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Biomedical subjects

K Mochida

Publications and source records attributed to K Mochida.

At least 19 recordsLinked to original sources
PubMed2003

New microinsemination techniques for laboratory animals.

Since the development of a reliable mouse intracytoplasmic sperm injection (ICSI) technique in 1995, microinsemination techniques have been widely applied in several laboratory species. As gametes and embryos have specific biological and biochemical features according to the species, technical improvements are necessary for successful microinsemination that subsequently leads to normal fetal development in several species. Recent advanced reproductive research involving genetic engineering often depends on microinsemination techniques that require a high degree of skill, and new human assisted reproductive technology (ART) requires experimental models using laboratory animals. The accumulation of technical improvements in these fields should accelerate the development of microinsemination techniques in mammals, including humans.

Animals↗
PubMed2001

Cancer chemopreventive activity of carotenoids in the fruits of red paprika Capsicum annuum L.

Capsanthin and related carotenoids isolated from the fruits of red paprika Capsicum annuum L. showed potent in vitro anti-tumor-promoting activity with inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Among them, capsanthin diester and capsorbin diester showed strong inhibitory effects. Furthermore, capsanthin , capsanthin 3'-ester and capsanthin 3,3'-diester , major carotenoids in paprika, exhibited potent anti-tumor-promoting activity in an in vivo mouse skin two-stage carcinogenesis assay using 7, 12-dimethylbenz[a]anthracene as an initiator and TPA as a promoter.

Animals↗
PubMed2001

Functional recovery and regeneration of descending tracts in rats after spinal cord transection in infancy.

STUDY DESIGN: The functional recovery of rats that underwent spinal cord transection in infancy was evaluated by multimodal examination (functional tests, electrophysiologic evaluation, tract-tracing) to determine the basis for the recovery. OBJECTIVES: To determine whether the hind limb function in rats that underwent spinal cord transection in infancy is regained completely, which descending tracts regenerate after the transection, and whether the functional recovery is correlated with axonal reconnection. SUMMARY OF BACKGROUND DATA: It is widely accepted that a newborn rat recovers its hind limb function after spinal cord transection even without any specific treatments. This functional recovery might be attributed to possible regeneration of some descending pathways, although there is a counterargument that well-trained spinal cord reflexes may bring about functional compensation. METHODS: The thoracic spinal cord of infant rats was completely transected at Th10 when they were 2 weeks of age. Multimodal functional tests and electrophysiologic studies were performed 5 weeks later. Some recovered rats (i.e., those able to walk after the transection) underwent spinal cord retransection, with subsequent reevaluation of locomotion and muscle-evoked potentials. At 6 weeks after the initial transection, tract-tracing studies were performed in some animals. RESULTS: A motor performance score detected the functional differences between the control and the recovered rats. Muscle-evoked potentials of hind limbs after electrical stimulation to the brain were recorded in some of the recovered rats, but never in the unrecovered rats. Moreover, the muscle-evoked potentials of the recovered rats disappeared after spinal cord retransection that resulted in loss of voluntary movement. Morphologic studies in two rats provided evidence that reconnection of rubrospinal, vestibulospinal, and reticulospinal tracts had occurred, whereas corticospinal regeneration was not detected. CONCLUSIONS: It can be concluded that the hind limb function of rats that underwent spinal cord transection in infancy was partially regained; that axonal regeneration of the rubrospinal, vestibulospinal, or reticulospinal tracts was demonstrated, whereas the reconnection of the corticospinal tract was not observed; and that the axonal regeneration of these tracts is involved in the functional recovery.

Animals↗
PubMed2001

The transcription factor FoxH1 (FAST) mediates Nodal signaling during anterior-posterior patterning and node formation in the mouse.

FoxH1 (FAST) is a transcription factor that mediates signaling by transforming growth factor-beta, Activin, and Nodal. The role of FoxH1 in development has now been investigated by the generation and analysis of FoxH1-deficient (FoxH1(-/-)) mice. The FoxH1(-/-) embryos showed various patterning defects that recapitulate most of the defects induced by the loss of Nodal signaling. A substantial proportion of FoxH1(-/-) embryos failed to orient the anterior-posterior (A-P) axis correctly, as do mice lacking Cripto, a coreceptor for Nodal. In less severely affected FoxH1(-/-) embryos, A-P polarity was established, but the primitive streak failed to elongate, resulting in the lack of a definitive node and its derivatives. Heterozygosity for nodal renders the FoxH1(-/-) phenotype more severe, indicative of a genetic interaction between FoxH1 and nodal. The expression of FoxH1 in the primitive endoderm rescued the A-P patterning defects, but not the midline defects, of FoxH1(-/-) mice. These results indicate that a Nodal-FoxH1 signaling pathway plays a central role in A-P patterning and node formation in the mouse.

