The Bacterial Community in the Western Region of North Pacific Intermediate Water.

The bacterial composition in North Pacific Intermediate Water (NPIW) was investigated in three different years and compared with that in other seawaters around Japan. The results indicated that bacterial composition was surprisingly stable at the same point in a mesopelagic water mass throughout the years and supported previous physicochemical observations that NPIW is distributed to Kumejima, Japan.

Genome Sequence of the Psychrophilic Bacterium Tenacibaculum ovolyticum Strain da5A-8 Isolated from Deep Seawater.

Some bacterial species of the genus Tenacibaculum, including Tenacibaculum ovolyticum, have been known as fish pathogens in the sea. So far, the only published genome sequence for this genus is for Tenacibaculum dicentrarchi, which could also be a fish pathogen. Strain da5A-8, showing 100% identity to the 16S rRNA gene sequence of T. ovolyticum DSM 18103(T), was isolated from seawater at a depth of 344 m in Kochi, Japan, and grew optimally at 10 to 20°C. The genome sequence of strain da5A-8 revealed the possible virulence genes commonly observed in the genus Tenacibaculum.

Increased MFG-E8 expression and its implications in the vascular pathophysiology of cocaine abuse.

The aim of this study was to examine the possible involvement of smooth muscle cell remodeling and the induction of MFG-E8 (milk fat globule protein epidermal growth factor-VIII) in vascular pathophysiology during cocaine administration in cultured cells and rats. Cocaine exerts bifurcate effects on vascular cells; it stimulates vasoconstriction through enhancement of catecholamine release at low doses, while it suppresses cardiovascular functions through inhibition of ion channels at high doses. Short-term exposure to a high concentration of cocaine (3 mM, 24 hr) resulted in cell death of A7r5 rat aorta-derived smooth muscle cells. On the other hand, long-term exposure of the same cells to a low concentration (0.3 mM, ~7 days) resulted in a transient increase in MFG-E8 expression followed by an increased tendency toward cyclin D1, PCNA (proliferating cell nuclear antigen), and CDK4 (cyclin-dependent protein kinase-4) expression. Interestingly, autophagy was not induced, but rather was impaired, in cocaine-treated cells. Increased expressions of MFG-E8, PCNA, and CDK4 were also observed in the aortic vascular cells of rats administered cocaine (50 mg/kg, 2 days, i.v.), confirming that cocaine induced MFG-E8 expression in vivo. Taken together, the results show that MFG-E8 is induced in vascular cells exposed to cocaine, and that this induction is likely to be involved in the vascular toxicity elicited by cocaine abuse.

Aurantiacicella marina gen. nov., sp. nov., a myxol-producing bacterium from surface seawater.

A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped bacterium, strain 2A-8T, was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain produced myxol as a major carotenoid. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Flavobacteriaceae and was related most closely to the genus Aquimarina (91.0-94.4 % 16S rRNA gene sequence similarity to the type strains of species of this genus). The DNA G+C content was 35 mol%. The major fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and five unidentified lipids. Menaquinone 6 was detected as the sole isoprenoid quinone. On the basis of phenotypic, genotypic and chemotaxonomic data, strain 2A-8T represents a novel genus and species, for which the name Aurantiacicella marina gen. nov., sp. nov. is proposed. The type strain of Aurantiacicella marina is 2A-8T ( = NBRC 111187T = KCTC 42676T).

Verbal regulation of grip force in preschoolers.

The purpose of this study was to clarify the developmental processes in verbal regulation by preschool children. Participants were 152 typically developing children (74 boys, 78 girls) between 4 and 6 years of age (M = 5.3, SD = .8), and 30 healthy adults (15 men, 15 women) between 19 and 26 years of age (M = 20.8, SD = 1.4). In Exp. 1, the task was to regulate grip force based on quantitative instruction which implies using a scale for regulation. Participants were required to produce a half-grip force of the maximum (Task 1). In Exp. 2, the task was grip-force regulation based on nonquantitative instruction. The participants were asked to respond with a slightly weaker grip force than the maximum (Task 2) and then a further weaker grip force (Task 3) than that used on Task 2. The regulation rates produced the extent of regulation and suggest regulation by quantitative instruction may develop earlier than by nonquantitative instruction. Also, precise grip-force regulation based on the semantic aspect of instruction may be difficult for young children. The developmental changes in the rate of performance especially observed in children of 4 to 6 years indicate that the tendency to use too much grip force disappears during this preschool period. In addition, too little grip force in regulation may reflect the developmental process toward fine grasping movements.

