Stim1 Regulates Enamel Mineralization and Ameloblast Modulation.

Loss-of-function mutations in the Ca2+ release-activated Ca2+ channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE) and result in ectodermal dysplasia with amelogenesis imperfecta. However, because of the limited availability of patient tissue, analyses of enamel mineralization or possible changes in ameloblast function or morphology have not been possible. Here, we generated mice with ectodermal tissue-specific deletion of Stim1 ( Stim1 cKO [conditional knockout]), Stim2 ( Stim2 cKO), and Stim1 and Stim2 ( Stim1/2 cKO) and analyzed their enamel phenotypes as compared with those of control ( Stim1/2fl/fl) animals. Ablation of Stim1 and Stim1/2 but not Stim2 expression resulted in chalky enamel and severe attrition at the incisor tips and molar cusps. Stim1 and Stim1/2 cKO, but not Stim2 cKO, demonstrated inferior enamel mineralization with impaired structural integrity, whereas the shape of the teeth and enamel thickness appeared to be normal in all animals. The gene expression levels of the enamel matrix proteins Amelx and Ambn and the enamel matrix proteases Mmp20 and Klk4 were not altered by the abrogation of SOCE in Stim1/2 cKO mice. The morphology of ameloblasts during the secretory and maturation stages was not significantly altered in either the incisors or molars of the cKO animals. However, in Stim1 and Stim1/2 cKO incisors, the alternating modulation of maturation-stage ameloblasts between the smooth- and ruffle-ended cell types continued beyond the regular cycle and extended to the areas corresponding to the zone of postmodulation ameloblasts in the teeth of control animals. These results indicate that SOCE is essential for proper enamel mineralization, in which Stim1 plays a critical role during the maturation process.

Metal levels in sugar cane (Saccharum spp.) samples from an area under the influence of a municipal landfill and a medical waste treatment system in Brazil.

In July 2003, duplicated samples of roots, stems and leaves of sugar cane (Saccharum spp.) were collected in 25 points of an area under direct influence of the municipal landfill site (MLS) and medical waste treatment system (MWTS) of Ribeirao Preto, São Paulo, Brazil. Cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), manganese (Mn), lead (Pb) and zinc (Zn) were determined by atomic absorption spectrophotometry. The following concentrations (mg/kg) were found in roots: Cd, 0.22+/-0.12; Cr, 64.3+/-48.7; Cu, 140.6+/-27.7; Hg, 0.04+/-0.02; Mn, 561.6+/-283.3; Pb, 7.9+/-2.1 and Zn, 177.4+/-64.9. For some metals, these levels are higher than the concentrations previously reported for different plants, reaching, in some cases, values that might be considered toxic for vegetables. Metal levels in stems were 80-90% of those found in roots, while the concentrations detected in leaves were significantly lower than those in roots. The present results suggest that MLS and MWTS activities might have been increasing metal concentrations in edible tissues of sugar cane grown in the area under their influence. Moreover, the traditional agricultural practices in the production of sugar cane could be also another determinant factor to reach the current metal levels. The results of this study indicate that sugar cane is a crop that is able to grow in areas where metals in soils are accumulated.

Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation.

DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.

Evolution of CHR-2 SINEs in cetartiodactyl genomes: possible evidence for the monophyletic origin of toothed whales.

Short interspersed repetitive elements (SINEs) are a kind of retroposons dispersed among the eukaryotic genomes. Previously, we isolated and characterized a new SINE family, named CHR-2, members of which are distributed in the genomes of cetaceans, hippopotamuses, and ruminants. We analyzed systematically more than a hundred members of the CHR-2 SINEs, which were isolated from the genomes of cetaceans and cow, together with the additional data available in the DNA databases, and showed that these SINEs are divided into at least five distinct subfamilies that share diagnostic nucleotides and/or deletions. A hybridization analysis clearly demonstrated that, among these five subfamilies, two subfamilies, named CD and CDO, are specific to cetaceans and toothed whales, respectively. We reconstruct the evolutionary history of the CHR-2 SINEs during evolution of cetartiodactyl genomes.

Maximum likelihood analysis of the complete mitochondrial genomes of eutherians and a reevaluation of the phylogeny of bats and insectivores.

