Biomedical subjects

T Kuwana

Publications and source records attributed to T Kuwana.

At least 19 recordsLinked to original sources

PubMed2004

Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR.

1. The present study was carried out to determine whether primordial germ cells isolated from embryonic blood can enter the bloodstream and successfully migrate to the germinal ridges of recipient embryos after transfer to stage X blastoderms, and also whether they can differentiate into blood cells, as is suggested in mice. 2. Primordial germ cells were transfected in vitro by lipofection and then transferred to stage X blastoderms. The introduced GFP gene was efficiently expressed in the gonads of 6-d incubated embryos. 3. Freshly collected primordial germ cells were transferred to stage X blastoderms. The fate of the transferred primordial germ cells was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in White Leghorn and Barred Plymouth Rock chickens used in this study. The transferred donor primordial germ cell-derived cells were detected in the gonads, but not in the blood cells, of 17-d incubated embryos by PCR. 4. This procedure for primordial germ cell manipulation could provide a novel method of producing germline chimaeric chickens. 5. In conclusion, our findings indicate that primordial germ cells isolated from embryonic blood can migrate to the germinal ridges of recipient embryos after being transferred to stage X blastoderms. Although these transferred primordial germ cells differentiated into germ cells, no differentiation into blood cells was observed.

Animals↗

PubMed2003

Purification of avian circulating primordial germ cells by nycodenz density gradient centrifugation.

1. Primordial germ cells (PGCs) are the progenitor cells for gametes. In aves, PGCs show a unique migration pathway, that is, they circulate temporarily through the bloodstream during early development. 2. In this study we developed a method to purify circulating primordial germ cells (cPGCs) in quail and chicks by Nycodenz density gradient centrifugation. 3. The process consisted of primary and secondary purification. In primary purification, cPGCs were enriched at the interface of 8 and 12% Nycodenz fractions. In secondary purification, cPGCs were harvested from 8% Nycodenz fraction at a purity of 90% and from 10% Nycodenz fraction at a purity of 70%. The recovery rate of cPGCs was over 70%. 4. This method would facilitate research on cPGCs' culture and the production of transgenic birds using cPGCs.

Animals↗

PubMed2001

Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway.

Cytotoxic T lymphocytes kill virus-infected and tumor cell targets through the concerted action of proteins contained in cytolytic granules, primarily granzyme B and perforin. Granzyme B, a serine proteinase with substrate specificity similar to the caspase family of apoptotic cysteine proteinases, is capable of cleaving and activating a number of death proteins in target cells. Despite the ability to engage the death pathway at multiple entry points, the preferred mechanism for rapid induction of apoptosis by granzyme B has yet to be clearly established. Here we use time lapse confocal microscopy to demonstrate that mitochondrial cytochrome c release is the primary mode of granzyme B-induced apoptosis and that Bcl-2 is a potent inhibitor of this pivotal event. Caspase activation is not required for cytochrome c release, an activity that correlates with cleavage and activation of Bid, which we have found to be cleaved more readily by granzyme B than either caspase-3 or caspase-8. Bcl-2 blocks the rapid destruction of targets by granzyme B by blocking mitochondrial involvement in the process.

Amino Acid Sequence↗

PubMed2001

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.

Animals↗

PubMed2001

Localization of primordial germ cells or their precursors in stage X blastoderm of chickens and their ability to differentiate into functional gametes in opposite-sex recipient gonads.

This study was performed to determine the distribution of primordial germ cells and their precursors in stage X blastoderm of chickens. The blastoderm (Barred Plymouth Rock chickens) isolated from the yolk was separated into three portions: the central disc, the marginal zone and the area opaca. The dissociated blastodermal cells derived from the central disc, marginal zone and area opaca were transferred into a recipient blastoderm (White Leghorn chicken) from which a cell cluster was removed from the centre of the central disc. The manipulated embryos were cultured in host eggshells until hatching. The chicks were raised until sexual maturity and test mated with Barred Plymouth Rock chickens to assess the donor cell contribution to the recipient germline. Germline chimaeric chickens were produced efficiently (46.7%, 7/15) when the blastodermal cells derived from the central disc were transferred into recipient embryos of the same sex, whereas no germline chimaeric chickens were produced when the blastodermal cells derived from the marginal zone or area opaca were transferred into recipient embryos of the same sex (0/12). Germline chimaeric chickens were also produced by transfer of blastodermal cells derived from the central disc (6.7%, 1/15), marginal zone (10.0%, 1/10) or area opaca (11.1%, 1/9) into recipient embryos of the opposite sex. It is concluded that primordial germ cells are induced during or shortly after stage X and that the cells derived from the central disc have the highest potential to give rise to germ cells. Cells derived from the marginal zone and area opaca can also give rise to germ cells, although the frequency is low.

