Eukaryotic diversity in late Pleistocene marine sediments around a shallow methane hydrate deposit in the Japan Sea.

Marine sediments contain eukaryotic DNA deposited from overlying water columns. However, a large proportion of deposited eukaryotic DNA is aerobically biodegraded in shallow marine sediments. Cold seep sediments are often anaerobic near the sediment-water interface, so eukaryotic DNA in such sediments is expected to be preserved. We investigated deeply buried marine sediments in the Japan Sea, where a methane hydrate deposit is associated with cold seeps. Quantitative PCR analysis revealed the reproducible recovery of eukaryotic DNA in marine sediments at depths up to 31.0 m in the vicinity of the methane hydrate deposit. In contrast, the reproducible recovery of eukaryotic DNA was limited to a shallow depth (8.3 m) in marine sediments not adjacent to the methane hydrate deposit in the same area. Pyrosequencing of an 18S rRNA gene variable region generated 1,276-3,307 reads per sample, which was sufficient to cover the biodiversity based on rarefaction curves. Phylogenetic analysis revealed that most of the eukaryotic DNA originated from radiolarian genera of the class Chaunacanthida, which have SrSO4 skeletons, the sea grass genus Zostera, and the seaweed genus Sargassum. Eukaryotic DNA originating from other planktonic fauna and land plants was also detected. Diatom sequences closely related to Thalassiosira spp., indicative of cold climates, were obtained from sediments deposited during the last glacial period (MIS-2). Plant sequences of the genera Alnus, Micromonas, and Ulmus were found in sediments deposited during the warm interstadial period (MIS-3). These results suggest the long-term persistence of eukaryotic DNA from terrestrial and aquatic sources in marine sediments associated with cold seeps, and that the genetic information from eukaryotic DNA from deeply buried marine sediments associated with cold seeps can be used to reconstruct environments and ecosystems from the past.

Pathologic and immunologic profiles of a limited form of neuromyelitis optica with myelitis.

BACKGROUND: Neuromyelitis optica (NMO) is a demyelinating syndrome characterized by myelitis and optic neuritis. Detection of anti-NMO immunoglobulin G antibody that binds to aquaporin-4 (AQP4) water channels allows the diagnosis of a limited form of NMO in the early stage with myelitis, but not optic neuritis. However, the detailed clinicopathologic features and long-term course of this limited form remain elusive. METHODS: We investigated 8 patients with the limited form of NMO with myelitis in comparison with 9 patients with the definite form. RESULT: All patients with limited and definite form showed uniform relapsing-remitting courses, with no secondary progressive courses. Pathologic findings of biopsy specimens from the limited form were identical to those of autopsy from the definite form, demonstrating extremely active demyelination of plaques, extensive loss of AQP4 immunoreactivity in plaques, and diffuse infiltration by macrophages containing myelin basic proteins with thickened hyalinized blood vessels. Moreover, the definite form at the nadir of relapses displayed significantly higher amounts of the inflammatory cytokines interleukin (IL)-1beta and IL-6 in CSF than the limited form and multiple sclerosis. CONCLUSION: This consistency of pathologic findings and uniformity of courses indicates that aquaporin 4-specific autoantibodies as the initiator of the neuromyelitis optica (NMO) lesion consistently play an important common role in the pathogenicity through the entire course, consisting of both limited and definite forms, and NMO continuously displays homogeneity of pathogenic effector immune mechanisms through terminal stages, whereas multiple sclerosis should be recognized as the heterogeneous 2-stage disease that could switch from inflammatory to degenerative phase. This report is a significant description comparing the pathologic and immunologic data of limited NMO with those of definite NMO.

Chronic myositis with cardiomyopathy and respiratory failure associated with mild form of organ-specific autoimmune diseases.

We report the four patients with chronic myositis characterized by a very slow progression with cardiomyopathy and frequently with marked respiratory muscle weakness associated with other organ-specific autoimmune diseases such as primary biliary cirrhosis. The histopathology of the muscle showed many degenerative and regenerative fibers, but inflammatory-cell infiltration were minimal. The patients showed favorable response to high-dose corticosteroid treatment. Because of these clinical features, these patients are sometimes misdiagnosed as muscular dystrophy and not treated properly. It is important to distinguish this type of treatable myositis.

Progress in pursuit of therapeutic A2A antagonists: the adenosine A2A receptor selective antagonist KW6002: research and development toward a novel nondopaminergic therapy for Parkinson's disease.

