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The Tbx6 T-box genes are expressed in somite precursor cells of vertebrate embryos and are essential for the differentiation of paraxial mesoderm. However, it is unclear how spatial regulation of the gene expression is controlled and how the genes function to promote muscle differentiation. The Tbx6-related gene As-T2 of the ascidian Halocynthia roretzi is first expressed very transiently in endodermal cells around the 32- approximately 44-cell stage, is then expressed distinctly and continuously in muscle precursor cells, and later in epidermal cells situated in the distal tip region of the elongating tail. We now show that inhibition of As-T2-mediated transcriptional activation by microinjection of As-T2/En(R) into one-cell embryos resulted in suppression of the expression of the muscle-specific actin gene (HrMA4) and myosin heavy chain gene (HrMHC), but the injection did not affect the differentiation of endodermal cells or tail tip cells, suggesting that the primary function of As-T2 is associated with muscle cell differentiation. The 5' flanking region of As-T2 contains two promoter modules that regulate its specific expression: a distal module that responsible for its specific expression in the tail, and a proximal module required for its muscle-specific expression. Around the proximal module, there are two putative T protein-binding motifs (TTCACACTT). Co-injection of an As-T2/lacZ construct with or without the T-binding motifs together with As-T2 mRNA revealed that these motifs are essential for autoregulatory activation of the gene itself. In addition, we found that the minimal promoter regions of HrMA4 and HrMHC contain T-binding motifs. Co-injection of HrMA4/lacZ or HrMHC/lacZ containing the T-binding motifs along with As-T2 mRNA revealed that As-T2 protein binds to these motifs to upregulate the gene activity. Taking into account the recent finding of maternal molecules for muscle differentiation, we propose a model for a genetic cascade that includes As-T2 as a regulator of muscle cell differentiation in the ascidian embryo.

In early Ciona embryos, nuclear accumulation of beta-catenin is most probably the first step of endodermal cell specification. If beta-catenin is mis- and/or overexpressed, presumptive notochord cells and epidermal cells change their fates into endodermal cells, whereas if beta-catenin nuclear localization is downregulated by the overexpression of cadherin, the endoderm differentiation is suppressed, accompanied with the differentiation of extra epidermal cells ( Imai, K., Takada, N., Satoh, N. and Satou, Y. (2000) Development 127, 3009-3020). Subtractive hybridization screens of mRNAs between beta-catenin overexpressed embryos and cadherin overexpressed embryos were conducted to identify potential beta-catenin target genes that are responsible for endoderm differentiation in Ciona savignyi embryos. We found that a LIM-homeobox gene (Cs-lhx3), an otx homolog (Cs-otx) and an NK-2 class gene (Cs-ttf1) were among beta-catenin downstream genes. In situ hybridization signals for early zygotic expression of Cs-lhx3 were evident only in the presumptive endodermal cells as early as the 32-cell stage, those of Cs-otx in the mesoendodermal cells at the 32-cell stage and those of Cs-ttf1 in the endodermal cells at the 64-cell stage. Later, Cs-lhx3 was expressed again in a set of neuronal cells in the tailbud embryo, while Cs-otx was expressed in the anterior nervous system of the embryo. Expression of all three genes was upregulated in beta-catenin overexpressed embryos and downregulated in cadherin overexpressed embryos. Injection of morpholino oligonucleotides against Cs-otx did not affect the embryonic endoderm differentiation, although the formation of the central nervous system was suppressed. Injection of Cs-ttf1 morpholino oligonucleotides also failed to suppress the endoderm differentiation, although injection of its synthetic mRNAs resulted in ectopic development of endoderm differentiation marker alkaline phosphatase. By contrast, injection of Cs-lhx3 morpholino oligo suppressed the endodermal cell differentiation and this suppression was rescued by injection of Cs-lhx3 mRNA into eggs. In addition, although injection of delE-Ci-cadherin mRNA into eggs resulted in the suppression of alkaline phosphatase development, injection of delE-Ci-cadherin mRNA with Cs-lhx3 mRNA rescued the alkaline phosphatase development. These results strongly suggest that a LIM-homeobox gene Cs-lhx3 is one of the beta-catenin downstream genes and that its early expression in embryonic endodermal cells is responsible for their differentiation.