Animals↗
PubMed2001

Two-step regulation of left-right asymmetric expression of Pitx2: initiation by nodal signaling and maintenance by Nkx2.

Pitx2 is left--right (L--R) asymmetrically expressed initially in the lateral plate and later in primordial visceral organs. The transcriptional regulatory mechanisms that underlie L--R asymmetric expression of Pitx2 were investigated. Mouse Pitx2 has a left side-specific enhancer (ASE) that mediates both the initiation and maintenance of L--R asymmetric expression. This element contains three binding sites for the transcription factor FAST. The FAST binding sites function as Nodal-responsive elements and are sufficient for the initiation but not for the maintenance of asymmetric expression. The maintenance requires an Nkx2-5 binding site also present within the ASE. These results suggest that the left-sided expression of Pitx2 is directly initiated by Nodal signaling and is subsequently maintained by Nkx2. Such two-step control may represent a general mechanism for gene regulation during development.

Animals↗
PubMed2001

Fertilization without spermatozoa.

Mammalian spermatozoa first acquire the ability to fertilize oocytes as they pass through the epididymis to mature. Due to recent advances in microinsemination techniques, not only mature spermatozoa, but also immature sperm cells at certain stages in the testis, have been used to construct diploid zygotes, some of which subsequently develop to normal offspring. Using round spermatids, the most youngest haploid male germ cells, normal births have been reported in the mouse, rabbit, and human. Furthermore, in the mouse, secondary and primary spermatocytes also support full term development after incorporation into immature or mature homologous oocytes. Spermatogenic cells of several species can be cryopreserved easily in simple cryoprotectant solutions. Thus, the microinsemination techniques using spermatogenic cells give us a way to treat infertility, and provide valuable information on gametogenesis, including spermatogenesis, meiosis, and genomic imprinting.

Animals↗
PubMed2000

Keratin 9 is a component of the perinuclear ring of the manchette of rat spermatids.

Previous work in our laboratory has shown that a 62- to 64-kDa protein was a major component of the perinuclear ring of manchettes fractionated from rat spermatids. Mass spectrometry analysis of this protein indicated the presence of a glycine-rich domain homologous to human keratin 9 (K9). Several antibodies to K9, raised against synthetic peptides of human K9, recognized the 64- to 62-kDa protein in the perinuclear ring of the manchette as well as in keratinocytes of the suprabasal layer of the rat and human footpad/sole epidermis in both immunoblotting and immunocytochemical experiments. Based on these data, human-derived K9 primers were used to clone rat K9 cDNA from epidermis by RT-PCR. Rat-specific K9 primers were then used to perform a two-step (nested) PCR to amplify the K9-specific rat testicular RNA and to obtain cDNA to demonstrate K9 gene expression in rat testis. The deduced amino acid sequence of rat K9 cDNA contains 618 amino acids with an estimated molecular mass of 63,020 Da, in agreement with that obtained by electrophoretic fractionation of rat manchette and epidermis footpad proteins. The deduced protein structure correlates with the recognizable pattern of keratins: a rod domain of 304 amino acids with well-conserved initiation and termination sequences (MQNLNSRLASY and EIETYRKLLEG, respectively), flanked by glycine/serine-rich head and tail domains of 141 and 173 amino acids, respectively. A high content of phenylalanine was detected in the head domain and a repetitive motif (SGGSYGGGS) in the tail domain. A comparison with human keratin 9 showed an overall nucleotide and amino acid similarity of 75%. An increased level of K9 transcripts was detected in a cDNA library prepared from fractionated round spermatids. Results of this study show that rat testis expresses K9 and that this protein is a component the perinuclear ring of the manchette of rat spermatids.

Amino Acid Sequence↗
PubMed2000

Structural features of the 26S proteasome complex isolated from rat testis and sperm tail.

