Pore configuration landscape of granular crystallization.

Uncovering grain-scale mechanisms that underlie the disorder-order transition in assemblies of dissipative, athermal particles is a fundamental problem with technological relevance. To date, the study of granular crystallization has mainly focussed on the symmetry of crystalline patterns while their emergence and growth from irregular clusters of grains remains largely unexplored. Here crystallization of three-dimensional packings of frictional spheres is studied at the grain-scale using X-ray tomography and persistent homology. The latter produces a map of the topological configurations of grains within static partially crystallized packings. Using numerical simulations, we show that similar maps are measured dynamically during the melting of a perfect crystal. This map encodes new information on the formation process of tetrahedral and octahedral pores, the building blocks of perfect crystals. Four key formation mechanisms of these pores reproduce the main changes of the map during crystallization and provide continuous deformation pathways representative of the crystallization dynamics.

Rapid induction of the growth hormone gene transcription by glucocorticoids in vitro: possible involvement of membrane glucocorticoid receptors and phosphatidylinositol 3-kinase activation.

The regulation of transcription of the growth hormone (GH) gene by glucocorticoids was studied in MtT/S cells, a cell line derived from an oestrogen-induced mammotrophic tumour in the rat, and in the primary culture of the anterior pituitary gland of adult mice. The levels of the GH heteronuclear RNA (GH hnRNA), which are mainly determined by the transcription rate, increased by 25-fold during 24 h in response to dexamethasone (DEX; 1 µM) in MtT/S cells that were cultured in the medium containing charcoal-stripped serum for 7 days. The stimulatory effect of DEX on the GH hnRNA levels was detectable as early as 30 min. This rapid effect of DEX did not require on-going protein synthesis, whereas it was considered that DEX requires the presence of unknown cellular proteins produced independently of DEX stimulation. By contrast, on-going protein synthesis was required for DEX action when incubated for 6 h, as has been observed in the previous studies. The specific inhibitor of glucocorticoid receptor, RU486, inhibited both rapid (30 min) and delayed (6 h) the effects of glucocorticoids on GH hnRNA levels. Membrane impermeable glucocorticoid, corticosterone-bovine serum albumin conjugate (CSBSA), was found to have effects similar to those of DEX and free corticosterone (CS), suggesting that glucocorticoids regulate GH gene transcription at least in part through the membrane bound receptors. From pharmacological studies, it was suggested that phosphatidylinositol 3-kinase (PI3K) activation is involved in the rapid effects but not in the delayed effects of glucocorticoids. This also suggests that the delayed effects of glucocorticoids depend on mechanisms other than the activation of PI3-kinase. Finally, both rapid and delayed effects of CS and CSBSA were observed not only in MtT/S cells, but also in the mouse pituitary cells in primary culture. Therefore, it is possible that the membrane initiated action of glucocorticoids is involved in the regulation of GH transcription in normal pituitary cells, as well as in pituitary tumour cells.

Epidermal growth factor-activated extracellular signal-regulated kinase suppresses growth hormone expression and stimulates proliferation in MtT/ E cells.

The mechanism for the inhibition of growth hormone (GH) expression by the epidermal growth factor (EGF) was examined in two clonal cell lines, MtT/E and MtT/S. The former has a negligible basal level of GH, whereas the latter has a high basal GH. The treatment of MtT/E cells with retinoic acid resulted in a significant increase in GH mRNA and subsequently GH. This stimulatory response to retinoic acid was strongly suppressed by EGF. This suppression was associated with an increase in the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2). The MEK [mitogen-activated protein kinase (MAPK) kinases that activate ERK1 and ERK2] inhibitor, PD98059, clearly inhibited the phosphorylation of Erk1/2 and restored the stimulatory effects of retinoic acid. These results suggest that the inhibitory effects of EGF on GH expression are mediated by MAPK activation in these cells. By contrast to the GH-producing clones examined previously, EGF showed a marked stimulation of proliferation of the MtT/E cells through a mechanism dependent on MAPK activation. On the other hand, the inhibitory effect of EGF on GH expression was less pronounced and the stimulation of cellular proliferation was not seen in MtT/S cells, even though it induced Erk-phosphorylation similar to that seen in MtT/E. The distinct difference in the response to EGF between these two GH cell lines appears to be attributed to differences in the function of MAPK cascade in each cell line. This may reflect the developmental stage of the cells from which MtT/E and MtT/S are derived.

Oxidative stress by biphenyl metabolites induces inhibition of bacterial cell separation.