The Pax6 gene plays a developmental role in various metazoans as the master regulatory gene for eye patterning. Pax6 is also spatially regulated in particular regions of the neural tube. Because the amphioxus has no neuromeres, an understanding of Pax6 expression in the agnathans is crucial for an insight into the origin of neuromerism in the vertebrates. We have isolated a single cognate cDNA of the Pax6 gene, LjPax6, from a Lampetra japonica cDNA library and observed the pattern of its expression using in situ hybridization. Phylogenetic analysis revealed that LjPax6 occurs as an sister group of gnathostome Pax6. In lamprey embryos, LjPax6 is expressed in the eye, the nasohypophysial plate, the oral ectoderm and the brain. In the central nervous system, LjPax6 is expressed in clearly delineated domains in the hindbrain, midbrain and forebrain. We compared the pattern of LjPax6 expression with that of other brain-specific regulatory genes, including LjOtxA, LjPax2/5/8, LjDlx1/6, LjEmx and LjTTF1. Most of the gene expression domains showed conserved pattern, which reflects the situation in the gnathostomes, conforming partly to the neuromeric patterns proposed for the gnathostomes. We conclude that most of the segmented domains of the vertebrate brain were already established in the ancestor common to all vertebrates. Major evolutionary changes in the vertebrate brain may have involved local restriction of cell lineages, leading to the establishment of neuromeres.

A set of 3423 expressed sequence tags derived from the Ciona intestinalis tailbud embryos was categorized into 1213 independent clusters. When compared with DNA Data Bank of Japan database, 502 clusters of them showed significant matches to reported proteins with distinct function, whereas 184 lacked sufficient information to be categorized (including reported proteins with undefined function) and 527 had no significant similarities to known proteins. Sequence similarity analyses of the 502 clusters in relation to the biosynthetic function, as well as the structure of the message population at this stage, demonstrated that 390 of them were associated with functions that many kinds of cells use, 85 with cell-cell communication and 27 with transcription factors and other gene regulatory proteins. All of the 1213 clusters were subjected to whole-mount in situ hybridization to analyze the gene expression profiles at this stage. A total of 387 clusters showed expression specific to a certain tissue or organ; 149 showed epidermis-specific expression; 34 were specific to the nervous system; 29 to endoderm; 112 to mesenchyme; 32 to notochord; and 31 to muscle. Many genes were also specifically expressed in multiple tissues. The study also highlighted characteristic gene expression profiles dependent on the tissues. In addition, several genes showed intriguing expression patterns that have not been reported previously; for example, four genes were expressed specifically in the nerve cord cells and one gene was expressed only in the posterior part of muscle cells. This study provides molecular markers for each of the tissues and/or organs that constitutes the Ciona tailbud embryo. The sequence information will also be used for further genome scientific approach to explore molecular mechanisms involved in the formation of one of the most primitive chordate body plans.

