Relation Between Asymmetry of Prefrontal Activity and Autonomic Nervous System in Post-stroke Patients with a Disorder of Consciousness.

Early mobilization and rehabilitation of stroke patients can improve functional recovery. However, effects of mobilization on the responses of the autonomic nervous system (ANS) and emotions in post-stroke patients are not yet clear, particularly in patients with disorders of consciousness. We evaluated the effects of standing load on the ANS and emotions of post-stroke patients (n = 8, 70.8 ± 11.8 years) with a disorder of consciousness by measuring the prefrontal cortex (PFC) activity, which plays important roles in regulation of the ANS and emotions. Employing near infrared spectroscopy (NIRS), we measured concentration changes of oxygenated haemoglobin (oxy-Hb) in the PFC for 10 min at rest. Then, subjects were placed in supine position on a tilt table, and standing loads were given by 30°, 45° and 60° every 5 min. We assessed left-right asymmetry of the PFC activity at rest by calculating the Laterality Index at Rest (LIR; LIR>0 indicates right dominant activity while LIR<0 indicates left dominant activity). We observed fluctuations of oxy-Hb in the PFC at rest, indicating activation of the PFC. Interestingly, 75% (six patients) of the patients exhibited right dominant PFC activation at rest. These patients exhibited an increase of oxy-Hb during standing load; four patients suffered from orthostatic hypotension. Finally, there was a significant positive correlation between LIR before the standing test and oxy-Hb changes during the standing test (r = 0.78, p = 0.02). These results suggest that the standing load might act on post-stroke patients as a stressor, which caused an increase of PFC activity (i.e., rCBF). NIRS may be useful to assess feasibility to start rehabilitation of post-stroke patients with a disorder of consciousness.

Effects of Motor Imagery on Cognitive Function and Prefrontal Cortex Activity in Normal Adults Evaluated by NIRS.

Recent near-infrared spectroscopy (NIRS) studies demonstrated that physical exercise enhances working memory (WM) performance and prefrontal cortex (PFC) activity during WM tasks in normal adults. Interestingly, the effects of rehabilitation (i.e. physiotherapy) on post-stroke patients could be enhanced by motor imagery (MI), an active process during which the specified action is reproduced within WM without any actual physical movement. However, it is not known whether MI can enhance cognitive function and associated brain activity. To clarify these issues, we evaluated the effect of MI on WM performance and PFC activity during WM tasks in normal adults, employing NIRS. We studied 10 healthy adults. The present study was a crossover comparison test; the MI training and control condition (rest) were applied to the subjects at random. The Time Up and Go method was used for MI training: the subject sat on a chair and conducted MI for 3 min, three times. Neuronal activity (oxyhemoglobin concentration) in the bilateral PFC was measured using 2-CH NIRS during WM tasks. We found that MI improved the behavioral performance of WM compared with the control (p < 0.01). NIRS revealed that MI enhanced PFC activity induced by the WM task compared with the control task (p < 0.01). These results suggest that MI can improve cognitive function and increase associated PFC activity in normal adults.

Effects of Physical Exercise on Working Memory and Prefrontal Cortex Function in Post-Stroke Patients.

Physical exercise enhances prefrontal cortex activity and improves working memory performance in healthy older adults, but it is not clear whether this remains the case in post-stroke patients. Therefore, the aim of this study was to examine the acute effect of physical exercise on prefrontal cortex activity in post-stroke patients using near-infrared spectroscopy (NIRS). We studied 11 post-stroke patients. The patients performed Sternberg-type working memory tasks before and after moderate intensity aerobic exercise (40 % of maximal oxygen uptake) with a cycling ergometer for 15 min. We measured the NIRS response at the prefrontal cortex during the working memory task. We evaluated behavioral performance (response time and accuracy) of the working memory task. It was found that physical exercise improved behavioral performance of the working memory task compared with the control condition (p < 0.01). In addition, NIRS analysis indicated that physical exercise enhanced prefrontal cortex activation, particularly in the right prefrontal cortex (p < 0.05), during the working memory task compared with the control condition. These findings suggest that the moderate-intensity aerobic exercise enhances prefrontal cortex activity and improves working memory performance in post-stroke patients.

Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid.

Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the pieces of evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterized by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbor human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.

Mutagenesis by 8-oxoguanine: an enemy within.

The presence of reactive oxygen species in cells ensures that the oxidatively damaged base 8-oxoguanine will be generated at high frequency in the DNA of all living organisms. DNA damage threatens genomic integrity: enzymes have evolved that protect prokaryotes and eukaryotes from the mutagenic effect of this ubiquitous lesion.

A 12 month in vivo study on the response of bone to a hydroxyapatite-polymethylmethacrylate cranioplasty composite.

We investigated the osteoconductivity and biocompatibility in vivo of a new hydroxyapatite-polymethylmethacrylate (HA-PMMA) composite developed for use as an implant material for cranioplasty, which is expected to have the good osteoconductivity of HA together with the strength and ease of handling of PMMA. The HA-PMMA composites were implanted in eight full-grown beagles and then 6, 12, 24 weeks and 1 year after implantation, the animals were sacrificed and the implanted materials removed along with the surrounding tissues. Extirpated specimens were studied using an optical microscope and micro-computed tomography (micro-CT). Fibrous connective tissue was prominent in the interface of the composite at 6 weeks. New bone formation was seen around the implant, 12 and 24 weeks after operation. At 1 year, new bone filled in the interface of the HA-PMMA composite and adhered to the surrounding autogenous bone. Mixing HA and PMMA did not interfere with the osteoconductivity of the HA component. In micro-CT findings, the new bone growing on the HA-PMMA composite could be seen attaching preferentially to HA particles exposed at the composite surface, rather than the PMMA. This study demonstrated that this HA-PMMA composite is a good candidate for cranial bone implants due to its good osteoconductivity and biocompatibility.

The effect of hematoma removal for reducing the development of brain edema in cases of putaminal hemorrhage.

INTRODUCTION: Surgical intervention in putaminal hemorrhage has been a controversial issue. The aim of this research is to evaluate the benefits of surgery for reducing the development of brain edema. MATERIALS AND METHODS: Sixteen cases of putaminal hemorrhage were examined. Eight patients were treated conservatively (C group), and the other 8 patients were treated surgically (S group). Head CT scans were performed on the day of onset (day 0) in C group or performed just after surgery (day 0) in S group, and performed again once per period on days 1-7, 8-14, and 15-21. The volume of the mass including hematoma and edema (H + E) was measured using CT scans and the (H + E)/H0 ratios were calculated (H0; hematoma volume on day 0). The (H + E)/H0 ratios for each period were compared statistically between the 2 groups using a t-test. RESULTS: The mean values of(H + E)/H0 ratios at each period were 2.19, 2.63, 2.53 in C group, and 1.29, 1.29, 0.66 in S group. The values in S group were significantly lower as compared with C group in every period (p < 0.01, < 0.05, < 0.01). CONCLUSIONS: Hematoma volume reduction by surgery reduced the development of brain edema.

Progressive expression of vascular endothelial growth factor (VEGF) and angiogenesis after chronic ischemic hypoperfusion in rat.

Cerebrovascular stenosis caused by arteriosclerosis induces failure of the cerebral circulation. Even if chronic cerebral hypoperfusion does not induce acute neuronal cell death, cerebral hypoperfusion may be a risk factor for neurodegenerative diseases. The purpose of this study was to determine if vasodilation, expression of VEGF, and neovascularization are homeostatic signs of cerebral circulation failure after permanent common carotid artery occlusion (CCAO) in the rat. Neuronal cell death in neocortex was observed 2 weeks after CCAO and gradually increased in a time-dependent manner. The diameter of capillaries and expression of VEGF also increased progressively after CCAO. Moreover, we observed unusual irregular angiogenic vasculature at 4 weeks. In conclusion, chronic hypoperfusion results in mechanisms to compensate for insufficiency in blood flow including vasodilation, VEGF expression, and neovascularization in the ischemic region. These results suggest that angiogenesis might be induced in adult brain through the support of growth factors and transplantation of vascular progenitor cells, and that neovascularization might be a therapeutic strategy for children and adults with diseases such as vascular dementia.