Animals↗

PubMed2000

Anti-cathepsin G antibodies in the sera of patients with ulcerative colitis.

The presence of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCAs) and that of antibodies against cathepsin G, a target antigen for P-ANCAs, was determined in the sera of patients with ulcerative colitis (UC), relative to the endoscopic severity and disease activity. P-ANCAs were detected by indirect immunofluorescent assay (IIF) on ethanol-fixed human neutrophils. Antibodies to cathepsin G were detected by an enzyme-linked immunosorbent assay (ELISA) and Western blotting. P-ANCAs were detected by IIF in 62.5% of 32 patients with active UC. Anti-cathepsin G antibodies were detected in 40.6% of 32 patients with active UC, and their prevalence was significantly higher in patients with severe colitis, as determined by endoscopy, than in those with mild or moderate colitis (P < 0.05). The prevalence and titers of anti-cathepsin G antibodies were significantly higher during the active than the inactive phase of the disease (P < 0.05). Measurement of titers of anti-cathepsin G antibodies by ELISA in the serum is useful for evaluating the activity of UC.

Adolescent↗

PubMed2000

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

Apoptosis↗

PubMed2000

The 'harmless' release of cytochrome c.

Release of cytochrome c from the mitochondria plays an integral role in apoptosis; however, the mechanism by which cytochrome c is released remains one of the conundrums that has occupied the field. Recently, evidence has emerged that the commitment to death may be regulated downstream of cytochrome c release; therefore the mechanism of release must be subtle enough for the cell to recover from this event. In this review, we discuss the evidence that cytochrome c release is mediated by Bcl-2 family proteins in a process that involves only outer membrane permeability but leaves inner membrane energization, protein import function and the ultrastructure of mitochondria intact. Cell Death and Differentiation (2000) 7, 1192 - 1199.

Animals↗

PubMed1999

Study on the concentration of circulating primordial germ cells (cPGCs) in early chick embryos.

Experiments were conducted to elucidate the factor that influences the concentration of circulating primordial germ cells (cPGCs) in two-day old chick embryos. The concentration of cPGCs was observed to be highest at stage 14 (66.9 +/- 23.2 microliters) and decreased thereafter. However, considerable egg to egg variations in cPGC concentration, especially at stages 13, 14, 15, and 16 were observed. After conducting experiments to elucidate the source of egg to egg variation in cPGC concentration among embryos, it was revealed that there are hens that lay eggs which contain either constantly high (more than 80 PGCs/microliter) or constantly low (less than 30 PGCs/microliter) concentration of cPGCs. The results obtained from the present experiments showed that one of the major source of egg to egg variation in the concentration of cPGCs was due to the individual differences among females that produced the eggs.

Animals↗

PubMed1999

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol.

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.

Adenylate Kinase↗

PubMed1999

Differentiation of donor primordial germ cells into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells isolated from embryonic blood.

This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.

Animals↗

PubMed1999

Migratory mechanisms of chick primordial germ cells toward gonadal anlage.