Evaluation of postmortem serum and cerebrospinal fluid levels of thyroid-stimulating hormone with special regard to fatal hypothermia.

The aim of the present study was to undertake, during routine forensic work, a comprehensive analysis of the serum and cerebrospinal fluid (CSF) levels of thyroid-stimulating hormone (TSH) and to examine hypophyseal TSH immunopositivity in relation to the cause of death, with particular regard to fatal hypothermia. Medicolegal autopsy cases (n=120; within 48 h postmortem; survival time, <24 h), including cases of blunt injury (n=9), sharp instrument injury (n=8), fire fatality (n=18), mechanical asphyxiation (n=10), drowning (n=21), poisoning (n=6), hypothermia (n=10), and acute ischemic heart disease (n=38), were examined. Serum and CSF TSH concentrations were measured using an electrochemiluminescence immunoassay. TSH immunoreactivity in adenohypophysis was quantitatively analyzed. Serum and CSF TSH levels were significantly lower in cases of hypothermia than in the other groups (p<0.05 and p<0.001, respectively). TSH immunopositivity in adenohypophysis was significantly lower in cases of hypothermia, but exhibited a large case-to-case variation for poisoning. These observations suggest that a decrease in serum and CSF TSH levels in hypothermia is related to hypothalamic adenohypophyseal dysfunction.

Morphological and functional alterations in the adenohypophysis in cases of brain death.

In order to examine the function of the adenohypophysis during brain death, levels of adrenocorticotropic hormone (ACTH), growth hormone (GH), and thyroid stimulating hormone (TSH) were investigated during forensic autopsy. Cases examined were those of brain death (n=12; within 24h postmortem; time to cardiac death after cerebral death was diagnosed, approximately 4-25 days), including those in which the cause of death was head injury (subdural hematoma or brain contusion, n=10) and asphyxia (strangulation, n=2). The concentrations of ACTH and TSH were measured by enzyme chemiluminescent immunoassay (ECLIA), and that of GH by radioimmunoassay (RIA). The immunoreactivities of ACTH, GH, and TSH in the adenohypophysis were observed and analyzed with electron microscopy. Morphological studies revealed partial necrosis of the central anterior lobe, but preservation of its periphery. Immunohistochemical staining revealed the appearance of peripheral adenohypophysis with each hormone. Ultrastructural findings for the pituitary and hypothalamus indicated swelling of the mitochondria and dilation of both the smooth endoplasmic reticulum and Golgi apparatus. Furthermore, in most cases, concentrations of the anterior pituitary hormones in the serum and cerebrospinal fluid (CSF) were generally within the clinical reference range. These results indicate that the pituitary is partially preserved after brain death.

Immunohistochemical distribution of chromogranin A in medicolegal autopsy materials.

Chromogranin A (CgA) was recently reported as a marker of various stress responses. The aim of this study was to investigate the immunohistochemical distribution of CgA in human tissues in medicolegal autopsy cases as a basis for postmortem investigation of stress responses. The autopsy cases (n=30, within 48 h postmortem) comprised cases of mechanical asphyxia (n=15: strangulation, n=8; hanging, n=7) and acute myocardial infarction/ischemia (AMI, n=15). Routinely formalin-fixed paraffin-embedded tissue sections, including those of the hypothalamus, pituitary gland, cardiac muscle, lungs, liver, kidneys, spleen, skeletal muscle, skin, thyroid gland, submandibular gland, pancreas, and adrenal gland, were stained with polyclonal anti-human CgA antibodies and CgA positivity was quantitatively examined. Localization of CgA immunopositivity was clearly demonstrated in specific cell components in all tissue sections. CgA was mainly observed in the anterior lobe of the pituitary, adrenal medulla, neurons and some gliocytes in the hypothalamus, submandibular gland, follicular epithelial cells and connective tissue in the thyroid gland and pancreatic islet cells. CgA immunopositivity showed no significant difference between mechanical asphyxia and AMI cases. Positivity was slightly higher in adenohypophysis, adrenal medullar, and pancreatic islet cells (approximately 50-80%) than in the thyroid and submandibular glands (approximately 30-60%); however, a large case difference was observed in hypothalamic CgA immunopositivity (0-100%). These findings suggest that hypothalamic CgA immunopositivity can be used as a marker for investigating individual differences in stress responses during the death process. Further investigation of other causes of death is needed.

