Spin-dependent acoustic response in the nonunitary A1 and A2 phases of superfluid 3He under high magnetic fields.

The transverse acoustic impedance of superfluid ^{3}He was measured in the A1 and A2 phases up to 13 T to investigate the surface states in nonunitary superfluids. The temperature dependence of the impedance was much larger in the A1 phase than in the A2 phase. This nonsymmetric behavior indicates that momentum exchange with walls for spin-down surface states is quite different from that for spin-up surface states. The spin-dependent response might be a reflection of an essential feature of the nonunitary states where gap amplitudes depend on spin states. Weak-coupling theories ignore any spin-dependent processes and do not account for the nonsymmetric behavior.

Magnetic response of odd-frequency s-wave Cooper pairs in a superfluid proximity system.

We investigate the magnetic response of a dirty-normal-Fermi-liquid-spin-triplet-superfluid proximity system consisting of liquid 3He and aerogel. In contrast to bulk superfluids, Pauli spin susceptibility in the proximity system exceeds its normal-state value locally around the interface. This enhanced Pauli paramagnetism originates from odd-frequency s-wave pairing arising due to spatial inhomogeneity. A characteristic observable signature of the paramagnetic effect can be found in the spin susceptibility temperature dependence.

New anomaly in the transverse acoustic impedance of superfluid 3He-B with a wall coated by several layers of 4He.

We measured the transverse acoustic impedance of superfluid 3He-B with a wall coated by several layers of 4He. The coating is known to enhance the specularity in quasiparticle scattering by the wall. We found a new anomaly, a bump and a peak, in the temperature dependence of the transverse acoustic impedance. This agrees with a theoretical calculation using a partially specular wall boundary condition. The new anomaly is shown to arise from a change in the surface density of states by coating and the scattering of thermally occupied surface bound states to other states. The change is towards the density of states of Majorana cone in the specular limit.

Observation of surface Andreev bound States of superfluid 3He by transverse acoustic impedance measurements.

Complex transverse acoustic impedance of the superfluid (3)He-B was measured at the frequencies of 10 to 80 MHz at 17.0 bar by a cw bridge method. The observed temperature dependence was well explained by the quasiclassical theory with random S-matrix model for a diffusive surface. The temperature dependence was influenced by pair breaking and by quasiparticle density of states at the surface, which was drastically modified from the bulk one by the presence of surface Andreev bound states.

Gypsy embryo specifies ovule curvature by regulating ovule/integument development in rice.

The embryo position in a seed is stable in most plant species, indicating the existence of a strict regulatory mechanism that specifies the embryo position in the seed. To elucidate this mechanism, we analyzed the gypsy embryo (gym) mutant of rice, in which the position of the mature embryo in the seed is altered at a low frequency. Analyses of early embryogenesis and ovule development showed that the ectopic embryo was derived from an ill-positioned egg cell, which resulted from the incomplete curvature of the ovule. Although the development of both the inner and outer integuments was impaired, the ovule curvature was associated closely with the extent of inner integument growth. Therefore, inner integument development controls ovule curvature in rice. The expression patterns of OSH1 and OsMADS13 indicated that, in gym, a small number of indeterminate cells are maintained on the style side of the ovule and then in the integument primordium at a low frequency. The prolonged survival of these indeterminate cells disturbs normal integument development. The gym fon2 double mutant suggests that GYM and FON2 are involved redundantly in floral meristem determinacy. Possible functions of the GYM gene and the ovule developmental mechanism are discussed.

A genetic and physical map of the region containing PLASTOCHRON1, a heterochronic gene, in rice ( Oryza sativa L.).