We have previously cloned a cDNA encoding TBP-1, a protein present in the rat spermatid manchette and outer dense fibers of the developing sperm. TBP-1 contains a heptad repeat of six-leucine zipper fingers at the amino terminus and highly conserved ATPase and DNA/RNA helicase motifs toward the carboxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory complex of the 26S proteasome, an ATP-dependent multisubunit protease found in most eukaryotic cells. We now report the isolation of the 26S proteasome from rat testis and sperm tail and its visualization by whole-mount electron microscopy using negative staining. The 26S proteasome from rat testis was fractionated by Sephacryl S-400/Mono-Q chromatography using homogenates suspended in a 10% glycerol-supplemented buffer. Chromatographic fractions were analyzed by immunoblotting using a specific anti-TBP-1 serum. During the purification of Sak57, a keratin filament present in outer dense fibers from epididymal sperm, we detected a substantial amount of 26S proteasomes. Intact 26S proteasomes from rat testis display a rod-shaped particles about 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alphabetabetaalpha), capped by two polar 19S regulatory complexes, each identified by an element known as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subunit of the 19S regulatory cap. Rat sperm preparations displayed both dissociated 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasomes in the perinuclear-arranged manchette are in a suitable location for recognition, sequestration, and degradation of accumulating ubiquitin-conjugated somatic and transient testis-specific histones during spermiogenesis. In the sperm tail, the 26S proteasome may have a role in the remodeling of the outer dense fibers and other tail components during epididymal transit.

Animals↗
PubMed2000

Follicle selection in cyclic guinea pigs with active immunization against inhibin alpha-subunit.

Experiments were conducted to elucidate the mechanisms of active immunization against inhibin on ovarian follicular development and selection in guinea pigs. Estrous cycle was synchronized in experimental guinea pigs by implanting progesterone containing tubes. Antibodies that bound 125I-labeled bovine inhibin were produced by all guinea pigs receiving the inhibin vaccine (recombinant ovine alpha-subunit in oil emulsion) without any effects on duration of the estrous cycle. Active immunization against inhibin increased the plasma concentrations of progesterone during the luteal phase and the plasma concentrations of estradiol but failed to increase the plasma concentration of follicle-stimulating hormone (FSH) during preovulatory period. The treatment also increased the number of corpora lutea (from 1.3+/-0.3 to 7.0+/-1.6 per each ovary), and preovulatory sized follicles (from 1.8+/-0.6 to 7.0+/-1.6 per each ovary), and follicles stained positively for inhibin alpha-subunit (from 2.3+/-0.5 to 6.3+/-1.3 per each ovary) significantly. The results indicate that active immunization against inhibin enhances ovulation rate by affecting the follicle selection and only dominant follicle can be stained for inhibin alpha-subunit in guinea pigs. This study is firstly to provide direct evidence that inhibins play important role in follicle selections in guinea pigs.

Animals↗
PubMed2000

Left-right asymmetric expression of lefty2 and nodal is induced by a signaling pathway that includes the transcription factor FAST2.

The left-right (L-R) asymmetric expression of lefty2 and nodal is controlled by a left side-specific enhancer (ASE). The transcription factor FAST2, which can mediate signaling by TGF beta and activin, has now been identified as a protein that binds to a conserved sequence in ASE. These FAST2 binding sites were both essential and sufficient for L-R asymmetric gene expression. The Fast2 gene is bilaterally expressed when nodal and lefty2 are expressed on the left side. TGF beta and activin can activate the ASE activity in a FAST2-dependent manner, while Nodal can do so in the presence of an EGF-CFC protein. These results suggest that the asymmetric expression of lefty2 and nodal is induced by a left side-specific TGF beta-related factor, which is most likely Nodal itself.

Animals↗
PubMed2000

Chemotherapy-induced acral erythema: report of a case and immunohistochemical findings.