Cell-cell separation of a polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 was monitored by flow cytometry. When monohydroxy metabolites of biphenyl (BP) (2-hydroxybiphenyl and 3-hydroxybiphenyl) were added to the culture, cell-cell separation of strain TK102 was inhibited at stationary phase. This inhibition was reproduced on non-PCB degrading bacteria such as Pseudomonas putida PpY101 and Escherichia coli MV1184, but was not observed on Pseudomonas aeruginosa PAO1. An opportunistic pathogen, P. aeruginosa PAO1, produces exopolysaccharide, which is known to scavenge damaging chemicals such as reactive oxygen species (ROS). The higher level of ROS and lipid peroxidants were detected in the cells treated by monohydroxybiphenyls. Fat-soluble vitamin E, which is a lipid radical scavenger, maintained bacterial cell separation during monohydroxybiphenyls treatment. Our results demonstrated that intracellular oxidative stress played an important role in the inhibition of bacterial cell separation during BP metabolism. This study shows that metabolites of environmental pollutants, such as monohydroxylated BP, inhibit bacterial cell separation by oxidative stress.

Cytological affinities and interfertilities between Lolium temulentum and L. persicum (Poaceae) accessions.

Interspecific crossing between L. temulentum L. and L. persicum Boiss. & Hohen. ex Boiss. was performed to clarify their interfertility based on the results of chromosome pairing, pollen fertility and seed set. Both parents were normal with a high percentage of chromosome association of ring bivalents in contrast to rod bivalents at metaphase I, pollen fertility and seed set, but F1 hybrids showed different proportions of them for each crossing combination. Chromosome affinity expressed by pairing was certainly a factor affecting the pollen fertility or seed set in F1 hybrids, but it was not the most important. The positive correlation was generally found between pollen fertility and seed set of F1 hybrids. The L. persicum accession with relatively high interfertility with L. temulentum was supposed to be derived from natural hybridization between L. temulentum and L. persicum. The degree of cytogenetic differentiation between L. temulentum and L. persicum existed because of lower chromosomal pairing, pollen fertility and seed set, but their F1 hybrids were partially fertile.

Novel collagen sponge reinforced with polyglycolic acid fiber produces robust, normal hair in murine hair reconstitution model.

The hair reconstitution assay is a useful system for studying cell-cell and epithelial-mesenchymal interaction. The current method consists of transplantation of both epidermal and dermal cells, using a silicone chamber placed on an athymic nude mouse. However, because of leakage and tilting of the grafted cells, the rate and area of hair growth vary depending on the chamber. We modified this method by using a collagen sponge as a scaffold and compared two types of collagen sponges, each having different tensile strengths. A conventional collagen sponge disturbed normal hair follicle formation; in contrast, a collagen sponge containing polyglycolic acid (PGA) fiber supported proper restructuring of skin and hair follicles. These data suggested the usefulness of PGA fiber-containing collagen sponges for hair reconstitution in research and clinical applications.

Dynamic behavior of microtubules during dynein-dependent nuclear migrations of meiotic prophase in fission yeast.

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.

Mitotic specific phosphorylation of serine-1212 in human DNA topoisomerase IIalpha.

It is known that topoisomerase IIalpha is phosphorylated by several kinases. To elucidate the role of phosphorylation of topoisomerase IIalpha in the cell cycle, we have examined the cell cycle behavior of phosphorylated topoisomerase IIalpha in HeLa cells using antibodies against several phospho-oligopeptides of this enzyme. Here we demonstrate that serine1212 in topoisomerase IIalpha is phosphorylated only in the mitotic phase. Using an antibody against an oligopeptide containing phosphoserine-1212 in topoisomerase IIalpha (PS1212), subcellular localization of topoisomerase IIalpha phosphorylated at serine1212 was examined by indirect immunofluorescence staining, and compared with that of overall topoisomerase IIalpha. Serine1212-phosphorylated topoisomerase IIalpha was localized specifically on mitotic chromosomes, but not on interphase chromosomes; this result contrasts with overall topoisomerase IIalpha which was observed on chomosomes in both interphase and mitosis. Serine1212-phosphorylated topoisomerase lIalpha first appeared on chromosome arms in prophase, became concentrated on the centromeres in metaphase, and disappeared in early telophase. In addition, ICRF-193, a catalytic inhibitor of topoisomerase II, prevented accumulation of serine1212-phosphorylated topoisomerase IIalpha at the centromeres. These results indicate that serine1212 of topoisomerase IIalpha is phosphorylated specifically during mitosis, and suggest that the serine1212-phosphorylated topoisomerase IIalpha acts on resolving topological constraint progressively from the chromosome arm to the centromere during metaphase chromosome condensation.