The complete mitochondrial genomes of two microbats, the horseshoe bat Rhinolophus pumilus, and the Japanese pipistrelle Pipistrellus abramus, and that of an insectivore, the long-clawed shrew Sorex unguiculatus, were sequenced and analyzed phylogenetically by a maximum likelihood method in an effort to enhance our understanding of mammalian evolution. Our analysis suggested that (1) a sister relationship exists between moles and shrews, which form an eulipotyphlan clade; (2) chiropterans have a sister-relationship with eulipotyphlans; and (3) the Eulipotyphla/Chiroptera clade is closely related to fereuungulates (Cetartiodactyla, Perissodactyla and Carnivora). Divergence times on the mammalian tree were estimated from consideration of a relaxed molecular clock, the amino acid sequences of 12 concatenated mitochondrial proteins and multiple reference criteria. Moles and shrews were estimated to have diverged approximately 48 MyrBP, and bats and eulipotyphlans to have diverged 68 MyrBP. Recent phylogenetic controversy over the polyphyly of microbats, the monophyly of rodents, and the position of hedgehogs is also examined.

Retroposon analysis of major cetacean lineages: the monophyly of toothed whales and the paraphyly of river dolphins.

SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram, and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: (i) Odontocetes are monophyletic; (ii) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; (iii) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and (iv) the rapid radiation of extant cetacean lineages occurred some 28-33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.

CetSINEs and AREs are not SINEs but are parts of cetartiodactyl L1.

Short interspersed repetitive elements (SINEs) are widely distributed among the genomes of eukaryotes. We proposed previously that a SINE should be defined by the presence of a region homologous to a tRNA or to 7SL RNA, together with A-box and B-box promoter sequences, in order to distinguish SINEs from other short repetitive sequences, such as short segments of LINEs (long interspersed repetitive elements; Okada et al. Gene 205, 229-243, 1997). Numerous SINE sequences have been deposited to date in DNA databases. In some cases, however, designation of a particular sequence is problematic when the short repetitive sequence has been defined as a SINE without reference to the presence or absence of promoter elements specific for RNA polymerase III. We demonstrate here that four different sequences, namely, ARE1p, ARE2p, CetSINE1, and CetSINE2, each of which has been reported as a SINE, are, in fact, only partial sequences of members of a new subfamily of L1. We also demonstrate that members of this subfamily are distributed specifically among the genomes of cetartiodactyls.

Consistency of SINE insertion topology and flanking sequence tree: quantifying relationships among cetartiodactyls.

Short interspersed nuclear elements (SINEs) have been used to generate unambiguous phylogenetic topologies relating eukaryotic taxa. The irreversible nature of SINE retroposition is supported by a large body of comparative genome data and is a fundamental assumption inherent in the value of this qualitative method of inference. Here, we assess the key assumption of unidirectional SINE insertion by comparing the SINE insertion-derived topology and the phylogenetic tree based on seven independent loci of five taxa in the order Cetartiodactyla (Cetacea + Artiodactyla). The data sets and analyses were largely independent, but the loci were, by definition, linked, and thus their consistency supported an irreversible pattern of SINE retroposition. Moreover, our analyses of the flanking sequences provided estimates of divergence times among cetartiodactyl lineages unavailable from SINE insertion analysis alone. Unexpected rate heterogeneity among sites of SINE-flanking sequences and other noncoding DNA sequences were observed. Sequence simulations suggest that this rate heterogeneity may be an artifact resulting from the inaccuracies of the substitution model used.

Monophyletic origin of the order chiroptera and its phylogenetic position among mammalia, as inferred from the complete sequence of the mitochondrial DNA of a Japanese megabat, the Ryukyu flying fox (Pteropus dasymallus).

Complete sequences of mitochondrial DNA (mtDNA) are useful for the reconstruction of phylogenetic trees of mammals and, in particular, for inferring higher-order relationships in mammals. In this study, we determined the complete sequence (16,705 bp) of the mtDNA of a Japanese megabat, the Ryukyu flying fox (Pteropus dasymallus). We analyzed this sequence phylogenetically by comparing it with the complete sequence of mtDNAs of 35 mammals in an effort to reevaluate the enigmatic relationship between Megachiroptera and Microchiroptera and the relationships between them and other mammals. Maximum-likelihood analysis of 12 concatenated mitochondrial proteins from 36 mammals strongly suggested the monophyly of the order Chiroptera and its close relationship to Fereuungulata (Carnivora + Perissodactyla + Cetartiodactyla). We estimated that megabats and microbats diverged approximately 58 MyrBP and discussed the origin and early evolution of Chiroptera based on our findings.

Interordinal relationships and timescale of eutherian evolution as inferred from mitochondrial genome data.