After appearing at the germinal crescent region, chick primordial germ cells (PGCs) migrate toward the presumptive gonads (pG) till stage 19 (Hamburger and Hamilton, 1951). This study seeks to elucidate the roles of passive and active factors in the PGC-migration, physical trapping of circulating PGCs by the capillary network and PGC attraction by chemotactic factor from presumptive gonads. Firstly, latex beads/pollens (the same size or larger than PGCs) were injected into the embryonic bloodstream at stage 13-19 (when PGCs are in the migrating and settlement phase to the presumptive gonad) in ovo in order to determine whether the PGCs passively reach pG. Most of such particles accumulated in the head region (60%), whereas the remainder did the same in the gonadal region (23% at the peak) at stage 16 when both the head and gonadal regions are rich in capillary plexus. After 3 days, most particles in the gonadal region were located at the angles of dorsal mesentery near the developing gonads where many extra-gonadal PGCs had been located, and a few particles were detected close to the gonad. These results suggest that one of the mechanisms of PGC-migration to the developing gonads is an autonomous trapping of PGCs by the capillary network quite close to the germinal epithelium (GE) and passive translocation by morphogenetic movement. Secondly, the attraction for PGCs by the gonadal anlage proper was examined in ovo using chick and quail embryos. Grafts of quail gonadal anlage containing gonadal epithelium and neighbouring mesenchymal tissue were excised from the quail embryo at stages 12 to 16 (staging by Zacchei, 1961). With the aims of eliminating the influence of surrounding tissue, the quail graft was ectopically transplanted into the posterior to the optic vesicle of 8 to 17 somite chick embryo from the point of a posterior region to the auditory vesicle by a fine tungsten needle under the illumination by the method of Hara (1971). Then the region posterior to the level of presumptive vitelline arteries was surgically excised in ovo. After a 48 hrs.-incubation, the host PGCs which lost their own gonadal anlage as a target organ accumulated in the transplanted quail gonadal anlage originating from the embryo at PGC-migrating periods. This result strongly suggested the presence of some attractive factor that may be emitted from the gonadal anlage proper. Furthermore, it was demonstrated that the PGCs in vitro showed no contact inhibition in relation to other PGCs or fibroblasts in their moving pathway.

Animals↗

PubMed1998

Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c.

Apoptosis often involves the release of cytochrome c from mitochondria, leading to caspase activation. However, in apoptosis mediated by CD95 (Fas/APO-1), caspase-8 (FLICE/MACH/Mch5) is immediately activated and, in principle, could process other caspases directly. To investigate whether caspase-8 could also act through mitochondria, we added active caspase-8 to a Xenopus cell-free system requiring these organelles. Caspase-8 rapidly promoted the apoptotic program, culminating in fragmentation of chromatin and the nuclear membrane. In extracts devoid of mitochondria, caspase-8 produced DNA degradation, but left nuclear membranes intact. Thus, mitochondria were required for complete engagement of the apoptotic machinery. In the absence of mitochondria, high concentrations of caspase-8 were required to activate downstream caspases. However, when mitochondria were present, the effects of low concentrations of caspase-8 were vastly amplified through cytochrome c-dependent caspase activation. Caspase-8 promoted cytochrome c release indirectly, by cleaving at least one cytosolic substrate. Bcl-2 blocked apoptosis only at the lowest caspase-8 concentrations, potentially explaining why CD95-induced apoptosis can often evade inhibition by Bcl-2.

Animals↗

PubMed1998

Determination of L-phenylalanine based on an NADH-detecting biosensor.

An enzyme carbon paste electrode containing three different enzymes was developed for the determination of L-phenylalanine. This sensor is based on the enzymatic/electrochemical recycling of tyrosinase in combination with salicylate hydroxylase and L-phenylalanine dehydrogenase (PADH). The enzymes salicylate hydroxylase and tyrosinase were coimmobilized first in a carbon paste electrode for the sensitive detection of NADH. The principle of the bienzyme scheme is as follows: the first enzyme, salicylate hydroxylase, converts salicylate to catechol in the presence of oxygen and NADH. The second enzyme, tyrosinase, then oxidizes the catechol to o-quinone, which is electrochemically detected and reduced back to catechol at the electrode at an Eappl = -50 mV vs Ag/AgCl. This results in an amplified signal due to the recycling of the catechol and o-quinone between tyrosinase and the surface of the electrode. Prior to adding PADH, the salicylate hydroxylase-tyrosinase carbon paste electrode was characterized in terms of its sensitivity to NADH, pH dependence, buffer composition, interferences, and stability. Interference from ascorbic acid and uric acid was found to be minimal. Human serum was used to investigate whether this bienzyme system was suitable for the detection of NADH in serum and blood samples. The sensitivity for NADH was increased by a factor of 33 times using the bienzyme amplification scheme (electroreduction of o-quinone at Eappl = -50 mV) as opposed to the salicylate hydroxylase single-enzyme system (at which catechol would have been oxidized at Eappl = +150 mV vs Ag/AgCl). The detection limit for NADH achieved by the bienzyme carbon paste electrode was 1 vs 100 microM for the single-enzyme carbon paste electrode. The salicylate hydroxylase-tyrosinase system was then coupled with phenylalanine dehydrogenase for L-phenylalanine determination. This multienzyme sensor was able to achieve a linear range of 20-150 microM and a detection limit of 5 microM for L-phenylalanine. The sensitivity is sufficient since the reference clinical range for L-phenylalanine is 78-206 microM.