Live observation of fission yeast meiosis in recombination-deficient mutants: a study on achiasmate chromosome segregation.

Regular segregation of homologous chromosomes during meiotic divisions is essential for the generation of viable progeny. In recombination-proficient organisms, chromosome disjunction at meiosis I generally occurs by chiasma formation between the homologs (chiasmate meiosis). We have studied meiotic stages in living rec8 and rec7 mutant cells of fission yeast, with special attention to prophase and the first meiotic division. Both rec8 and rec7 are early recombination mutants, and in rec7 mutants, chromosome segregation at meiosis I occurs without any recombination (achiasmate meiosis). Both mutants showed distinct irregularities in nuclear prophase movements. Additionally, rec7 showed an extended first division of variable length and with single chromosomes changing back and forth between the cell poles. Two other early recombination deficient mutants (rec14 and rec15) showed very similar phenotypes to rec7 during the first meiotic division, and the fidelity of achiasmate chromosome segregation slightly exceeded the expected random level. We discuss possible regulatory mechanisms of fission yeast to deal with achiasmate chromosome segregation.

A conserved protein, Nuf2, is implicated in connecting the centromere to the spindle during chromosome segregation: a link between the kinetochore function and the spindle checkpoint.

The centromere is crucial for the proper segregation of chromosomes in all eukaryotic cells. We identified a centromeric protein, Nuf2, which is conserved in fission yeast, human, nematode, and budding yeast. Gene disruption of nuf2+ in the fission yeast Schizosaccharomyces pombe caused defects in chromosome segregation and the spindle checkpoint: the mitotic spindle elongated without segregating the chromosomes, indicating that spindle function was compromised, but that this abnormality did not result in metaphase arrest. Certain nuf2 temperature-sensitive mutations, however, caused metaphase arrest with condensed chromosomes and a short spindle, indicating that, while these mutations caused abnormalities in spindle function, the spindle checkpoint pathway remained intact. Metaphase arrest in these cells was dependent on the spindle checkpoint component Mad2. Interestingly, Nuf2 disappeared from the centromere during meiotic prophase when centromeres lose their connection to the spindle pole body. We propose that Nuf2 acts at the centromere to establish a connection with the spindle for proper chromosome segregation, and that Nuf2 function is also required for the spindle checkpoint.

Telomere binding of the Rap1 protein is required for meiosis in fission yeast.

Telomeres are essential for chromosome integrity, protecting the ends of eukaryotic linear chromosomes during cell proliferation. Telomeres also function in meiosis; a characteristic clustering of telomeres beneath the nuclear membrane is observed during meiotic prophase in many organisms from yeasts to plants and humans, and the role of the telomeres in meiotic pairing and the recombination of homologous chromosomes has been demonstrated in the fission yeast Schizosaccharomyces pombe and in the budding yeast Saccharomyces cerevisiae. Here we report that S. pombe Rap1 is a telomeric protein essential for meiosis. While Rap1 is conserved in budding yeast and humans, schemes for telomere binding vary among species: human RAP1 binds to the telomere through interaction with the telomere binding protein TRF2; S. cerevisiae Rap1, however, binds telomeric DNA directly, and no orthologs of TRF proteins have been identified in this organism. In S. pombe, unlike in S. cerevisiae, an ortholog of human TRF has been identified. This ortholog, Taz1, binds directly to telomere repeats [18] and is necessary for telomere clustering in meiotic prophase. Our results demonstrate that S. pombe Rap1 binds to telomeres through interaction with Taz1, similar to human Rap1-TRF2, and that Taz1-mediated telomere localization of Rap1 is necessary for telomere clustering and for the successful completion of meiosis. Moreover, in taz1-disrupted cells, molecular fusion of Rap1 with the Taz1 DNA binding domain recovers telomere clustering and largely complements defects in meiosis, indicating that telomere localization of Rap1 is a key requirement for meiosis.

T cell-mediated immune response enhances the severity of myocarditis in secondary cardiotropic virus infection in mice.