Detection of mutagenicity of the colon carcinogen 1,2-dimethylhydrazine by the host-mediated assay and its correlation to carcinogenicity.

Mutagenic potential of 1,2-dimethylhydrazine (DMH) was investigated in the host-mediated assay with mice used as hosts. This assay revealed potent mutagenicity of this colon carcinogen for Salmonella typhimurium G46. The mutagenicity of DMH was inhibited by pretreatment of mice with disulfiram. In addition, mouse strain and sex differences influenced the mutation induction by DMH: Mutation induction was significantly lower in C57BL/6 mice than in outbred ICR mice of either sex and was generally higher in male than in females of either C57BL/6 or ICR mice.

Assay for mutagenicity of bile in Sprague-Dawley rats treated subcutaneously with intestinal carcinogens.

To investigate the mode of action of sc injected intestinal carcinogens, the mutagenicity assay of bile collected from noninbred Sprague-Dawley rats treated sc with carcinogens was conducted in the presence and absence of beta-glucuronidase. The bile samples from rats inoculated with 4-aminobiphenyl were mutagenic for Salmonella typhimurium TA100 only in the presence of beta-glucuronidase, whereas those from the 3,2'-dimethyl-4-aminobiphenyl-treated rats did not require the enzyme for mutagenicity toward strain TA100. On the contrary, the assays with S. typhimurium G46 and TA100 of bile from rats inoculated with 1,2-dimethylhydrazine, azoxymethane, or methylazoxymethanol acetate failed to reveal mutagenicity whether beta-glucuronidase was added or not, though these carcinogens were highly mutagenic for strain G46 in the Salmonella-microsome mutagenicity test and/or in the host-mediated assay.

Estimation of annual pollutant loadings in two small catchments and examination of their differences caused by regional properties.

A series of runoff surveys was conducted for more than one year in two small catchments of the Kamo River basin (75.4 km2) and the Takano River basin (66.8 km2) in Kyoto, Japan, which adjoin each other, and may have the same precipitation pattern. The investigation consisted of a high-frequency periodic survey, a long-term regular survey and a storm event survey. The survey results were compared with the regional properties of the basins, and the following results were obtained. (1) Pollutant loadings were successfully estimated as two portions of base discharge and storm events discharge from the survey results. (2) Estimated annual loading of the sites was 2.9-4.5, 1.3-1.8, 17-27, 1.3-2.2, 0.076-0.97 t/km2/y, respectively for COD(Mn), DOC, SS, TN and TP. (3) 52-53% of the whole flow, which was caused by rainfall events, conveyed 81-87, 68-73, 92-95, 64-67, 76-81% of the whole loading, respectively for COD(Mn), DOC, SS, TN and TP. (4) Differences of regional properties in two basins cause different runoff patterns, but the differences in runoff patterns also depend on the rainfall patterns. In general, a more urbanized basin receives early and strong influence of precipitation on the storm event runoff.

Analysis of parameter variations in L-Q equations for river runoff processes from the viewpoint of spatial and temporal conditions.

This study aims to find out variation characteristics of the parameters of 'a' and 'b' in L = a x Q(b), an empirical equation for run-off loading (L) and flow rate (Q), by evaluating the effects of flow conditions and regional properties of the watersheds on the values. We selected the Kamo River basin (155 km2) as a study field, and conducted various kinds of investigations, such as 80 day high frequency observations, continuous monitoring for more than 2 years, storm event surveys, and simultaneous surveys of 39 stations. Then, we obtained 7-170 data in each of 39 sampling stations. The main results obtained are as follows: (1) L-Q equation with a range of 'a' can express L-Q relation in most of the WQIs (water quality indices); (2) 'a' receives temporal (flow condition) effects more in SS, VSS and Al, while it receives regional effect more in inorganic carbon, TN, Ca and Fe; (3) both of flow change in storm events, and base flow levels affect the L-Q relation, and their effects can classify the WQIs into several groups; (4) the effects of regional properties were obviously observed in 'a', and quantitatively evaluated, especially for density of population.