Evaluation of postmortem S100B levels in the cerebrospinal fluid with regard to the cause of death in medicolegal autopsy.

Previous studies have suggested the usefulness of the postmortem serum S100B level as a marker of the severity of brain damage. In this study, we investigated the S100B level in the cerebrospinal fluid (CSF) in serial autopsy cases (n=216, within 3 days postmortem), including those of blunt injury (n=34: fatal head injury, n=20; others, n=14), sharp instrument injury (n=9), mechanical asphyxiation (n=19), drowning (n=11), fire fatality (n=26), intoxication (n=20), hypothermia (cold exposure, n=16), hyperthermia (heat stroke, n=9), acute cardiac death (n=52) and pneumonia (n=20). The CSF S100B level showed a moderate postmortem time-dependent increase for acute cardiac death (r=0.58, p<0.0001) and asphyxia (r=0.741, p<0.001). In cases of survival time within 48 h, drowning and hypothermia usually showed a lower CSF S100B level (around 500 ng/ml), and the level was higher for delayed head injury death, asphyxia, intoxication, and hyperthermia (around 1500 ng/ml) (p<0.05). In fatal head injury cases, however, CSF S100B did not correlate with the survival time or postmortem interval. A CSF S100B level of >2000 ng/ml in the early postmortem period might be considered a biochemical sign of fatally severe brain damage.

Potential risk factors for sudden cardiac death: an analysis of medicolegal autopsy cases.

Chronic kidney disease and elevated serum C-reactive protein (CRP) have been suggested as clinical risk factors for cardiac attacks. The present study investigated postmortem blood urea nitrogen (BUN), creatinine (Cr) and CRP levels in the peripheral blood of sudden cardiac death cases. Adult autopsy cases of ischemic heart diseases (n=153, >20 years of age), including acute myocardial infarction (AMI, n=71), recurrent myocardial infarction (RMI, n=47), acute ischemic heart disease without infarction (AIHD, n=27) and chronic ischemic heart disease (CIHD, n=8), were examined and compared with chronic congestive heart disease (CHD, n=24), spontaneous cerebral hemorrhage (SCH, n=17) and mechanical asphyxiation (n=32). BUN was slightly higher for RMI and CHD, although Cr was slightly higher for SCH. CRP was higher for AMI than for AIHD. The correlation between BUN and Cr levels was significant for AMI, AIHD and CHD, but insignificant for RMI and CIHD. Heart weight was larger for all heart diseases and SCH than for asphyxiation, and was larger for RMI and CHD but lower for AIHD and CIHD among them. Body mass index (BMI) was slightly higher for AMI, RMI, AIHD and CHD, remaining within the reference interval in most cases, but was lower for CIHD. These findings suggest different risk factors or etiologies, including active atherosclerosis, latent renal failure, dehydration and cardiac hypertrophy, for sudden deaths due to these heart diseases.

Postmortem serum levels of pulmonary surfactant-associated proteins A and D with regard to the cause of death in medicolegal autopsy.

Pulmonary surfactant-associated proteins A and D (SP-A and -D) are tissue-specific components. Previous studies showed an increase in the postmortem serum SP-A level due to acute pulmonary alveolar damage and acute respiratory distress. The present study comparatively investigated serum SP-A and SP-D levels with regard to the cause of death in serial medicolegal autopsy cases (n=679, within 48 h postmortem). SP-A and SP-D levels were usually higher in left cardiac blood than at other sites, independent of postmortem interval. The left-to-right difference was significantly larger for mechanical asphyxiation, drowning, intoxication and spontaneous cerebral hemorrhage. Both SP-A and -D levels in bilateral cardiac blood were significantly higher for drowning and secondary pulmonary damage involving ARDS after traumas, but were lower for hypothermia (cold exposure). SP-A was predominantly elevated in fire fatality and delayed deaths from injury and fires, while pneumonia showed a predominant elevation of SP-D. These findings suggest that comparative analysis of serum SP-D and SP-A is useful for investigating primary or secondary pulmonary alveolar damage in the death process.