Research and development of the adenosine A2A receptor selective antagonist KW6002 have focused on developing a novel nondopaminergic therapy for Parkinson's disease (PD). Salient pharmacologic features of KW6002 were investigated in several animal models of PD. In rodent and primate models, KW6002 provides symptomatic relief from parkinsonian motor deficits without provoking dyskinesia or exacerbating existing dyskinesias. The major target neurons of the A2A receptor antagonist were identified as GABAergic striatopallidal medium spiny neurons. A possible mechanism of A2A receptor antagonist action in PD has been proposed based on the involvement of striatal and pallidal presynaptic A2A receptors in the "dual" modulation of GABAergic synaptic transmission. Experiments with dopamine D2 receptor knockout mice showed that A2A receptors can function and anti-PD activities of A2A antagonists can occur independent of the dopaminergic system. Clinical studies of KW6002 in patients with advanced PD with L-dopa-related motor complications yielded promising results with regard to motor symptom relief without motor side effects. The development of KW6002 represents the first time that a concept gleaned from A2A biologic research has been applied successfully to "proof of concept" clinical studies. The selective A2A antagonist should provide a novel nondopaminergic approach to PD therapy.

Changes in antioxidative mechanisms in elderly patients with non-insulin-dependent diabetes mellitus. Investigation of the redox dynamics of alpha-tocopherol in erythrocyte membranes.

BACKGROUND: Recently, it has been suggested that the onset and aggravation of diabetes are closely related to free radicals. Also, vitamin E is a lipophilic free radical scavenger that is localized mainly in biomembranes. OBJECTIVE: The purpose of this study was to clarify the defensive mechanisms against oxidative stress by investigating the differences in the redox dynamics of alpha-tocopherol in plasma and erythrocyte membranes between elderly patients with non-insulin-dependent diabetes mellitus (NIDDM) and healthy elderly subjects. METHODS: Total, alpha-, beta-, gamma- and delta-tocopherol and alpha-tocopherolquinone concentrations were determined by high-performance liquid chromatography with a redox detection mode using a series of four coulometric working electrodes. RESULTS: The alpha-tocopherolquinone/alpha-tocopherol ratio in plasma and erythrocyte membranes was not different between the two groups. Both the alpha-tocopherol concentrations in erythrocyte membranes and ratio of alpha-tocopherol in erythrocyte membranes to alpha- tocopherol in plasma was significantly lower in elderly NIDDM patients than in healthy subjects. CONCLUSION: These findings suggest that alpha-tocopherol is used normally in both plasma and erythrocyte membranes and alpha-tocopherol uptake in erythrocyte membranes is significantly decreased in elderly NIDDM patients. The functional disorder of the antioxidative activity of alpha-tocopherol in erythrocyte membranes due to impairment of this transfer mechanism may be associated with the pathogenesis of NIDDM.

Caspase-1 activity as a possible predictor of apoptosis induced by cisplatin in gastric cancer cells.

Recent studies have shown that caspases, which are cystein proteases, elevate endonuclease activity and induce apoptosis. Caspase-1, an interleukin-1beta converting enzyme, has been reported to be related with anti-cancer drug induced apoptosis as well as with caspase-3. To elucidate the caspase-1 activity, which might be a predictor for the effect of chemotherapy, we examined the changes of caspase-1 activity induced after exposure to cisplatin (CDDP) in six gastric cancer cell lines. A high correlation between the 50% inhibitory concentration (IC50) and caspase-1 activity ratio was shown (r=0.83, p=0.041) (caspase-1 activity ratio: the caspase-1 activity of cells at 4 h after CDDP treatment/the caspase-1 activity of untreated cells). Further, we examined the correlation between caspase-1 activity and apoptosis induced by CDDP in two cell lines that have very different CDDP sensitivities; OCUM-2M and OCUM-2M/DDP (IC50; 0. 85+/-0.4 microg/ml and 9.0+/-1.2 microg/ml, respectively). The apoptotic index of OCUM-2M was significantly higher than that of OCUM-2M/DDP (19.8+/-3.8% vs. 4.5+/-1.2%, respectively; p=0.0005). In both cell lines, caspase-1 activity began to increase immediately after exposure to CDDP and peaked at approximately 4 h after cessation of exposure to CDDP, and gradually decreased thereafter. The caspase-1 activity of OCUM-2M was approximately 1.8-times higher than that of OCUM-2M/DDP at 4 h after exposure to CDDP. Taken together, our results indicate that evaluating the changes of caspase-1 activity after exposure to CDDP may be useful to predict apoptosis following CDDP treatment in gastric cancer cells.

Mechanical tensile properties of the aortic wall in the premature rat exposed to the microgravity environment during space flight for 16 days.