The rice heterochronic gene plastochron1, pla1, shows shorter plastochron and ectopic expression of the vegetative program during the rice reproductive phase resulting in aberrant panicle formation. A genetic and physical map was constructed to isolate the causal gene for the pla1 syndrome. Small-scale mapping was carried out to determine the approximate map position of the pla1 locus, and then a high-resolution genetic map was made for pla1-1, one of the pla1 alleles, using an F(2) population comprising 578 pla1-1 homozygous plants. In a high-resolution genetic map, the pla1-1 locus was found to map between RFLP markers C961 and R1738A on chromosome 10, within a 3.6-cM genetic distance. A physical map encompassing the pla1-1 locus was constructed by overlapping Bacterial Artificial Chromosome (BAC) clones through chromosome walking. PCR-based RFLP markers from BAC-end clones were developed and mapped relative to the pla1 locus. Physical map construction using BAC clones indicated that a BAC clone, B44A10 (167-kb), contained the pla1 locus within 74-kb corresponding to a 0.52-cM genetic distance. Gene prediction of 74-kb region carrying the pla1 locus suggested several candidate genes for the pla1 gene. Identification of a candidate gene for pla1 will be made by sequence analysis of allele variation and cDNA screening.

Shoot organization genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice.

The mechanism regulating the pattern of leaf initiation was analyzed by using shoot organization (sho) mutants derived from three loci (SHO1, SHO2, and SHO3). In the early vegetative phase, sho mutants show an increased rate of leaf production with random phyllotaxy. The resulting leaves are malformed, threadlike, or short and narrow. Their shoot apical meristems are relatively low and wide, that is, flat shaped, although their shape and size are highly variable among plants of the same genotype. Statistical analysis reveals that the shape of the shoot meristem rather than its size is closely correlated with the variations of plastochron and phyllotaxy. Rapid and random leaf production in sho mutants is correlated with the frequent and disorganized cell divisions in the shoot meristem and with a reduction of expression domain of a rice homeobox gene, OSH1. These changes in the organization and behavior of the shoot apical meristems suggest that sho mutants have fewer indeterminate cells and more determinate cells than wild type, with many cells acting as leaf founder cells. Thus, the SHO genes have an important role in maintaining the proper organization of the shoot apical meristem, which is essential for the normal initiation pattern of leaf primordia.

Relationship between copper, zinc and metallothionein in hepatocellular carcinoma and its surrounding liver parenchyma.

BACKGROUND/AIM: Accumulation of copper (Cu) in hepatocellular carcinoma (HCC), especially in small tumors, is greater than that in the surrounding liver parenchyma. Metallothionein (MT) is considered to be present as Cu-MT, Zn,Cu-MT or Zn-MT. The aim of this study was to determine the presence and localization of Cu-MT and Zn-MT in HCC and surrounding liver parenchyma. METHODS: In 16 HCC patients, surgically resected specimens including HCC and surrounding liver parenchyma were evaluated. RESULTS: The level of Cu present in small HCC (<4 cm in diameter) was significantly greater than that in the surrounding liver parenchyma (p<0.05). However, the level of Cu in large HCC (>4 cm in diameter) was similar to that in the surrounding liver parenchyma. Analysis by Sephadex G-75 gel filtration revealed that the peak fraction due to Cu was identical to that due to MT in 14 (87.5%) of 16 HCC, the peak fraction due to Cu and Zn was identical to that due to MT in 2 (12.5%) HCC, and the peak fraction due to Zn was identical to that of MT in none of 16 HCC. CONCLUSIONS: Accumulation of Cu in small HCC, in which Cu was present as Cu-MT or Zn, Cu-MT, was greater than that in the surrounding liver parenchyma. Cu accumulation and the presence of MT in the liver may be related to carcinogenesis of HCC, because of the similarity of these findings in the experimental data of Long-Evans rats with a cinnamon-like coat color who develop HCC spontaneously.

Molecular analysis of the NAC gene family in rice.

Genes that encode products containing a NAC domain, such as NO APICAL MERISTEM (NAM) in petunia, CUP-SHAPED COTYLEDON2 (CUC2) and NAP in Arabidopsis thaliana, have crucial functions in plant development. We describe here molecular aspects of the OsNAC genes that encode proteins with NAC domains in rice (Oryza sativa L.). Sequence analysis revealed that the NAC genes in plants can be divided into several subfamilies, such as the NAM, ATAF, and OsNAC3 subfamilies. In rice, OsNAC1 and OsNAC2 are classified in the NAM subfamily, which includes NAM and CUC2, while OsNAC5 and OsNAC6 fall into the ATAF subfamily. In addition to the members of these subfamilies, the rice genome contains the NAC genes OsNAC3, OsNAC4 (both in the OsNAC3 subfamily), OsNAC7, and OsNAC8. These results and Southern analysis indicate that the OsNAC genes constitute a large gene family in the rice genome. Each OsNAC gene is expressed in a specific pattern in different organs, suggesting that this family has diverse and important roles in rice development.