Chemotherapy-induced acral erythema (CAE) is an uncommon and distinct reaction seen in patients receiving high-dose chemotherapy. The exact pathogenic mechanisms of this disorder are still unknown. We report a 27-year-old woman who presented with red, swollen and painful macules on both palms, clinically consistent with this disease. Histological examination demonstrated vacuolar degeneration of the basal cell layer and spongiotic blisters in the epidermis, especially in the atrophied eccrine ducts and papillary oedema with mild perivascular infiltration of mononuclear and hypersegmented neutrophils. Immunohistochemistry showed that the infiltrating mononuclear cells were CD3-CD16+CD56+ leucocyte function antigen-1+, possibly natural killer cells. The eccrine ducts expressed HLA-DR and intracellular adhesion molecule-1 (ICAM-1). Our findings suggest that cell-to-cell interaction between NK cells and keratinocytes in the eccrine apparatus may induce CAE and may be involved in the pathogenesis of the skin reaction in our patient and possibly in this disease.

Adrenal Cortex Hormones↗
PubMed2000

Recent advances in the microinsemination of laboratory animals.

Microinsemination techniques were applied to laboratory animals, including mice, Mongolian gerbils, mastomys, guinea pigs and cynomolgus monkeys. After micro-insemination with spermatozoa or round spermatids, their oocytes were successfully fertilized and subsequently developed into 4-cells to blastocysts, depending on species. The efficiencies of microinsemination techniques for producing fertilized oocytes were comparable with, or superior to those of IVF in these species.

Animals↗
PubMed2000

beta-cyanoalanine production by marine bacteria on cyanide-free medium and its specific inhibitory activity toward cyanobacteria.

In screening the culture broth of marine bacteria collected at Yap (Micronesia), Palau (Belau), and Okinawa (the southwest islands of Japan) for antimicroalgal activity, 37 out of 2,594 bacterial isolates tested were found to produce anticyanobacterial substances against Oscillatoria amphibia NIES-361. One strain, C-979, identified as a Vibrio sp., was selected and cultured in 2.4 liters of marine broth 2216 to identify the bioactive compound produced by the strain. The purified very hydrophilic compound (16.4 mg) was determined to be beta-cyano-L-alanine (L-CNAla) by instrumental analyses and the application of the advanced Marfey method. L-CNAla did not inhibit the growth of bacteria, yeast, or eukaryotic microalgae, but some cyanobacteria were found to be sensitive to L-CNAla at a concentration of 0.4 to 25 microg/ml. The effect of L-CNAla on some other environmental organisms, including invertebrates and a macroalgae, is discussed. CNAla production in marine broth was examined by thin-layer chromatography for the 37 bacterial isolates which produced an anticyanobacterial substance. The broth of 36 of these strains contained CNAla, suggesting the wide distribution of CNAla production by marine bacteria. This is the first report on bacteria that produce CNAla without a supply of the cyanide ion in the medium.

Alanine↗
PubMed2000

Development of embryos in superovulated guinea pigs following active immunization against the inhibin alpha-subunit.

Embryo recovery and subsequent embryonic development from guinea pigs treated with or without inhibin vaccines were compared to determine the effect of active immunization against the inhibin alpha-subunit. Twenty female guinea pigs of the Hartley strain were injected 3 times either with 1 ml inhibin vaccine (recombinant ovine inhibin a-subunit in oil emulsion: 50 microg/ml, inhibin-immunized group), or 1 ml placebo (saline in oil emulsion; control group) at 4 week intervals. After one estrous cycle following the last injection, females were naturally mated and embryos were collected at 11:00 hr of day 6 of pregnancy (Day 1: sperm in the vaginal smear) for culture in vitro. Active immunization increased the number of corpora lutea (12.6+/-3.0 vs. 4.6+/-0.2, P<0.05), recovered embryos (9.8+/-1.9 vs. 3.6+/-0.4, P<0.01) and normal embryos (7.8+/-1.4 vs. 3.6+/-0.4, P<0.05), although estrous cycle length was not affected (P>0.05). During subsequent 8 day culture in vitro, most of the recovered embryos formed trophoblast outgrowth; 100% (14/14) and 88.2% (15/17) in control and immunized groups, respectively. High levels of inhibin antibody titers were sustained in the inhibin-immunized guinea pigs at least for 5 months after the last injection while no antibody titer was detected in the control animals. These results indicate that active immunization against the inhibin a-subunit is a long-acting and efficient method to induce superovulation with normal embryonic development in the guinea pig.

Animals↗
PubMed1999

Quinone reductase inhibitors block SAPK/JNK and NFkappaB pathways and potentiate apoptosis.