In this report, we showed that a previous enterovirus exposure in ordinary mice with normal T cell function, but not in T cell-deficient mice, can influence development of myocardial inflammation with a second virus exposure. Inoculation of 4-week-old male BALB/c-nu/+ (euthymic and normal T cell function) mice with amyocarditic Coxsackie virus B1 (CB1), followed by inoculation 28 days later with myocarditic variant of Coxsackie virus B3 (CB3-m) resulted in more intense myocardial inflammation and injury than was seen in BALB/c-nu/+ inoculated with CB1, followed by inoculation with non-enterovirus, i.e., encephalomyocarditis virus (EMC) or influenza A virus and in age-matched BALB/c-nu/+ mice secondary inoculated with CB3-m alone. In contrast, this phenomenon of the enhancement of the severity of myocarditis by a secondary CB3-m inoculation was not seen in BALB/c-nu/nu (athymic and T cell- deficient) mice. Interestingly, inoculation of BALB/c-nu/+ mice with CB1, followed by inoculation 28 days later with another amyocarditic variant of Coxsackie virus B3 (CB3-o), resulted in more severe myocarditis than was seen in age-matched BALB/c-nu/+ mice secondary inoculated CB3-o alone. Myocardial-activated T cells and elevated serum interleukin-6 were involved in the exacerbation of the disease during the reinfection. T cell-mediated immune responses to a conserved antigenic epitope among the enteroviruses may be involved in the exacerbation of myocardial inflammatory disease during a second enterovirus infection.

CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells.

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.

Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom Phaeodactylum tricornutum.

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO(2)-grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO(2) conditions. Ethoxyzolamide (0.4 mM), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mM acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to beta-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO(2)-binding site appeared to be unique among the beta-CAs so far reported.

A novel meiosis-specific protein of fission yeast, Meu13p, promotes homologous pairing independently of homologous recombination.

Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation. We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination. Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe. Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not. We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion. Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe. Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.

Identification of human endomucin-1 and -2 as membrane-bound O-sialoglycoproteins with anti-adhesive activity.

Using a signal sequence trap method and database search, we identified a series of human cDNAs encoding two structurally related type I membrane proteins of approximately 25 kDa with multiple glycosylation motifs. These genes, termed endomucin-1/-2, are expressed in several human tissues including heart, kidney, and lung. Exogenously expressed human endomucin-1/-2 proteins were modified into 80-120 kDa glycoproteins, which were susceptible to O-sialoglycoprotein endopeptidase digestion. Transient overexpression of endomucin-1/-2 reduced the number of adhesion plaques and reduced cell attachment to the substrate. This phenotype was suppressed by laminin or the protein kinase inhibitor staurosporine. Our findings suggest that human endomucin-1/-2 negatively regulate cell adhesion to the extracellular matrix.

mRNA sequence of the Xenopus laevis paxillin gene and its expression.

Paxillin is a cytoskeletal protein found in structures of focal adhesions where cells adhere to the extracellular matrix. We isolated paxillin cDNA from the Xenopus laevis ovary. The cDNA sequence encodes a protein of 539 amino acids with four LIM and five LD motifs. 80% of the amino acids of frog paxillin are shared by human and chicken paxillins. Northern analysis showed that the frog gene is expressed in the spleen, kidney, testis and ovary. Immunocytochemistry showed that paxillin protein is accumulated in the nucleus as well as in the periphery of the cytoplasm of the A6 cell. This intriguing result shows that paxillin, which has been characterized as a cytoskeletal protein, is capable of translocating to the nucleus.

How do meiotic chromosomes meet their homologous partners?: lessons from fission yeast.

Homologous chromosome pairing is required for proper chromosome segregation and recombination during meiosis. The mechanism by which a pair of homologous chromosomes contact each other to establish pairing is not fully understood. When pairing occurs during meiotic prophase in the fission yeast, Schizosaccharomyces pombe, the nucleus oscillates between the cell poles and telomeres remain clustered at the leading edge of the moving nucleus. These meiosis-specific activities produce movements of telomere-bundled chromosomes. Several lines of evidence suggest that these movements facilitate homologous chromosome pairing by aligning homologous chromosomes and promoting contact between homologous regions. Since telomere clustering and nuclear or chromosome movements in meiotic prophase have been observed in a wide range of eukaryotic organisms, it is suggested that telomere-mediated chromosome movements are general activities that facilitate homologous chromosome pairing.

Schizosaccharomyces pombe taf1+ is required for nitrogen starvation-induced sexual development and for entering the dormant GO state.

Environmental change, such as nutritional starvation, induces physiological and morphological alterations that enable fission yeast cells to survive. We isolated a novel gene, taf1+, required for the response to nitrogen starvation in the fission yeast Schizosaccharomyces pombe. taf1 disruptants could not mate upon nitrogen starvation, but could upon carbon starvation. taf1 disruptants had a defect in inducing stell+ expression under nitrogen starvation conditions. Furthermore, they lost viability quickly in nitrogen-depleted medium. Unlike wild-type cells, starved taf1-cells had nuclear chromatin that were flat and adhered to the cell periphery. These results indicate that tqf1+ is required for nitrogen starvation-induced sexual development and entering the dormant G0 state.