Initiation of shoot apical meristem in rice: characterization of four SHOOTLESS genes.

The regulatory mechanism of shoot apical meristem (SAM) initiation is an important subject in developmental plant biology. We characterized nine recessive mutations derived from four independent loci (SHL1-SHL4) causing the deletion of the SAM. Radicles were produced in these mutant embryos. Concomitant with the loss of SAM, two embryo-specific organs, coleoptile and epiblast, were lost, but the scutellum was formed normally. Therefore, differentiation of radicle and scutellum is regulated independently of SAM, but that of coleoptile and epiblast may depend on SAM. Regeneration experiments using adventitious shoots from the scutellum-derived calli showed that no adventitious shoots were regenerated in any shl mutant. However, small adventitious leaves were observed in both mutant and wild-type calli, but they soon became necrotic and showed no extensive growth. Thus, leaf primordia can initiate in the absence of SAM, but their extensive growth requires the SAM. An in situ hybridization experiment using a rice homeobox gene, OSH1, as a probe revealed that shl1 and shl2 modified the expression domain of OSH1, but normal expression of OSH1 was observed in shl3 and shl4 embryos. Accordingly, SHL1 and SHL2 function upstream of OSH1, and SHL3 and SHL4 downstream or independently of OSH1. These shl mutants are useful for elucidating the genetic program driving SAM initiation and for unraveling the interrelationships among various organs in grass embryos.

Isolation and characterization of a rice homebox gene, OSH15.

In many eukaryotic organisms including plants, homeobox genes are thought to be master regulators that establish the cellular or regional identities and specify the fundamental body plan. We isolated and characterized a cDNA designated OSH15 (Oryza sativa homeobox 15) that encodes a KNOTTED-type homeodomain protein. Transgenic tobacco plants overexpressing the OSH15 cDNA showed a dramatically altered morphological phenotype caused by disturbance of specific aspects of tobacco development, thereby indicating the involvement of OSH15 in plant development. We analyzed the in situ mRNA localization of OSH15 through the whole plant life cycle, comparing the expression pattern with that of another rice homeobox gene, OSH1. In early embryogenesis, both genes were expressed as the same pattern at a region where the shoot apical meristem would develop later. In late embryogenesis, the expression pattern of the two genes became different. Whereas the expression of OSH1 continued within the shoot apical meristem, OSH15 expression within the shoot apical meristem ceased but became observable in a ring shaped pattern at the boundaries of some embryonic organs. This pattern of expression was similar to that observed around vegetative or reproductive shoots, or the floral meristem in mature plants. RNA in situ localization data suggest that OSH15 may play roles in the shoot organization during early embryogenesis and thereafter, OSH15 may be involved in morphogenetic events around the shoot apical meristem.

A recessive heterochronic mutation, plastochron1, shortens the plastochron and elongates the vegetative phase in rice

We describe two recessive alleles of a rice heterochronic gene, plastochron1-1 (pla1-1) and pla1-2, that reduce the length of the plastochron to approximately half that of the wild type. Because the onset of the reproductive phase in pla1 was not temporally affected, the number of leaves produced in the vegetative phase was nearly twice that produced in the wild type. Panicle development was severely disturbed in pla1 mutants. In pla1-1, many primordia of primary rachis branches were converted into vegetative shoots. These ectopic shoots repeated the initiation of panicle development and the conversion of primary rachis branches into shoots. In the weak allele pla1-2, however, only the basal one or two primordia developed as vegetative shoots, and the remaining primordia developed to produce a truncated panicle. These results indicate that both vegetative and reproductive programs are expressed simultaneously during the reproductive phase of pla1; however, the degree varied depending on the strength of the allele. Accordingly, pla1 is a heterochronic mutation that extends the vegetative period. The shoot apical meristem of pla1 was larger than that of the wild type, although the shape was not modified. An in situ hybridization experiment using the histone H4 gene as a probe revealed that cell divisions are accelerated in the pla1 meristem. The PLA1 gene is considered to regulate the duration of the vegetative phase by controlling the rate of leaf production in the meristem.