Under microgravity environment, blood shifts headward and thereafter decrease in volume to adapt to the environment, which could affect cardiovascular hemodynamics and their regulatory mechanisms. Baroreceptor sensitivity is known to be reduced in newborn animals and to gradually increase with development. The baroreceptor is a stretch receptor; therefore its function is closely related to the rheological properties and fine structure of the aortic wall in which the baroreceptor lies. The mechanical and histological properties could be altered under microgravity conditions in the process of development with change in circulatory function. In the present study, we investigated the mechanical tensile characteristics and histological structure of the aortic wall in the proximal thoracic aorta of premature rats bred in the microgravity environment of the space shuttle for 16 days.

Age-related changes in alpha-tocopherol dynamics with relation to lipid hydroperoxide content and fluidity of rat erythrocyte membrane.

Age-related changes in alpha-tocopherol dynamics in plasma and erythrocyte membranes of 10- to 120-week-old rats were investigated by high-performance liquid chromatography (HPLC) with redox detection mode. Furthermore, changes in lipid hydroperoxide content and fluidity of erythrocyte membrane with age were assessed using chemiluminescence-HPLC and Fourier transform infrared spectrophotometer, respectively. A slight increase in the alpha-tocopherolquinone/alpha-tocopherol ratio in erythrocyte membrane and a decrease in the alpha-tocopherol in erythrocyte membrane/alpha-tocopherol in plasma ratio were observed. A significant increase in lipid hydroperoxide content and a marked decrease in the fluidity of erythrocyte membrane were seen with age. These findings suggest that alpha-tocopherol uptake in erythrocyte membrane declines, and utilization rate of alpha-tocopherol in erythrocyte membrane increases age-dependently. These changes, which enhanced lipid peroxidation and consequently reduced membrane fluidity, may be caused by the impairment of this transfer mechanism.

[Age-related change in the alpha-tocopherolquinone/alpha-tocopherol ratio in the rat erythrocyte membrane].

alpha-Tocopherol (alpha-Toc), a lipophilic phenolic antioxidant that is localized mainly in the biomembrane, protects cells against oxidation-associated cytotoxicity by prevention of membrane lipid peroxidation, maintenance of the redox balance intracellular thiols and stabilization of the membrane structure. We investigated the age-related changes in redox dynamics of alpha-Toc in plasma and erythrocyte membrane of an elderly (66 weeks old) and young group (10 weeks old). Total, alpha-, beta + gamma-, delta-Toc and alpha-tocopherolquinone (alpha-TocQ) in plasma and erythrocyte membrane were determined by high-performance liquid chromatography (HPLC) with a series of multiple coulometric working electrodes (CWE). Rat venous blood sample was divided into plasma and erythrocyte layers by centrifugation, and then erythrocyte membrane sample was prepared according to the method of Dodge et al. under a stream of nitrogen. In plasma, total and alpha-Toc concentrations were increased, and beta + gamma-, delta-Toc and alpha-TocQ concentrations were decreased age-dependently. In the erythrocyte membrane, total, alpha-TocQ concentrations and three fractions of tocopherols decreased age-dependently. Also, a decrease in the alpha-TocQ/alpha-Toc ratio in erythrocyte membrane was observed in the elderly group. These findings suggest that the alpha-Toc uptake in erythrocyte membrane and utilization rate of alpha-Toc in erythrocyte membrane decline age-dependently. This decline may promote membrane lipid peroxidation. alpha-Toc redox dynamics in erythrocyte membrane were useful to investigate the pathophysiology of aging mechanisms related to oxidative stress.

Redox dynamics of alpha-tocopherol in erythrocyte membranes of elderly patients with asymptomatic primary hyperlipidemia.

We investigated the redox dynamics of alpha-tocopherol in plasma and erythrocyte membranes in elderly patients with asymptomatic primary hyperlipidemia divided into three groups (hypercholesterolemia, hypertriglyceridemia and low high-density lipoprotein cholesterol levels) and in healthy elderly subjects to assess the antioxidative status of alpha-tocopherol. alpha-Tocopherol and alpha-tocopherolquinone were determined by high-performance liquid chromatography using a redox detection mode. In the erythrocyte membrane, there was no difference in the alpha-tocopherol concentration between hyperlipidemic and healthy subjects. The alpha-tocopherolquinone/alpha-tocopherol ratio in plasma and erythrocyte membrane, and the alpha-tocopherol in erythrocyte membrane/alpha-tocopherol in plasma ratio were significantly lower in elderly patients with hypercholesterolemia or hypertriglyceridemia. These findings suggest that the uptake ratio in erythrocyte membranes and the antioxidative activity of alpha-tocopherol in both plasma and erythrocyte membranes are decreased in elderly hyperlipidemic patients. These decreases may promote membrane lipid peroxidation or accelerate atherosclerosis.

