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Biomedical subjects

Y Okazaki

Publications and source records attributed to Y Okazaki.

At least 91 records · Page 5Linked to original sources
PubMed1999

Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display.

We have developed a method for high-efficiency window separation of cDNA display by increasing the specificity of priming in reverse transcription. In the conventional method, two-base anchored oligo(dT) primers (5'dT16VN3', where N is any base and V is G, A or C) are used to make windows for the display of transcripts. However, reverse transcriptase often extends misprimed oligonucleotides. To avoid mispriming from dT16VN primers, we have developed two new technologies. One is higher temperature priming with reverse transcriptase thermoactivated by the disaccharide trehalose. The other is the use of competitive oligonucleotide blockers that hybridize to the non-selectively primed mRNAs, preventing the mispriming from the VN site. These methods were combined to improve restriction landmark cDNA scanning (RLCS), resulting in the elimination of the redundant signals that appear in different windows. This was achieved by the increased specificity of initiation of reverse trans-cription from the beginning of poly(A) sites. This method paves the way for the precise visualization of transcripts to allow expression profiles in individual tissues and at each developmental stage to be understood.

Animals↗
PubMed1999

Metallothionein does not protect mouse endocrine cells from damage induced by alloxan injection.

Effects of metallothionein (MT) on pancreatic endocrine cells of mice, injected with alloxan and at different zinc status were studied. Mice were given drinking water containing four different concentrations of zinc (0, 0.05, 0.1 or 0.5%) for 18 days, and alloxan was injected once on the 14th day. When zinc was added to the drinking water, pancreatic zinc and MT contents increased after injection of alloxan, but did not change with injection of vehicle alone, except in the group of mice drinking 0.5% zinc in water. However, plasma glucose level was increased in all the alloxan injected groups, and was independent of their zinc status. In mice given water with 0.5% of zinc, both pancreatic zinc and MT contents were higher than control mice given water alone. There was no difference in zinc and MT contents of the pancreas in mice drinking 0.5% zinc in groups injected with either alloxan or vehicle. The increase in plasma alpha-amylase activity, an indicator of pancreatic exocrine toxicity, was observed only in mice drinking water with 0.5% zinc after injection of both alloxan and vehicle. Histochemically, degranulation of zymogen and duct-like structures of exocrine cells and atrophy and disappearance of islet cells were observed in alloxan-injected mice drinking 0.5% zinc in water. The zymogen degranulation was observed on the vehicle-injected mice drinking 0.5% zinc in water. MT was immunohistochemically detected in the exocrine cells of both alloxan- and vehicle-injected mice given 0.5% zinc in water. No MT was detected in islet cells of mice in any group. The results show that an increase of zinc content may be followed by induction of MT synthesis in the pancreas of mice given increasing amounts of zinc in drinking water. However, MT dose not provide any protection against damage caused by alloxan to endocrine cells of the pancreas.

Alloxan↗
PubMed1999

Whole blood platelet aggregation in humans and animals: a comparative study.

BACKGROUND: Many animal species are used to evaluate the performance and blood compatibility of cardiovascular devices, but interspecies differences in platelet activity have not been well characterized. This study measures platelet response to six agonists in human, dog, and calf blood. MATERIALS AND METHODS: We used whole blood impedance lumi-aggregometry to measure platelet aggregation and ATP release in blood samples from adult humans (n = 19), mongrel dogs (n = 19), and Holstein calves (n = 7). The agonists were collagen, ristocetin, arachidonic acid, thrombin, and three concentrations of both ADP and epinephrine. RESULTS: Only collagen (1 microg/ml) and ADP (5, 10, and 20 microM) caused aggregation and ATP release in all samples. Canine platelets responded to all six agonists at all doses. Human platelets responded to everything except epinephrine at 2 and 100 microM. Bovine platelets responded only to collagen, ADP, and thrombin. In bovine platelets, aggregation from collagen and ATP release from thrombin were significantly lower than the corresponding responses in human and canine blood. The aggregation induced by 10 microM ADP was significantly higher in canine than in human platelets. CONCLUSION: Human, canine, and bovine platelets have very different responses to agonists. In these models, collagen (1 microg/ml) and ADP (10 microM) are the agonists of choice for investigating whole blood platelet aggregation because they provide the most consistent results between species. For ATP release, 1 U/ml thrombin is the recommended agonist and the dose for all three species.

Adenosine Diphosphate↗
PubMed1999

High accumulation of calcium and phosphorus in human iliac arteries.

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.

Aged↗
PubMed1999

Element content of human umbilical artery and vein in umbilical cord.