Evaluation of postmortem calcium and magnesium levels in the pericardial fluid with regard to the cause of death in medicolegal autopsy.

Previous studies have suggested the usefulness of postmortem serum calcium (Ca) and magnesium (Mg) for investigating cause of death. The present study investigated their levels in the pericardial fluid of serial autopsy cases of adults within 48 h postmortem (n=385), including fatalities from blunt injury (n=57), sharp instrument injury (n=9), mechanical asphyxiation (n=28), salt- and freshwater drowning (n=14 and n=61, respectively), fire fatality (n=35), intoxication (n=23), hypothermia (cold exposure, n=12), hyperthermia (heat stroke, n=7), acute cardiac death (ACD, n=86), pneumonia (n=9) and spontaneous cerebral hemorrhage (n=11). The pericardial Ca level was independent of the postmortem interval, showing a value similar to that of the clinical reference range in cases other than saltwater drowning, while the Mg level was higher than the clinical reference range and showed a mild postmortem time-dependent increase. Pericardial Ca was significantly higher for saltwater drowning than other groups, and a lower level was seen for hyperthermia, and some cases of blunt injury and intoxication. The Mg level was also significantly higher for saltwater drowning than the other groups, and showed a higher level for sharp instrument injury, but a lower level for hypothermia. The Mg/Ca ratio was higher for sharp instrument injury and saltwater drowning, but was lower for hypothermia. These findings suggest that postmortem pericardial Ca and Mg can be used to investigate the cause of death, especially for saltwater drowning, hypothermia and hyperthermia.

Analyses of cardiac blood cells and serum proteins with regard to cause of death in forensic autopsy cases.

To investigate hematological and serum protein profiles of cadaveric heart blood with regard to the cause of death, serial forensic autopsy cases (n=308, >18 years of age, within 48 h postmortem) were examined. Red blood cells (Rbc), hemoglobin (Hb), platelets (Plt), white blood cells (Wbc), total protein (TP) and albumin (Alb) were examined in bilateral cardiac blood. Blood cell counts, collected after turning the bodies at autopsy, approximated to the clinical values. Postmortem changes were not significant for these markers. In non-head blunt injury cases, Rbc counts, Hb, TP and Alb levels in bilateral cardiac blood were lower in subacute deaths (survival time, 1-12 h) than in acute deaths (survival time <1 h). Wbc counts of left cardiac blood were significantly higher for non-head injury than for head injury in subacute deaths. In fire fatality cases, Plt count was markedly higher with an automated hematology analyzer than by using a blood smear test, suggesting Rbc fragmentation caused by deep burns, while increases in Wbc count and decreases in Alb levels were seen for subacute deaths. For asphyxiation, Rbc count, Hb, TP and Alb levels in bilateral cardiac blood were higher than other groups, and TP and Alb levels in the right cardiac blood were higher for hanging than for strangulation. These findings suggest that analyses of blood cells and proteins are useful for investigating the cause of death.

Analysis of postmortem biochemical findings with regard to the lung weight in drowning.