Mutagenesis induced by a single 1,N6-ethenodeoxyadenosine adduct in human cells.

To study the genotoxic properties of 1,N6-ethenodeoxyadenosine (epsilondA) in human cells, a novel site-specific mutagenesis approach was developed, in which a single DNA adduct was uniquely placed in either strand of a shuttle plasmid vector. The analysis of progeny plasmid derived from the modified strand shows that epsilondA, when incorporated into the position of the second A of 5'-CAA (codon 61 of the ras gene), is mutagenic in human cells, inducing A-->T, A-->G, and A-->C mutations. The efficient induction of A-->T transversions in experiments using modified double- and singlestranded DNA substrates supports the hypothesis that A:T-->T:A transversions in human and animal tumors induced by vinyl compounds reflect misinsertion of dAMP opposite this adduct. Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand. EpsilondA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G-->T transversions in HeLa cells. In Escherichia coli, epsilondA did not significantly miscode (<0.27%) even in the presence of induced SOS functions.

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies.

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water's surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Cerebellar microfolia and other abnormalities of neuronal growth, migration, and lamination in the Pit1dw-J homozygote mutant mouse.

The Snell dwarf mouse (Pit1dw-J homozygote) has a mutation in the Pit1 gene that prevents the normal formation of the anterior pituitary. In neonates and adults there is almost complete absence of growth hormone (GH), prolactin (PRL), thyroxin (T4), and thyroid-stimulating hormone (TSH). Since these hormones have been suggested to play a role in normal development of the central nervous system (CNS), we have investigated the effects of the Pit1dw-J mutation on the cerebellum and hippocampal formation. In the cerebellum, there were abnormalities of both foliation and lamination. The major foliation anomalies were 1) changes in the relative size of specific folia and also the proportional sizes of the anterior vs posterior cerebellum; and 2) the presence of between one and three microfolia per half cerebellum. The microfolia were all in the medial portion of the hemisphere in the caudal part of the cerebellum. Each microfolium was just rostral to a normal fissure and interposed between the fissure and a normal gyrus. Lamination abnormalities included an increase in the number of single ectopic granule cells in the molecular layer in both cerebellar vermis (86%) and hemisphere (40%) in comparison with the wild-type mouse. In the hippocampus of the Pit1dw-J homozygote mouse, the number of pyramidal cells was decreased, although the width of the pyramidal cell layer throughout areas CA1-CA3 appeared to be normal, but less densely populated than in the wild-type mouse. Moreover, the number of granule cells that form the granule cell layer was decreased from the wild-type mouse and some ectopic granule cells (occurring both as single cells and as small clusters) were observed in the innermost portion of the molecular layer. The abnormalities observed in the Pit1dw-J homozygote mouse seem to be caused by both direct and indirect effects of the deficiency of TSH (or T4), PRL, or GH rather than by a direct effect of the deletion of Pit1.

Inhibition of carcinogenicities of 1,2-dimethylhydrazine and azoxymethane by pyrazole.

Inhibitory effects of pyrazole on the carcinogenicities of 2 large-bowel carcinogens, 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM), were examined, because our previous study revealed that pyrazole completely inhibited the induction of mutations by these carcinogens in the host-mediated mutation assay. ICR male mice were treated subcutaneously once a week for 20 weeks either with DMH or with AOM. Pyrazole was given orally to mice 2 h before treatment with the carcinogen. Pathological examinations were conducted 36 weeks after the first treatment. Treatment with DMH or AOM alone induced colorectal and/or anal tumorigenic lesions in 92% (23/25) mice of the DMH group and 100% (22/22) mice of the AOM group. By contrast, none of the animals in the combined treatment groups (carcinogen + pyrazole) developed those tumors. On the other hand, 50% (13/26) of mice treated with DMH alone and 78% (18/23) of mice treated with AOM alone developed vascular tumors. Pretreatment of mice with pyrazole reduced the percentage of mice bearing this type of tumor to about 30% of that in the carcinogen group with either carcinogen. These results clearly show that pyrazole has the ability to inhibit carcinogenicities of DMH and AOM, especially for the colorectum and anus, and indicate that the inhibition studies of mutation induction in vivo provide a useful tool for the screening for inhibitors of the carcinogenicities of DMH and AOM.