Inhibition in a microgravity environment of the recovery of Escherichia coli cells damaged by heavy ion beams during the NASDA ISS phase I program of NASA Shuttle/Mir mission no. 6.

We participated in a space experiment, part of the National Space Development Agency of Japan (NASDA) Phase I Space Radiation Environment Measurement Program, conducted during the National Aeronautics and Space Administration (NASA) Shuttle/Mir Mission No. 6 (S/MM-6) project. The aim of our study was to investigate the effects of microgravity on the DNA repair processes of living organisms in the in orbit. Heavy ion beam radiation- or ç-irradiation-damaged biological samples of Escherichia coli and the radioresistant bacterium Deinococcus radiodurans were prepared and placed in a biospecimen box, which was loaded into the RRMD III sensor unit of the Space Shuttle. Two identical sets of samples were left in the Spacehab's Payload Processing Facility (SPPF) in Florida, USA, as a control. (flight No. STS-84) was launched from NASA John F. Kennedy Space Center (KSC) in Florida, USA, on May 15, 1997. The mission duration was 9.22 days. An astronaut activated the biological samples in the biospecimen box in the Spacehab during orbit in order to start repair of the DNA damaged by heavy ion beams or ç-irradiation and the samples were incubated for 19 h 35 min at about 22ûC, the cabin temperature. The control specimens in the SPPF were subjected to the same treatment under terrestrial gravity. After returned to earth, we investigated cell recovery by comparing the repair of the radiation-damaged DNA of E. coli and D. radiodurans in the microgravity environment in space with that on Earth. The results indicated that the DNA repair process of E. coli, but not of D. radiodurans, cells was inhibited in a microgravity environment.

p53 protein overexpression as a predictor of the response to chemotherapy in gastric cancer.

This study was performed to evaluate p53 overexpression as a predictor of the response to chemotherapy of patients with gastric cancer. The subjects comprised 20 patients with Stage IV gastric cancer and three with locally recurrent lesions, all of whom were treated with 5-fluorouracil (5-FU) plus cisplatin (CDDP) for 4 weeks. Of the total 23 patients there were 10 responders; 2 showing complete response (CR) and 8, partial response (PR). Specimens obtained by endoscopic biopsy were immunohistochemically stained using anti-p53 protein and bcl-2 protein antibody. Of the 10 responders, 7 demonstrated negative p53 staining, and of the 13 nonresponders, 11 demonstrated positive p53 staining (P = 0.013). Tissue from 3 of the responders and 7 of the nonresponders that stained for bcl-2 were positive prior to chemotherapy; however, there was no association between bcl-2 staining and chemotherapeutic effect. In conclusion, immunohistochemical identification of p53 in pretreatment tissue may represent a useful predictor for chemotherapeutic outcome in patients with gastric cancer.

The antiproliferative effect of HGF on hepatoma cells involves induction of apoptosis with increase in intracellular polyamine concentration levels.

Hepatocyte growth factor (HGF) induced apoptosis and decreased the DNA synthesis in Hep G2 cells. In the HGF group interleukin-1 converting enzyme, ornithine decarboxylase (ODC) activity and intracellular polyamine concentrations were increased compared to those of the control group. Administration of the ODC inhibitor decreased polyamine concentration, and inhibited apoptotic changes in the cells. These changes were reversed by exogenous addition of polyamine. These findings suggest that one of the mechanisms by which HGF exerts its antiproliferative effect is induction of apoptosis and that increase in intracellular polyamine concentration may be one of the triggers of cell death.

Assessment of middle-distance running performance in sub-elite young runners using energy cost of running.

The purpose of this study was to assess the validity of v(amax) as an indicator of middle-distance running performance in sub-elite young runners, v(amax) being defined as the quotient maximal oxygen uptake (VO2max) divided by the net energy cost of running (Cr) on a treadmill at a submaximal running velocity (280 m x min[-1]). The VO2max, ventilatory threshold, v(amax), and Cr were assessed in 39 young male sub-elite runners having only small variations in performance level. The relationship between each variable and running performance (at 1500 m, 3000 m, and 5000 m) was evaluated. A trend toward a negative correlation existed between Cr and performance although this was not significant. The VO2max and v(amax) were significantly related to performance. The v(amax) accounted for around 50% of the variability in performance whereas other physiological variables selected in this study were responsible, at best, for approximately 39%. The results presented in this study suggested that v(amax) was a useful indicator of middle-distance running performance in sub-elite young runners with similar performance levels as well as in top elite athletes.