To elucidate the element content of newborn blood vessels, umbilical arteries and veins in human umbilical cords, which had the advantage of easy sampling, were examined by ICP-AES. Umbilical cords were removed after birth. Mothers' ages ranged from 26 to 35 yr. It was found that the content of sulfur was the highest in both umbilical arteries and veins, being higher than the content of calcium and phosphorus. With respect of the content of sulfur, calcium, and magnesium, there were significant differences between the arteries and veins.

Aged↗
PubMed1999

Age-independent constancy of mineral contents in human ribs.

On age relationships of mineral contents in human bones, the contents of the sixth rib and a piece of its compact bone were determined by inductively coupled plasma atomic emission spectrometry (ICPS). The ribs were resected from 21 subjects (14 men and 7 women) who died in age ranging from 65 to 93 yr. There were no age-dependent decreases in Ca and P contents of the ribs in the age range on ICPS. It was found that there were no age-dependent decreases in Ca and P in compact bones of ribs.

Age Factors↗
PubMed1999

Age-related changes of elements in human anterior cruciate ligaments and ligamenta capitum femorum.

To elucidate compositional changes of human ligaments by aging, the content of elements in anterior cruciate ligaments (ACLs) and ligamenta capitum femorum (LCFs) was analyzed by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 11 men and 7 women, ranging from 59 to 91 yr of age. With regard to the content of elements, the content of sulfur and iron was significantly higher in the LCFs than in the ACLs. It was found in the ACLs that the content of sulfur decreased gradually with aging, whereas the content of calcium, phosphorus, and magnesium increased progressively with aging. On the other hand, it was found in the LCFs that the content of magnesium decreased gradually with aging, where as the content of phosphorus increased progressively with aging. The common finding that the content of phosphorus increased with aging, but the content of iron decreased, was obtained in the ACL and LCF. Regarding sexual difference, it was found in both the ACLs and LCFs that the content of phosphorus was higher in women's ligaments than in men's.

Aged↗
PubMed1999

Age-related changes of elements in the human articular disk of the temporomandibular joint.

To elucidate compositional changes of the articular disk (AD) of the temporomandibular joint (TMJ) by aging, elements of the ADs resected from 18 cadavers were determined by inductively coupled plasma atomic-emission spectrometry. It was found that calcium contents of ADs in TMJs increased progressively with aging, whereas the sulfur contents of the ADs decreased slightly with aging. Regarding the content of phosphorus, the contents increased progressively with aging. The study revealed that age-related changes of calcium contents in the ADs of TMJs were similar to those in women's pubic symphyses, but not those in intervertebral disks and menisci.

Aged↗
PubMed1999

Application of the RLGS image analysis tool (RAT) to the construction of a genetic linkage map of recombinant inbred strain SMXA.

The construction of a genetic linkage map is the first, fundamental step to analyze the genetic properties of any organism. For this purpose, the restriction landmark genome scanning method (RLGS) can be used and has been shown to have high productivity in various genetic analyses. However, construction of a genetic linkage map by the RLGS method is laborious, because hundreds of spots must be scored, usually by visual observation. In order to reduce human involvement in the data processing, we developed an image analysis software, RAT (RLGS Analysis Tool). We evaluated its accuracy and feasibility by comparing the parental distribution patterns of RLGS spots obtained by RAT and by human observation, using Syrian hamster strain backcross progeny. We then used RAT to construct a genetic linkage map of the recombinant inbred strain SMXA. We were able to obtain 121 progenitor strain-specific spots that were assigned to a specific chromosome.

Animals↗
PubMed1999

Decreased expression of the mRNA for somatostatin in the periventricular nucleus of depression-model rats.

Expression of the mRNA for somatostatin (SRIF) in the periventricular nucleus (PeN), the level of SRIF in the stalk-median eminence (SME) and the concentration of growth hormone (GH) in the plasma were examined in depression-model rats in an attempt to confirm the hypothesis that SRIF neurons in the hypothalamus are hypofunctional in this model. We exposed male Wistar rats to intermittent walking stress for two weeks and then we measured their spontaneous running activity for 12 days. We divided the rats into a depression-model group and a partial-recovery group according to the spontaneous running activity of each rat after the termination of exposure to stress. Expression of SRIF mRNA in the PeN of the hypothalamus was monitored by in situ hybridization and relative levels were determined with an image analysis system. The relative level of expression of SRIF mRNA in the PeN was lower in rats in the depression-model group than in the control group and the partial-recovery group. The level of SRIF in the SME was lower and the plasma concentration of GH was higher in the depression-model group than in the other groups. Our findings suggest that reduced expression of mRNA for SRIF in the PeN might be associated with the pathophysiology of rats with this particular model of depression.

Adrenal Glands↗
PubMed1999

Further evidence of westernization of dementia prevalence in Nagasaki, Japan, and family recognition.