Effect of cyclic polylactates on tumor cells and tumor bearing mice.

We studied the effect of cyclic polylactates ranging in size from a degree of polymerization number of 3 to 13 on pyruvate kinase, lactic dehydrogenase, anaerobic glycolysis, growth of tumor cells and survival of tumor bearing mice. Pyruvate kinase and lactic dehydrogenase activities were both inhibited by cyclic polylactates, and the inhibition mechanism of cyclic polylactates on lactic dehydrogenase was noncompetitive. About half the anaerobic glycolytic activity of FM3A ascites tumor cells was inhibited and tumor cell growth was also effectively inhibited by cyclic polylactates. Mice, which were treated with cyclic polylactates after inoculation of FM3A ascites tumor cells lived significantly longer than mice, which were treated with vehicle or non mice.

How is embryo size genetically regulated in rice?

It is unclear how embryo size is genetically regulated in plants. Since cereals have a large persisting endosperm, it is expected that embryo size is affected by endosperm development. Nine single recessive mutations, four reduced embryo mutations representing three loci, REDUCED EMBRYO1, REDUCED EMBRYO2 and REDUCED EMBRYO3, four giant embryo mutations derived from a single locus GIANT EMBRYO, and one endospermless mutation endospermless1-2 were analyzed. Every reduced embryo mutation caused reduction of all the embryonic organs including apical meristems and the enlargement of the endosperm. The giant embryo mutants have a reduced endosperm and an enlarged scutellum. However, shoot and radicle sizes were not affected. All the reduced embryo and giant embryo mutations did not largely affect postembryonic development. Accordingly, the expression of genes analyzed are seed-specific. In reduced embryo and giant embryo mutations, abnormalities were detected in both embryo and endosperm as early as 2 days after pollination. endospermless1-1 resulted in an early loss of endosperm, yielding a giant embryo, suggesting that embryo growth was physically limited by the endosperm. A double mutant between giant embryo-2 and club-shaped embryo1-1, which has a normal endosperm and a minute undifferentiated embryo, resulted in a club-shaped embryo1-1 embryo and a reduced endosperm of giant embryo-2, indicating that GIANT EMBRYO regulates the endosperm development. Double mutants between giant embryo-2 and three reduced embryo mutants exhibited the reduced embryo phenotype in both embryo and endosperm, suggesting that reduced embryo mutations cause the enlarged endosperm. Further, a double mutant of reduced embryo3 and endospermless1-1 showed the enlarged embryo in endospermless seed. This confirms that reduced embryo3 does not regulate embryo size but enlarges endosperm size. Together with the results of the other double mutant analysis, REDUCED EMBRYO1, REDUCED EMBRYO2, REDUCED EMBRYO3 and GIANT EMBRYO are concluded to regulate endosperm development.

A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis.

Homeobox genes encode a large family of homeodomain proteins that play a key role in the pattern formation of animal embryos. By analogy, homeobox genes in plants are thought to mediate important processes in their embryogenesis, but there is very little evidence to support this notion. Here we described the temporal and spatial expression patterns of a rice homeobox gene, OSH1, during rice embryogenesis. In situ hybridization analysis revealed that in the wild-type embryo, OSH1 was first expressed at the globular stage, much earlier than organogenesis started, in a ventral region where shoot apical meristem and epiblast would later develop. This localized expression of OSH1 indicates that the cellular differentiation has already occurred at this stage. At later stages after organogenesis had initiated, OSH1 expression was observed in shoot apical meristem [except in the L1 (tunica) layer], epiblast, radicle, and their intervening tissues in descending strength of expression level with embryonic maturation. We also performed in situ hybridization analysis with a rice organless embryo mutant, orl1, that develops no embryonic organs. In the orl1 embryo, the expression pattern of OSH1 was the same as that in the wild-type embryo in spite of the lack of embryonic organs. This shows that OSH1 is not directly associated with organ differentiation, but may be related to a regulatory process before or independent of the organ determination. The results described here strongly suggest that, like animal homeobox genes, OSH1 plays an important role in regionalization of cell identity during early embryogenesis.