Extensive phylogenetic analyses of the updated sequence data of mammalian mitochondrial genomes were carried out using the maximum likelihood method in order to resolve deep branchings in eutherian evolution. The divergence times in the mammalian tree were estimated by a relaxed molecular clock of the mitochondrial proteins calibrated with multiple references. A Chiroptera/Eulipotyphla (i.e. bat/mole) clade and a close relationship of this clade to Fereuungulata (Carnivora+Perissodactyla+Cetartiodactyla) were reconfirmed with high statistical significance. However, a support for a monophyly of Fereuungulata relative to the Chiroptera/Eulipotyphla clade was fragile, and we suggest that the three branchings among Carnivora, Perissodactyla, Cetartiodactyla and Chiroptera/Eulipotyphla occurred successively in a short time period, estimated to be approximately 77Myr BP. The Chiroptera/Eulipotyphla divergence was estimated to roughly coincide with the Cretaceous-Tertiary boundary (65Myr BP). The monophyly of Rodentia, the Lagomorpha/Rodentia clade (traditionally called Glires), and the Afrotheria/Xenarthra clade were preferred over alternative relationships, but the supports of these clades were not strong enough to exclude other possibilities. Although several super-order taxa of eutherians were strongly supported by the analyses of the mitochondrial genome data, the branching order in the deepest part of the eutherian tree remained ambiguous from the data presently available.

Phylogenetic relationships among cetartiodactyls based on insertions of short and long interpersed elements: hippopotamuses are the closest extant relatives of whales.

Insertion analysis of short and long interspersed elements is a powerful method for phylogenetic inference. In a previous study of short interspersed element data, it was found that cetaceans, hippopotamuses, and ruminants form a monophyletic group. To further resolve the relationships among these taxa, we now have isolated and characterized 10 additional loci. A phylogenetic analysis of these data was able to resolve relationships among the major cetartiodactyl groups, thereby shedding light on the origin of whales. The results indicated (i) that cetaceans are deeply nested within Artiodactyla, (ii) that cetaceans and hippopotamuses form a monophyletic group, (iii) that pigs and peccaries form a monophyletic group to the exclusion of hippopotamuses, (iv) that chevrotains diverged first among ruminants, and (v) that camels diverged first among cetartiodactyls. These findings lead us to conclude that cetaceans evolved from an immediate artiodactyl, not mesonychian, ancestor.

Genealogy of families of SINEs in cetaceans and artiodactyls: the presence of a huge superfamily of tRNA(Glu)-derived families of SINEs.

Several novel (sub)families of SINEs were isolated from the genomes of cetaceans and artiodactyls, and their sequences were determined. From comparisons of diagnostic nucleotides among the short interspersed repetitive elements (SINEs) in these (sub)families, we were able to draw the following conclusions. (1) After the divergence of the suborder Tylopoda (camels), the CHRS family of SINEs was newly created from tRNA(Glu) in a common ancestor of the lineages of the Suina (pigs and peccaries), Ruminantia (cows and deer), and Cetacea (whales and dolphins). (2) After divergence of the Suina lineage, the CHR-1 SINE and the CHR-2 SINE were generated successively in a common ancestor of ruminants, hippopotamuses, and cetaceans. (3) In the Ruminantia lineage, the Bov-tA SINE was generated by recombination between the CHR-2 SINE and Bov-A. (4) In the Suina lineage, the CHRS-S SINE was generated from the CHRS SINE. (5) In this latter lineage, the PRE-1 family of SINEs was created by insertion of part of the gene for tRNA(Arg) into the 5' region of the CHRS-S family. The distribution of a particular family of SINEs among species of artiodactyls and cetaceans confirmed the most recent conclusion for paraphyly of the order Artiodactyla. The present study also revealed that a newly created tRNA(Glu)-derived family of SINEs was subjected both to recombination with different units and to duplication of an internal sequence within a SINE unit to generate, during evolution, a huge superfamily of tRNA(Glu)-related families of SINEs that are now found in the genomes of artiodactyls and cetaceans.

Restricted expression of the receptor serine/threonine kinase BMPR-IB in zebrafish.

Bone morphogenetic proteins (BMPs) comprise a rapidly expanding subclass of the transforming growth factor-beta superfamily. They are known to regulate a diverse range of developmental phenomena including cell differentiation, morphogenesis and apoptosis. In this study, we have isolated a zebrafish homolog of BMP type IB receptor (BMPR-IB) and examined the localization of the transcripts during embryogenesis. Whole-mount in situ hybridization analysis revealed that unlike other type I and type II receptors that mediate BMP signal, it is expressed in developing somite and in mid-hind brain region in a restricted manner.