The present study examined the prevalence of dementia in the Nagasaki Prefecture. The purposes of our investigation were (a) to study the relationship between aging and the prevalence of dementia and the ratio of Alzheimer's disease (AD) to vascular dementia (VD), (b) to understand the features of early-onset dementia as seen in patients from 60 to 65 years, and (c) to examine the recognition of dementia by family members. The subjects of the study, a total of 4,368, were all 60 years old and over and were residing in the three areas of Nagasaki Prefecture at the time of the investigation, August 1995. We adopted a two-stage design. The first-stage questionnaire that we developed was delivered to subjects, and we selected for the second stage those subjects who met the criteria outlined in the Methods section. The second-stage investigation was an interview by community nurses and psychiatrists. The prevalence of dementia in subjects 60 years and over was 6.2% (men: 5.9%; women: 6.8%). The prevalence increased with age. The AD/VD ratio was 1.4, and was similar to the recent trend in Japan in that the ratio has reversed to resemble the western pattern. In regard to the family members' recognition of illness, the higher the severity of dementia, the higher the recognition ratio of family members became. Only half of these subjects were recognized as having dementia by their family members. In conclusion, the westernization of the AD/VD ratio in Japan was proved. There was little study about family recognition of dementia. In this study, it was remarkable that only half of the subjects were recognized as having dementia by their family members.

Aged↗
PubMed1999

Age-depending effects of methotrexate treatment on systemic bone turnover in experimental adjuvant arthritis.

Adjuvant arthritis was induced in rats in the growth stage (aged 6 weeks) and those in the mature stage (aged 4 months), and changes in the systemic bone turnover and the effects of methotrexate (MTX, CAS 133073-73-1) were compared. After induction of adjuvant arthritis, the paw edema ratio and the urinary deoxypyridinoline (u-Dpy) level increased in both age groups. No marked changes were observed in the serum osteocalcin (s-OC) level in either group. In the 6-week-old rats, arthritis completely inhibited the bone mass, and strength of the femur and lumbar vertebral body. The 4-month-old rats showed more marked changes than the 6-week-old rats in the bone mass and strength of the lumbar, vertebral body. MTX administration (0.05, 0.1 and 0.2 mg/kg/day) resulted in significant dose-dependent inhibition of arthritis-induced changes, and the effects of MTX were similar between the two age groups. MTX was useful at each age. These results suggest that 4-month-old rats with arthritis are more appropriate as a model for evaluation of drugs for bone metabolic turnover in human chronic rheumatoid arthritis.

Absorptiometry, Photon↗
PubMed1999

The mechanisms of immune suppression by high-pressure stress in mice.

The effects of high-pressure stress on the induction of anti-sheep red blood cells (SRBC) and of plaque-forming cells (PFC), and on thymus weight, were studied in BALB/c mice in-vivo and in-vitro. The efficacy of high-pressure stress in suppressing PFC and thymic involution was maximum when the stress was applied 1 h day(-1) for 2 days before immunization with SRBC. Both effects were blocked by administration of indomethacin, atropine, naloxone or phentolamine before the first application of stress, whereas hexamethonium and propranolol had no such effect. Hexamethonium, naloxone and propranolol administered before the second application of high-pressure stress blocked both effects. Prostaglandin and acetylcholine given 24 h before application of high-pressure stress caused a marked reduction in PFC count, but not in thymus weight. The reduced PFC count caused by acetylcholine was blocked by pretreatment with indomethacin. When adrenaline was injected 24 h after application of high-pressure stress a marked reduction in PFC was observed, but without thymic involution. When adrenaline was injected 24 h after prostaglandin injection the PFC count was also markedly reduced, but not thymus weight. The decrease in PFC caused by two exposures to stress or one exposure to stress plus injection of adrenaline was blocked by diethylcarbamazine before the second exposure to stress or the injection of adrenaline. In addition, normal spleen cells, were induced as suppressor cells when incubated with the serum of stressed mice, but not when supplemented with anti-leukotriene C4, D4 antibody. These data suggest that mice fall into a pre-stress condition via the release of prostaglandin after the first stress, and then immunosuppression is induced in these prestressed mice via the release of leukotriene C4, D4, caused by the activation of the autonomic nervous system by the second exposure to stress.

Animals↗
PubMed1999

[Effects of allopurinol for oxidative injury of cisplatin-induced nephrotoxicity in mice].