A variety of environmental stresses stimulate the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEKK) > stress-activated protein kinase (SAPK)-ERK kinase (SEK) > SAPK/c-Jun NH(2)-terminal kinase (JNK) stress-activated protein kinase cascade and coordinately activate the transcription factor NFkappaB. Mechanisms of stress activation upstream of MEKK1 have not been precisely determined. Redox mechanisms involving sulfhydryls are likely because N-acetyl-cysteine at millimolar concentrations blocks stress signals. Because intracellular sulfhydryl concentrations can be regulated through redox cycling involving reactive quinones (1), we tested the ability of quinone reductase inhibitors to alter stress signaling. Several quinone reductases are inhibited by dicoumarol, a coumarin derivative. Dicoumarol prevented SAPK activation in vivo by chemical cell stressors and also prevented SAPK activation induced by expression of the tumor necrosis factor alpha (TNFalpha) receptor-associated protein TRAF2 but not by expression of truncated active MEKK1. Other coumarin derivatives failed to block SAPK activation, but other inhibitors of quinone reductases, particularly menadione, similarly blocked SAPK activation. Cells deficient in a major quinone reductase, NQO1, displayed hypersensitivity to dicoumarol stress inhibition, whereas SAPK in cells reconstituted with the NQO1 gene displayed relative dicoumarol resistance. Consistent with the proposed role of overlapping upstream signaling cascades in activation of NFkappaB, dicoumarol also blocked NFkappaB activation in primary macrophages stimulated with either lipopolysaccharide or TNFalpha. In addition, dicoumarol strongly potentiated TNFalpha-induced apoptosis in HeLa cells, probably by blocking the anti-apoptotic effect of NFkappaB. The ability of dicoumarol to simultaneously inhibit SAPK and NFkappaB activation and to potentiate apoptotic cell death suggests that SAPK is not an obligate participant in apoptosis. Dicoumarol, currently in clinical use as an oral anticoagulant, represents a potential therapeutic inhibitor of the SAPK and NFkappaB response.

Apoptosis↗
PubMed1999

Determination of left/right asymmetric expression of nodal by a left side-specific enhancer with sequence similarity to a lefty-2 enhancer.

The nodal gene is expressed on the left side of developing mouse embryos and is implicated in left/right (L-R) axis formation. The transcriptional regulatory regions of nodal have now been investigated by transgenic analysis. A node-specific enhancer was detected in the upstream region (-9.5 to -8.7 kb) of the gene. Intron 1 was also shown to contain a left side-specific enhancer (ASE) that was able to direct transgene expression in the lateral plate mesoderm and prospective floor plate on the left side. A 3. 5-kb region of nodal that contained ASE responded to mutations in iv, inv, and lefty-1, all genes that act upstream of nodal. The same 3. 5- kb region also directed expression in the epiblast and visceral endoderm at earlier stages of development. Characterization of deletion constructs delineated ASE to a 340-bp region that was both essential and sufficient for asymmetric expression of nodal. Several sequence motifs were found to be conserved between the nodal ASE and the lefty-2 ASE, some of which appeared to be essential for nodal ASE activity. These results suggest that similar transcriptional mechanisms underlie the asymmetric expression of nodal and of lefty-2 as well as the earlier expression of nodal in the epiblast and endoderm.

Animals↗
PubMed1999

Distinct transcriptional regulatory mechanisms underlie left-right asymmetric expression of lefty-1 and lefty-2.

Both lefty-1 and lefty-2 genes are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) axis formation. With the use of transgenic analysis, the transcriptional regulatory regions of these genes responsible for their L-R asymmetric expression have now been investigated. The 9.5-kb upstream region of lefty-1 and the 5.5-kb upstream region of lefty-2 reproduced the expression pattern of the corresponding gene. Examination of deletion constructs revealed the presence of a left side-specific enhancer (ASE) that is essential and sufficient for lefty-2 asymmetric expression. In contrast, the asymmetric expression of lefty-1 was shown to be determined by a combination of bilateral enhancers and a right side-specific silencer (RSS). The 9. 5-kb region of lefty-1 and the 5.5-kb region of lefty-2 responded to iv and inv, upstream genes of lefty-1 and lefty-2. The regulation of lefty-2 by iv and inv was mediated by ASE. These results suggest that, in spite of the similarities between lefty-1 and lefty-2, different regulatory mechanisms underlie their asymmetric expression.

Animals↗