Direct Observation of Foam Film Rupture by Several Types of Antifoams Using a Scanning Laser Microscope.

It is well known that the stability of a pseudoemulsion film, a thin liquid film formed between an antifoam particle and air, can be important during defoaming action. We have compared the bursting behavior of a two-dimensional thin liquid film from an aqueous surfactant solution on a glass plate, in which six types of antifoam particles have been dispersed, using laser microscopic techniques. The used antifoams were a silicone oil, a mixed-type antifoam (mixture of silicone oil and hydrophobic solid particles), a hydrophobic silica, and a silicone-based solid antifoam (prepared by interfacial polymerization with oil and water). The antifoaming performance of these six types of antifoams, measured by the glass cylinder shaking test, was in the order: the mixed-type antifoam >== the silicone-based solid antifoam > the hydrophobic silica >== the silicone oil. Pseudoemulsion film rupture was observed at the film thickness less than 0.1 µm for both the silocone oil and the hydrophobic silica. In the case of particles with rough edges (the silicone-based antifoam), pseudoemulsion film on the top of the particles can be easily ruptured at a convex part of the solid surface. Furthermore, solid particles existing on the surface of an oil droplet in the mixed-type antifoams form marked projections and these projections give rise to distortion of the thinning film. As soon as the distortion of the thinning film took place on the top of the antifoam droplet, the pseudoemulsion film can be instantaneously ruptured. Referring to the antifoaming mechanisms for mixed-type antifoams, both steps from the pseudoemulsion film formation to the lens formation and from the counter pseudoemulsion film formation to the bridge formation would be very fast and cause a very high antifoaming efficiency. Copyright 1999 Academic Press.

In vivo analysis using variants of zebrafish BMPR-IA: range of action and involvement of BMP in ectoderm patterning.

It has been an intriguing problem whether the polypeptide growth factors belonging to the transforming growth factor-beta (TGF-beta) superfamily function as direct and long-range signaling molecules in pattern formation of the early embryo. In this study, we examined the mechanism of signal propagation of bone morphogenetic protein (BMP) in the ectodermal patterning of zebrafish embryos, in which BMP functions as an epidermal inducer and a neural inhibitor. To estimate the effective range of zbmp-2, we first performed whole-mount in situ hybridization analysis. The zbmp-2-expressing domain and the neuroectoderm, marked by otx-2 expression, were complementary, suggesting that BMP has a short-range effect in vivo. Moreover, mosaic experiments using a constitutively active form of a zebrafish BMP type I receptor (CA-BRIA) demonstrated that the cell-fate conversion, revealed by ectopic expression of gata-3 and repression of otx-2, occurred in a cell-autonomous manner, denying the involvement of the relay mechanism. We also found that zbmp-2 was induced cell autonomously within the transplanted cells in the host ectoderm, suggesting that BMP cannot influence even the neighboring cells. This result is consistent with the observation that there is no gap between the expression domains of zbmp-2 and otx-2. Taken together, we propose that, in ectodermal patterning, BMP exerts a direct and cell-autonomous effect to fate uncommitted ectodermal cells to become epidermis.

Stability Factors of Foam Film in Contrast to Fluctuation Induced by Humidity Reduction

We have examined the relationship between dynamic surface properties of aqueous SDS solutions and foam film stability by observing the dynamic surface tension (gammat ), elongation of the lamellae (L lamellae ), and the thickness of the lamellae measured by FT-IR. Three foam film models were used by controlling such factors as the Marangoni effect by changing the SDS concentration, the disjoining pressure with the addition of electrolytes, and the surface viscosity with the addition of glycerin. In order to obtain information about the most effective factor in foam film stability, the lifetime of these films was measured under the circumstances of low relative humidity, at which vigorous fluctuation occured in the foam film. Foam film stability was found to be remarkably enhanced by the Marangoni effect and surface shear viscosity in a thin aqueous film at a relative humidity between 60 and 75%. Under these conditions, mild fluctuation would occur in the films. On the other hand, foam film stability was found to be maintained by the disjoining pressure in a thin aqueous film at the humidity below 60%. Under these conditions, vigorous fluctuation would occur in the foam film. It is concluded that foam film stability of an ionic surfactant solution mainly depends on the disjoining pressure which generates a repulsion force between the two surfaces in contrast to the vigorous fluctuation induced by humidity reduction.

Conservation of BMP signaling in zebrafish mesoderm patterning.