To clarify drowning death, positive evidence for aspiration of the immersion medium and the subsequent fatal mechanism is necessary. This study investigated biochemical findings with regard to lung weight in drowning cases of adults (n=56, >18 years of age, <48 h postmortem: salt water, n=19; fresh water, n=21; brackish water, n=16), using acute cardiac death cases (n=240) as controls. The biochemical markers used in this study were urea nitrogen (UN), sodium (Na), chloride (Cl), calcium (Ca) and magnesium (Mg) in the blood and pericardial fluid (PCF). The left-to-right ratio of cardiac blood UN levels was lower for drowning, showing an inverse correlation to the total lung weight. There was a mild postmortem decrease in serum and PCF Na and Cl levels; however, left cardiac serum and PCF Na, Cl, Ca and Mg levels were higher for saltwater drowning, and left cardiac serum Na and Cl levels were lower for fresh water drowning. Correlation of the left cardiac serum level with lung weight was positive for Na, Cl and Mg in saltwater and brackish water drowning, and was also positive for Ca in saltwater drowning. There was an inverse correlation with lung weight for PCF Na and Cl levels in freshwater drowning. These findings suggest that analyses of serum and pericardial markers in relation to lung weight are useful for evaluating the composition and amount of aspirated medium when investigating drowning death.

Loss of yata, a novel gene regulating the subcellular localization of APPL, induces deterioration of neural tissues and lifespan shortening.

BACKGROUND: The subcellular localization of membrane and secreted proteins is finely and dynamically regulated through intracellular vesicular trafficking for permitting various biological processes. Drosophila Amyloid precursor protein like (APPL) and Hikaru genki (HIG) are examples of proteins that show differential subcellular localization among several developmental stages. METHODOLOGY/PRINCIPAL FINDINGS: During the study of the localization mechanisms of APPL and HIG, we isolated a novel mutant of the gene, CG1973, which we named yata. This molecule interacted genetically with Appl and is structurally similar to mouse NTKL/SCYL1, whose mutation was reported to cause neurodegeneration. yata null mutants showed phenotypes that included developmental abnormalities, progressive eye vacuolization, brain volume reduction, and lifespan shortening. Exogenous expression of Appl or hig in neurons partially rescued the mutant phenotypes of yata. Conversely, the phenotypes were exacerbated in double null mutants for yata and Appl. We also examined the subcellular localization of endogenous APPL and exogenously pulse-induced APPL tagged with FLAG by immunostaining the pupal brain and larval motor neurons in yata mutants. Our data revealed that yata mutants showed impaired subcellular localization of APPL. Finally, yata mutant pupal brains occasionally showed aberrant accumulation of Sec23p, a component of the COPII coat of secretory vesicles traveling from the endoplasmic reticulum (ER) to the Golgi. CONCLUSION/SIGNIFICANCE: We identified a novel gene, yata, which is essential for the normal development and survival of tissues. Loss of yata resulted in the progressive deterioration of the nervous system and premature lethality. Our genetic data showed a functional relationship between yata and Appl. As a candidate mechanism of the abnormalities, we found that yata regulates the subcellular localization of APPL and possibly other proteins.

Single-stranded DNA as an immunohistochemical marker of neuronal damage in human brain: an analysis of autopsy material with regard to the cause of death.

Single-stranded DNA (ssDNA) is a marker of apoptosis and programmed cell death, which appears prior to DNA fragmentation during delayed neuronal death. The present study investigated the immunohistochemical distribution of ssDNA in the brain to investigate apoptotic neuronal damage with regard to the cause of death in medicolegal autopsy cases (n=305). Neuronal immunopositivity for ssDNA was globally detected in the brain, independent of the age, gender of subjects and postmortem interval, and depended on the cause of death. Higher positivity was typically found in the pallidum for delayed brain injury death and fatal carbon monoxide intoxication, and in the cerebral cortex, pallidum and substantia nigra for drug intoxication. For mechanical asphyxiation, a high positivity was detected in the cerebral cortex and pallidum, while the positivity was low in the substantia nigra. The neuronal ssDNA increased during the survival period within about 24h at each site, depending on the type of brain injury, and in the substantia nigra for other blunt injuries. The neuronal positivity was usually lower for drowning and acute ischemic disease. Topographical analysis of ssDNA-positive neurons may contribute to investigating the cause of brain damage and survival period after a fatal insult.

[Immunohistochemical distribution of single-stranded DNA in the brain in medico-legal autopsy cases of carbon monoxide intoxication].