The effects of allopurinol (Allop) on the lipid peroxidation in the nephrotoxicity of an antitumor drug, cisplatin (CDDP) were studied in mice. CDDP was administered intraperitoneally to two groups (CDDP + Allop group and CDDP + CMC-Na group) at single doses of 10 mg/kg, and mice were sacrificed 3 days after CDDP administration. The body weights of the CDDP-administered group gradually decreased to approximately 78% of the values of the control group (saline + Allop group and saline + CMC-Na group) within 3 days. Plasma urea nitrogen and creatinine, especially in the CDDP + Allop group, increased after 3 days. Lipid peroxides in the blood and kidney were monitored by measuring the production of malondialdehyde (MDA), which increased in the CDDP + CMC-Na group. On the other hand, MDA levels in the CDDP + Allop group increased in the kidney but remained unchanged in the blood. Changes were observed in tissue glutathione (reduced form, GSH; oxidized form, GSSG) levels in the CDDP + Allop group but not in the CDDP + CMC-Na group. Histomorphological examination demonstrated the degeneration of the proximal tubuli in the CDDP-administered groups. Especially in the CDDP + Allop group, the increase of mesangium cells in the glomeruli was observed. From these results, it was suggested that Allop was not able to inhibit CDDP-induced lipid peroxidation in the kidney, and the kidney function became more severely impaired by the administration of Allop.

Allopurinol↗
PubMed1999

Trabecular bone turnover and bone marrow cell development in tail-suspended mice.

To clarify the relationship between the changes of trabecular bone turnover and bone marrow cell development during mechanical unloading and reloading, we performed experiments with tail-suspended mice. At 8 weeks of age, 150 male ddY mice were divided into three body weight-matched groups. Mice of group 1 were euthanized at the start of tail suspension (day 0) as a baseline control. The mice of group 2 were subjected to hindlimb unloading by tail suspension for 14 days and reloading for the subsequent 14 days. The mice of group 3 were normally loaded as age-matched controls. Mice of groups 2 and 3 were sacrificed at 7, 14, and 28 days after the start of the experiment. In the first experiment (histomorphometric study of tibiae), unloading for 7 and 14 days and reloading for the subsequent 14 days significantly decreased the bone volume compared with that in the age-matched controls, respectively. Unloading for 7 and 14 days also significantly reduced the bone formation rate (BFR/BS), respectively, but reloading for the subsequent 14 days restored BFR/BS to the control level. While the unloading for 7 and 14 days significantly increased both the osteoclast surface (Oc.S/BS) and the osteoclast number (Oc.N/BS), the reloading for the subsequent 14 days decreased Oc.S/BS and Oc. N/BS, respectively. In the second experiment (bone marrow cell culture study of tibiae), unloading for 7 and 14 days reduced the adherent stromal cell number, without significance. Unloading for 7 days significantly decreased the mineralized nodule formation. Reloading for the subsequent 14 days markedly increased the adherent stromal cell number and the mineralized nodule formation. Unloading for 7 days significantly increased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. These data clearly demonstrate that unloading reduces bone formation and increases bone resorption, and subsequent reloading restores reduced bone formation and suppresses increased bone resorption, closely associated with the changes in adherent stromal cell number, mineralized nodule formation, and the number of TRAP-positive multinucleated cells.

Animals↗
PubMed1999

Automated filtration-based high-throughput plasmid preparation system.

Current methods of plasmid preparation do not allow for large capacity automated processing. We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing. This system is based on our previously reported filtration method. In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-well microtiter plate from which sequence-ready DNA samples are collected. The plates are designed to allow all reagents to be injected from above the wells and the spent reagents to be aspirated from below. This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator. The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker. The plates move from the stacker to each of the 21 stages of the preparator. At specific stages, various reagents or chemicals are injected into the wells from above. Finally, the plates are collected in the second stacker. The optimal throughput of the preparator is 40,000 samples in 17.5 hr. Here, we describe a pilot experiment preparing 15,360 templates in 160 specially designed 96-well glass-filter plates. The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing.

Base Sequence↗
PubMed1998

Molecular cloning of pTAC12 an alternative splicing product of the CD3gamma chain as a component of the pre-T cell antigen-receptor complex.

We have reported that a 12-kDa molecule (pTAC12 as a pre-T cell receptor (TCR)-associated chain) was associated as a dimer with the pre-TCR complex as well as the clonotype-independent CD3 complex on the cell surface of immature thymocytes. We now report by protein sequencing and molecular cloning that pTAC12 is an alternatively spliced product of the CD3gamma chain lacking exon 4 containing the transmembrane region. The transcript of pTAC12 is expressed in most T cell lineages and parallels the expression of CD3gamma. However, the pTAC12 protein is expressed on the cell surface of immature thymocytes but not mature T cells, despite the fact that mature T cells express a low level of pTAC12 in association with the TCR complex within the cells. These results indicate that pTAC12 may play a special role for the transport/expression and assembly of the pre-TCR.CD3 complex as well as the clonotype-independent CD3 complex in immature thymocytes.

Alternative Splicing↗