To investigate the conservation of mechanisms for mesodermal patterning between zebrafish and Xenopus, we isolated two cDNA clones encoding bone morphogenetic protein (BMP)-related proteins from a zebrafish cDNA library. Based on their predicted amino acid sequences, these two clones were designated as zbmp-2 and zbmp-4. Whole-mount in situ hybridization analysis revealed that in gastrula embryo, both genes were localized in the ventral part of the embryo, consistent with the proposed function of Xenopus BMP-4 in ventral mesoderm specification. zbmp-4 expression, however, was also seen in the embryonic shield, the most dorsal mesodermal structure. To examine the ability of zbmp-2 to ventralize mesoderm, we injected synthetic mRNA into zebrafish embryos and found that overexpression of this gene eliminated dorsal structures including notochord at both morphological and molecular level. In contrast, expression of ventral marker gene eve1 was expanded to the dorsal side. These effects are analogous to the ventralization of embryos caused by ectopic xBMP-4 expression. Taken together, one may conclude that the developmental mechanisms for mesodermal patterning regulated by BMPs are evolutionarily conserved between amphibians and teleosts.

Performance of Meridian ImmunoCard Mycoplasma test in a multicenter clinical trial.

Serology is the principal laboratory method used to diagnose Mycoplasma pneumoniae infection. Meridian Diagnostics has developed the ImmunoCard Mycoplasma kit, a 10-min card-based enzyme-linked immunosorbent assay (ELISA) designed to detect immunoglobulin M (IgM) antibodies to M. pneumoniae. We compared the ImmunoCard with two M. pneumoniae IgM-specific assays (immunofluorescence assay [IFA] and ELISA) and a standard complement fixation (CF) procedure using 896 specimens submitted to clinical laboratories for M. pneumoniae serology. Equivocal results obtained by CF, IFA, or ELISA were resolved by testing with an additional method or by reviewing patient chart information. The ImmunoCard had sensitivities ranging from 74% compared with the ELISA to 96% compared with CF results with IFA. ImmunoCard specificities ranged from 85% compared with the IgM-specific ELISA to 98% compared with IgM-specific IFA results resolved with clinical chart review. We also compared the ImmunoCard results with consensus results of 694 specimens tested on at least two non-ImmunoCard methods because of the lack of a "gold standard" for M. pneumoniae serology. Overall, the ImmunoCard Mycoplasma IgM assay had 90% sensitivity, 93% specificity, and 92% agreement with the consensus results. The ImmunoCard is technically less complex and requires less equipment that the three other assays. Our results indicate that the ImmunoCard Mycoplasma IgM assay is a valid and simple procedure which can reduce technologist time (and, thus, labor cost) and turnaround time for laboratories analyzing small numbers of specimens (< 10 per batch) submitted for IgM anti-M. pneumoniae testing.

Health of infant is the main reason for breast-feeding in a WIC population in Hawaii.

OBJECTIVE: To determine factors influencing infant feeding method choices among women who received services from the Special Supplemental Food Program for Women, Infants, and Children (WIC) during pregnancy in Hawaii. DESIGN: A retrospective survey mailed to all resident mothers who had live births in Hawaii between January 1, 1989, and March 31, 1989, and who received WIC services during pregnancy. SETTING: The state of Hawaii. SUBJECTS: The subjects were 322 mothers who received WIC services during pregnancy for whom data were complete, taken from a sample of 2,013 women who had live births in the state of Hawaii (51% response rate) between January 1, 1989, and March 31, 1989, of whom 324 participated in WIC during pregnancy. MAIN OUTCOME MEASURES: Infant feeding method (exclusive breast, exclusive formula or mixed) at hospital discharge. STATISTICAL ANALYSES PERFORMED: The chi 2 analysis for differences among feeding method groups and multiple logistic regression to calculate odds ratios for independent effects of maternal characteristics and other influences on feeding method choice. RESULTS: Reasons for infant feeding choice, timing of the choice, maternal age, parity, and place of residence were the main factor influencing infant method choice. Health of the infant was the primary reason for choice of feeding method and was the strongest determinant of exclusive breast-feeding (odds ratio = 23.99; confidence interval = 9.75-59.02; P < .0001) and was negatively related to the choice to formula-feed (odds ratio = 0.03; confidence interval = 0.01-0.08; P < .0001). APPLICATIONS: Relationship between WIC and hospital lactation consultants could enhance follow up in the hospital and after returning home with WIC mothers who prenatally state an intention to breast-feed. Convenience and bonding are aspects of breast-feeding enjoyed by WIC mothers in Hawaii that could be used in breast-feeding promotion efforts.