic stroke (n=1), or intracranial bleeding (n=3) during follow-up periods (mean, 43.7 months). The annual risk for any stroke was 3.2%. Disease progression was associated with ischemic events or silent infarction in 4 of 5 patients. No cerebrovascular event occurred in the 6 patients who underwent surgical revascularization. CONCLUSIONS: The findings revealed that asymptomatic moyamoya disease is not a silent disorder and may potentially cause ischemic or hemorrhagic stroke. Asymptomatic patients with moyamoya disease should be carefully followed-up to further clarify their outcome and to establish the management guideline for them.

Population data of six STR loci using hexaplex PCR amplification and typing system, "Midi-6" in four regional populations in Japan.

The genetic differences of the allele frequency distributions for six STR loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290) among regions in Japan were examined using our recently designed hexaplex amplification and typing system, "Midi-6" newly named, to construct a database in the Japanese population. Genotypes at six loci were analyzed in 198, 200, 175, and 196 individuals from the area of Akita, Nagoya, Oita, and Okinawa, respectively, in Japan. The allele frequency distributions were significantly different (p<0.05) at from one to five loci among the four populations when compared pairwise. Significant differences were also observed at two or three loci between Oita- or Okinawa-Japanese and the "pooled" population (n=769), respectively. However, since F(ST) (theta) values were extremely low (<0.05), ranging from 0.0020 to 0.0118 for six loci, genetic differentiation within the pooled Japanese population was negligible. Therefore, it suggested that the data of the allele frequencies at six loci in the pooled population would be employed as the base of calculation for statistical probabilities.

Correlation between copper/zinc superoxide dismutase and the proliferation of neural stem cells in aging and following focal cerebral ischemia.

OBJECT: Neural stem cells (NSCs) have been demonstrated in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus (DG). Although aging rats manifest a decrease in NSCs, rats exposed to stress (for example, ischemia, epilepsy, radiation, and trauma) show an increase in these cells. In transgenic mice, the overexpression of human copper/zinc superoxide dismutase (SOD1), an endogenous antioxidant, has been reported to be a protective enzyme against transient focal cerebral ischemia. The authors investigated the correlation between SOD1 and the proliferation of NSCs in aging as chronic oxidative stress (Experiment 1) and acute oxidative stress induced by transient focal cerebral ischemia (Experiment 2) in mice. METHODS: Bromodeoxyuridine (BrdU) was used in the evaluation of NSCs. In Experiment 1, NSCs in the SVZ significantly increased in 16-month-old transgenic mice compared with wild-type mice (p = 0.0001). In Experiment 2, mice were subjected to 30-minute occlusions of the middle cerebral artery. The increase in NSCs in the DG in transgenic mice was significantly greater than that in wild-type mice (p < 0.05). CONCLUSIONS: Results in this study suggest that chronic and acute oxidative stress may inhibit the proliferation of NSCs and that SOD1 may play a key role in NSC proliferation.

Phylogenetic relationship of the populations within and around Japan using 105 short tandem repeat polymorphic loci.

We have analyzed 105 autosomal polymorphic short tandem repeat (STR) loci for nine East and South-eastern Asian populations (two Japanese, five Han Chinese, Thai, and Burmese populations) and a Caucasian population using a multiplex PCR typing system. All the STR loci are genomewide tetranucleotide repeat markers of which the total number of observed alleles and the observed heterozygosity were 756 and 0.743, respectively, for Japanese populations. Phylogenetic analysis for these allele frequency data suggested that the Japanese populations are more closely related with southern Chinese populations than central and/or northern ones. STRUCTURE program analysis revealed the almost clearly divided and accountable population structure at K=2-6, that the two Japanese populations always formed one group separated from the other populations and never belong to different groups at K> or =3. Furthermore, our new allele frequency data for 91 loci were analyzed with those for 52 worldwide populations published by previous studies. Phylogenetic and multidimensional scaling (MDS) analyses indicated that Asian populations with large population size (six Han Chinese, three Japanese, two Southeast Asia) formed one distinct cluster and are closer to each other than other ethnic minorities in east and Southeast Asia. This pattern may be the caviar of comparing populations with greatly differing population sizes when STR loci were analyzed.