Histological analysis of the aortic nerve in the rat raised in space (Rapid communication on Neurolab Project).

To study development of the aortic nerve baroreflex under conditions of microgravity, we examined the cross section of the left aortic nerve (LAN), which is the afferent of the baroreflex, in the neonate rats aged 25 days raised in microgravity on the space shuttle Columbia (flight:FLT group) for 16 days. In this paper, we report a part of the result obtained from the data of the myelinated fibers of LAN analyzed with an electron microscope. Two kind of ground control groups were compared to the FLT group; one was asynchronous ground control (AGC) group where the rats were housed in the same cage as that on the shuttle, and the other was vivarium(VIV) group where the rats were housed in a commercial cage. The LANs in each group were extirpated the from rats perfused with a fixative and embedded for histological analysis. We observed the transverse sections of LAN and took pictures of several areas (magnified to x 2K to x 200K). No irregular myelination was found in all fibers of FLT group when they were compared with two control groups. The thickness of myelin of the maximally myelinated fibers were 0.55 +/- 0.17 micrometer in FLT(n=5), 0.45 +/- 0.10 micrometer in AGC(n=5), and O.47 +/- 0.06 micrometer meter in VIV(n=5). There was no significant difference among three groups (unpared t-test). The results suggest that there is no effect of space environment on the myelin formation of each nerve fiber in the aortic nerve.

[Causes, diagnosis, and treatment of anemia in the elderly].

Healthy elderly people are mildly anemic peripheral blood data on 3,583 healthy elderly people (1,590 men and 1,993 women aged 65 years or older) from among those undergoing medical examinations at our hospital in the 8 years from 1988 to 1995 were compiled into 5-year age groups. For both men and women the mean values of red blood cell count, hemoglobin, and hematocrit were slightly lower among older subjects. The main causes of this apparent reduction may be a decrease in the number of hematopoietic stem cells and regression of the hematopoietic microenvironment. Observation of arteries in specimens of hematopoietic bone marrow obtained from the spines of elderly people showed arteriosclerotic changes such as greater hypertrophy of the media than of the intima, and adventitial fibrous hypertrophy. The number of venous sinuses was low and the amount of adipose tissue was high compared to the bone marrow of younger people. The cell density and the ratio of hematopoietic tissue to fat tended to be lower in older subjects. The number of erythroid burst-forming units formed after 14 days in culture medium containing erythropoietin was 28 +/- 19 in 32 healthy elderly people, which was significantly lower than the number in 30 young people 54 +/- 30, (p < 0.005). The value for erythroid colony-forming units was 170 +/- 67 in eight healthy people, which was much lower than in young people, 276 +/- 54. In the elderly subjects, the plasma iron disappearance time (PIDT/2) was 60-80 min (mean: 71.9 min), which was similar to that in the young, but the percent red cell iron utilization was 67.6%-84.9% (mean: 79.7%), which was slightly lower than in younger people. When the diagnostic criterion for anemia in the elderly was set at a hemoglobin value of 11.0 g/ dl, about 13% of outpatients who came to our Geriatrics department were found to have anemia, and in most of them the anemia had resulted from another disease. In conclusion, anemia in the elderly is likely to be affected by reduction in the function of various organs and by the decreased reserves associated with aging. The causes of anemia are complex and diagnosis is often difficult. The present article gives a general outline of the diagnosis and treatment of common types of primary and secondary anemia in the elderly.

Synthesis and evaluation of 5-HT3 receptor antagonist [11C]KF17643.

For imaging CNS 5-HT3 receptors by PET, a high affinity 5-HT3 receptor ligand, endo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl 2-(n-propyloxy)-4-quinolinecarboxylate (KF17643), have been labeled with 11C. N-Methylation of the desmethyl compound with [11C]methyl iodide followed by HPLC separation produced [11C]KF17643 with the decay-corrected radiochemical yield of 19-28%, the specific activity of 7.5-49 GBq/mumol and the radiochemical purity of > 99% at 35-40 min from EOB. After i.v. injection of [11C]KF17643 into mice, it was taken by the brain at a high level and was stable for metabolism, but no sign for the 5-HT3 receptor selectivity was found in the brain tissues by the tissue sampling and autoradiography, probably because of large non-specific binding. The [11C]KF17643 was not suitable as a PET ligand for mapping the CNS 5-HT3 receptors.