Carcinogenicity of N-nitroso-ethylenethiourea in female mice.

The nitrosation product of ethylenethiourea (ETU), N-nitroso-ETU, was tested for tumorigenicity in female ICR mice. Ten weekly oral administrations of this compound (0.66-2.64 mg/dose) caused apparently dose-dependent increases in the number of mice with pulmonary and lymphocytic neoplasms.

Inhibitors for the mutagenicities of colon carcinogens, 1,2-dimethylhydrazine and azoxymethane, in the host-mediated assay.

Inhibitory effects of several chemicals on the mutagenicities of 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM) for Salmonella typhimurium G46 in the host-mediated assay were investigated. They were carbon disulfide (CS2), tetraethylthiuram disulfide (disulfiram, DSF), sodium diethyldithiocarbamate (SDDC), ethylene-bis(dithiocarbamato) manganese (Maneb), pyrazole (PZ), aminoacetonitrile hydrogen sulfate (AAN), and sodium selenite (SE). All the compounds, except for SE, inhibited the mutagenicities of DMH and AOM.

Ultrastructure and 18S rDNA sequence analysis of Wobblia lunata gen. et sp. nov., a new heterotrophic flagellate (Stramenopiles, Incertae sedis).

A new heterotrophic flagellate Wobblia lunata gen. et sp. nov. is described. This organism usually attaches to the substratum showing a wobbling motion, and sometimes glides on the substratum or swims freely in the medium. W. lunata has various features characteristic of the stramenopiles. These include a hairy flagellum with tripartite tubular hairs, a mitochondrion with tubular cristae, arrangement of flagellar apparatus components and a double helix in the flagellar transition zone. W. lunata shares a double helix with heterotrophic stramenopiles, including Developayella elegans, oomycetes, hyphochytrids, opalinids and proteromonads, and could be placed in the phylum Bigyra Cavalier-Smith. However, from 18S rDNA tree analysis, these organisms form two distantly-related clades in the stramenopiles, and Wobblia appears at the base of the stramenopiles. Evaluation of morphological features and comparison of 18S rDNA sequences indicate that W. lunata is a member of the stramenopiles, but it is distinct from any other stramenopiles so far described. Its phylogenetic position within the stramenopiles is uncertain and therefore W. lunata is described as a stramenopile incertae sedis.

MICROINJECTION OF TOAD SPERM INTO OOCYTES UNDERGOING MATURATION DIVISION.

Spermatozoa of Bufo bufo japonicus were briefly treated with Triton X-100 to remove their plasma membrane, and were injected into oocytes at various stages of maturation division. All the sperm injected into mature coelomic eggs transformed into pronuclei and synthesized DNA, as a normally fertilizing sperm does. The sperm injected into oocytes at the germinal vesicle (GV) stage did not show any change as long as the GV remained intact. In the oocytes which were induced to mature by progesterone, the injected sperm displayed characteristic features in synchrony with those of the resident female nucleus. These included the formation of several sperm-derived chromosomes in association with multipolar spindles in the oocytes from the stage of the germinal vesicle breakdown to the first polar spindle; the appearance of swollen, vesicular nuclei without concomitant DNA synthesis in those at the stage of the first polar body emission; and the reappearance of the condensed chromosomes with giant spindles in those at the stage of the second meiotic metaphase. Pricking of these last oocytes induced the formation of several male pronuclei and DNA synthesis. These results prove that the injection of detergent-treated sperm employed here provides an excellent means of studying the cytoplasmic state that regulates nuclear behavior.