The abnormal distribution of mossy fiber bundles and morphological abnormalities in hippocampal formation of dreher(J) (dr(J)/dr(J))mouse.

The organization of pyramidal cells and mossy fibers in the hippocampal formation of homozygous dreher(J) mutant mice was investigated using Timm's and Golgi methods. Five clear abnormalities were found: (1) some pyramidal cells were located below the infrapyramidal mossy fiber layer, (2) mossy fibers emerged in diffuse fashion from between the suprapyramidal and infrapyramidal mossy fiber layers, and their fibers invaded within the pyramidal cell layer, where they traveled as 3-6 small, usually quite short, bundles, (3) some normally situated pyramidal cells had unusual contacts with mossy fibers at two or three places on their apical and/or basal dendrites, (4) some normally situated pyramidal cells had abnormal dendritic trees typified by the occurrence of fine-caliber dendritic branches extending out of the apical dendrite or the apical portion of the soma, and (5) a few Timm positive fibers extending from the dentate hilus to the dentate molecular layer in both dreher(J) and control mice were observed. These abnormalities indicate that in the hippocampal formation a variety of cell populations and neuronal circuits can be indirectly modified by the dreher mutation.

Morphological abnormalities in the hippocampus of the weaver mutant mouse.

The lamination of the hippocampus in the homozygous B6CBA weaver mouse (wv/wv) was compared with that in normal B6CBA littermates (+/+) and C57BL/6J mice using Nissl and Timm's staining. In Nissl-stained preparations, the normal littermates exhibit a compact, regular arrangement of pyramidal cells in area CA3 of the hippocampus. In contrast, in homozygous weaver mutant mice, the pyramidal cell layer of area CA3 frequently appears to be thicker than normal with an apparent increase of neuropil, as evidenced by the presence of cell-free spaces within the layer. Also, small ectopic clusters of pyramidal cells and sometimes the subdivision of the pyramidal cell layer into 2 or 3 layers were found throughout the dorsoventral extent of the hippocampus. In Timm's stained preparations of the normal mouse hippocampus, two clearly separated bundles of axons were seen emerging from the hilus: one bundle running above the pyramidal cell layer of area CA3 (i.e., the suprapyramidal mossy fiber layer, SPMFL), and the second bundle running below the pyramidal cell layer (i.e., the infrapyramidal mossy fiber layer, IPMFL). In contrast, in some homozygous weaver mice, the origin of the mossy fiber bundles is clearly different from normal; specifically, mossy fibers emerge in a diffuse fashion from the area between suprapyramidal and infrapyramidal mossy fiber layers. In other weaver mice, short, discontinuous bundles diverge from the infrapyramidal mossy fiber layer and invade the thickened pyramidal cell layer. In addition, ectopic pyramidal cells are situated below the IPMFL in area CA3. The morphological changes observed in hippocampus of weaver mutants are likely to be secondary to a more basic genetic defect.

Immunohistochemical detection of aberrant p53 expression in hepatocellular carcinoma: correlation with cell proliferative activity indices, including mitotic index and MIB-1 immunostaining.

We analyzed the p53 expression immunohistochemically in 50 specimens of hepatocellular carcinoma (HCC) using two monoclonal antibodies (DO7 and PAb1801) and one polyclonal antibody (CM1), which recognize both wild and mutant type p53 proteins and can be used for paraffin-embedded sections. Fifteen of the 50 HCC specimens (30%) showed p53 expression localized at tumor nuclei, and this expression was significantly more frequent in HCCs with histologically lower differentiation. Except for serum titers of alpha-fetoprotein, the p53 expression had no statistically significant correlation with clinicopathological parameters, including hepatitis virus infection, tumor size, and background liver diseases. Conversely, the cell proliferative activities of tumor cells as assessed by mitotic index and immunostaining for MIB-1 were well correlated with the grade of histological differentiation. Moreover, MIB-1 immunostaining was shown to be useful in distinguishing well differentiated HCC from hepatocytes in chronic liver diseases. It also was shown that p53 expression was strongly associated with cell proliferative activity. Our results indicate that p53 expression takes place in the late stage of tumor progression and is related to the high malignant potential of HCCs.