To evaluate apoptotic neuronal damage by carbon monoxide (CO) in medico-legal autopsy cases, we investigated the immunohistochemical distribution of single-stranded DNA (ssDNA) as a marker of apoptosis and programmed cell death in the brain. Formalin-fixed paraffin-embedded brain tissue specimens, including cerebral cortex of frontal lobe, substantia nigra of the midbrain and pallidum, from medico-legal autopsy cases of fire fatality (n=63), including cases with blood carboxyhemoglobin (COHb) of a lower (<60%) and a higher (>60%) level (n = 39 and 24, respectively), and CO intoxication without burns (n = 6) were examined, in comparison with acute ischemic heart disease (IHD, n = 29) and asphyxiation due to strangulation (AS, n= 14). In the pallidum, neuronal immunopositivity for ssDNA was significantly higher in fire fatality with a higher COHb level than in IHD (p<0.0001), and CO intoxication cases showed significantly higher positivity than other groups excluding fire fatality with a higher COHb level (p< 0.05). In cases without cardiopulmonary resuscitation, ssDNA-positivity in the pallidum mildly correlated to COHb concentrations (r = 0.31, p<0.05), and the positivity was significantly higher in higher COHb (>60%) cases than in lower COHb (<30%) cases. In the cerebral cortex and substantia nigra of the midbrain, neuronal ssDNA-positivity showed no significant findings with regard to the cause of death and COHb concentration. These findings suggest that CO causes selective neuronal damage in the pallidum.

Postmortem serum nitrogen compounds and C-reactive protein levels with special regard to investigation of fatal hyperthermia.

The present study analyzed serum levels of urea nitrogen (UN), creatinine (Cr), and C-reactive protein (CRP), which are very stable during the early postmortem period, for investigation of the cause of death with special regard to hyperthermia (heat stroke) in serial medico-legal autopsy cases (n = 429), excluding fatal injury, intoxication, and fire fatality. In this series, mechanical asphyxiation, drowning, and sudden cardiac death cases (n = 56, n = 43, and n = 212, respectively) usually showed low levels within postmortem reference ranges for these serum markers, although UN and CRP levels were mildly elevated in cases of sudden cardiac death and cerebrovascular stroke. There were concomitant significant elevations in serum levels of UN (>50 mg/dL), Cr (>2 mg/dL), and CRP (>2 mg/dL) for chronic renal failure, gastrointestinal bleeding, pneumonia, and hypothermia (cold exposure). UN and CRP were especially high for chronic renal failure and pneumonia, respectively. However, hyperthermia cases showed an isolated elevation in the serum Cr level, suggesting an influence of systemic skeletal muscle damage. These serum markers may be practically useful for postmortem investigation of death due to hyperthermia (heat stroke), for which specific pathological and toxicological evidence may not be available.

Autoinduction of tumor necrosis factor-alpha in FRTL-5 rat thyroid cells.

Tumor necrosis factor-alpha (TNFalpha) may play a role in the development of autoimmune thyroiditis such as Hashimoto's thyroiditis. In the present study, we examined whether TNFalpha induced its own expression in FRTL-5 rat thyroid cells. Lipopolysaccharide (LPS) markedly increased TNFalpha mRNA levels in FRTL-5 cells as assessed by semiquantitative RT-PCR. In addition, LPS-stimulated cells released TNFalpha protein into the culture medium. Similarly, TNFalpha induced its own gene and protein expression in FRTL-5 cells as assessed by RT-PCR and metabolic labeling and immunoprecipitation of TNFalpha. The autoinduction of TNFalpha gene was also observed in TNFalpha-stimulated human thyroid epithelial cells. TNFalpha induction was specific to LPS and TNFalpha since interferon-alpha or amiodarone failed to increase TNFalpha mRNA levels in FRTL-5 cells. Human TNFalpha induced rat TNFalpha gene expression, indicating that type 1 TNF receptor (TNF-R) is involved in the autoinduction. TNFalpha did not increase either type 1 or type 2 TNF-R mRNA levels, suggesting that upregulation of TNF receptors is not involved in the autoinduction of TNFalpha. Although the biological significance of autoinduction of TNFalpha remains unclear, our results suggest that thyroid epithelial cells may participate in the development of autoimmune thyroiditis through production of TNFalpha. Furthermore, inhibition of TNFalpha production in the thyroid may represent a novel approach to mitigating inflammation in autoimmune thyroiditis.