Amino Acid Oxidoreductases↗

PubMed1998

Production of germ-line chimeras by transfer of cryopreserved gonadal primordial germ cells (gPGCs) in chicken.

Gonadal primordial germ cells (gPGCs) were collected from gonadal anlage of 5-day-old White Leghorn (WL) embryos. Collected gPGCs were suspended in freezing medium containing 10% dimethyl sulphoxide (DMSO). The cell-suspension was frozen at 1 degree C/min until the temperature reached -80 degrees C; cells were then immersed in liquid nitrogen at -196 degrees C and stored up to 3 mo. Approximately 100 frozen/thawed gPGCs were injected into the dorsal aorta of each Barred Plymouth Rock (BPR) embryo from which blood was drawn prior to germ-cell injection. The injected embryos were incubated until hatched, and hatched chicks were raised until sexually mature. Upon reaching sexual maturity, a progeny test was performed by mating recipient chicks with normal BPR of the opposite sex. Of 840 progeny produced from 4 gPGC recipients, 74 chicks (8.8%) were phenotypically white. Present results demonstrate that frozen/thawed gPGCs collected from gonadal anlage are capable of producing germ-line chimeras in chicken.

Animals↗

PubMed1998

Exit of major histocompatibility complex class II-invariant chain p35 complexes from the endoplasmic reticulum is modulated by phosphorylation.

The Iip35 isoform of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains an endoplasmic reticulum (ER) targeting motif, but in B cell lines the ER retention is ineffective and a fraction of Iip35 is transported through the Golgi complex associated with class II molecules. We found Iip35 (but not Iip33, the major form of Ii) to be phosphorylated in B cell lines, as well as in transfected HeLa cells. The phosphorylation of Iip35 was found to be necessary for the exit of Iip35-class II complexes out of the ER. This requirement suggests that phosphorylation may change the interaction with factors responsible for ER retention/retrieval, and we did find that phosphorylated Iip35 associates with 14-3-3 proteins, a family of adaptor proteins that are involved in coordinating signal transduction pathways. This finding raises the intriguing possibility that the exit of Ii-class II complexes from the ER is regulated by intracellular signaling events.

14-3-3 Proteins↗

PubMed1998

Donor primordial germ cell-derived offspring from recipient germline chimaeric chickens: absence of long-term immune rejection and effects on sex ratios.

1. Germline chimaeric chickens were produced by the transfer of primordial germ cells, and the generation of donor-derived offspring was examined for a maximum of 146 weeks. 2. The frequencies of donor-derived offspring from the chimaeras were 47% to 97%, and no apparent changes in frequency were observed with increasing age during the test period. 3. Differentiation of donor primordial germ cells into functional gametes appeared to be restricted to a degree at some developmental stage in the gonads of chimaeric chickens of the opposite sex.

Animals↗

PubMed1998

Expression of exogenous DNA in the gonads of chimaeric chicken embryos produced by transfer of primordial germ cell transfected in vitro and subsequent fate of the introduced DNA.

The expression and fate of exogenous DNA (lacZ gene), introduced into the gonads of chimaeric embryos and chickens that had been produced by transfer of primordial germ cells (PGCs) transfected in vitro, were examined. PGCs obtained from embryonic blood were transfected in vitro by lipofection and transferred to the partially sterilized recipient embryos. Expression of the lacZ gene was observed in the gonads of chimaeric embryos incubated for 3 days after the PGC injection (71.2%, 37/52). Introduction of the lacZ gene into the gonads of chimaeric embryos was confirmed by PCR analysis. The percentage of embryos with gonads positive for the lacZ gene was 95% (38/40) after 3 days of incubation after the PGC injection. The lacZ gene, however, appeared to persist episomally but was gradually lost during embryonic development. After 17 days of incubation after the PGC injection, the lacZ gene was detected in only 14.3% (3/21) of the embryos examined. Although the lacZ gene was detected in the gonads of two hatched chicks (11.1%), it was not detected in the gonads of chimaeric chickens at sexual maturity. Offspring derived from the lipofected PGCs were obtained from the chimaeric chickens at frequencies of 12.1-69.9% in males and 71.6-97.6% in females. The technique developed in the present work could be used to test the expression of exogenous DNA in the gonads of early chicken embryos and should facilitate the production of transgenic chickens.

Animals↗