A new series of CuCoZnAl layered double hydroxides (LDHs) with various Cu/Co atomic ratios were synthesized by a coprecipitation method at constant pH ≈ 9. The effects of Co on the structural properties and reducibility of both as­synthesized and calcined materials were investigated thoroughly by employing powder X­ray diffraction (XRD), ultraviolet–visible diffuse­reflectance spectroscopy (UV–Vis DRS), thermogravimetry–differential thermal analysis (TG–DTA), and temperature programmed reduction (TPR) methods. Characterization of as­synthesized materials using these techniques revealed the formation of well­crystallized hydrotalcite (HT)­like LDHs with a general formula [Cu_{1 − (x + y + z)}Co_xZn_yAl_z(OH)₂]^z+[(CO₃²⁻)_z/2·mH₂O]^z− as a major phase. Thermal analyses of materials indicated three stages of endothermic weight loss processes due to the loss of interlayer water and some loosely bound CO₃²⁻ (100–250 °C), loss of structural water and CO₃²⁻ (250–400 °C) and the loss of some strongly held CO₃²⁻ anions (above 500 °C). The structural and redox properties of mixed oxides obtained upon thermal activation were investigated by calcining the LDH precursors at 300, 450, 700 and 900 °C. The Cu­rich sample generated a mixture of CuO, ZnO and CuAl₂O₄ spinel, while Co­based spinel phases were observed at all temperatures in the case of the Co­rich sample. Materials containing both Cu and Co showed a mixture of CuO, Co­based spinel and CuAl₂O₄ spinel. The interesting observation of the present study was that the reducibility of Cu²⁺ species of the CuO was enhanced with increasing Co content. On the other hand, the reducibility of Co species decreased with increasing Cu content.

Differentiation of notochord cells and mesenchyme cells of the ascidian Halocynthia roretzi requires interactions with neighboring endodermal cells and previous experiments suggest that these interactions require fibroblast growth factor (FGF). In the present study, we examined the role of FGF in these interactions by disrupting signaling using the dominant negative form of the FGF receptor. An FGF receptor gene of H. roretzi (HrFGFR) is expressed both maternally and zygotically. The maternally expressed transcript was ubiquitously distributed in fertilized eggs and in early embryos. Zygotic expression became evident by the neurula stage and transcripts were detected in epidermal cells of the posterior half of embryos. Synthetic mRNA for the dominant negative form of FGFR, in which the intracellular tyrosine kinase domain was deleted, was injected into fertilized eggs to interfere with the possible function of HRFGFR: Injected eggs cleaved and gastrulated the same as the control embryos. Analyses of the expression of differentiation markers in the experimental embryos indicated that the differentiation of epidermal cells, muscle cells and endodermal cells was not affected significantly. However, manipulated embryos showed downregulation of notochord-specific Brachyury expression and failure of notochord cell differentiation, resulting in the development of tailbud embryos with shorted tails. The expression of an actin gene that is normally expressed in mesenchyme cells was also suppressed. These results suggest that FGF signals are involved in differentiation of notochord cells and mesenchyme cells in Halocynthia embryos. Furthermore, the patterning of a neuron-specific tubulin gene expression was disturbed, suggesting that the formation of the nervous system was directly affected by disrupting FGF signals or indirectly affected due to the disruption of normal notochord formation.

The effect of several surface active additives (called for brevity “foam boosters”) on the foaming properties of solutions of the anionic surfactant sodium dodecyl polyoxyethylene-3 sulfate (SDP3S) is studied in the presence of silicone oil. As foam boosters we study lauryl amide propyl betaine (LAPB), lauryl acid diethanol amide (LADA), lauryl alcohol (LA), and a nonionic surfactant, which is a glycerin derivative (denoted as Booster A). All these substances are used or have a potential to be used as commercial foam boosters in different formulations. The silicone oil is predispersed in the foaming solution in the form of micrometer-sized droplets, which act as foam breaking entities (antifoams). A set of several experimental methods is employed to clarify the main characteristics that are affected by the boosters. Foam tests show that LADA and Booster A enhance mainly the foaminess of the solutions and that LA improves mainly the foam stability, whereas the LAPB increases both. The enhanced foam stability in the presence of LAPB and LA correlates well with the higher barriers to oil drop entry, measured by the film trapping technique (FTT). No correlation of the film stability with the so-called entry, E, spreading, S, and bridging, B, coefficients of the silicone oil is observed. Optical observations of foam films show that the boosting effect cannot be explained by a reduced rate of the foam film thinning. Only the addition of LA leads to deceleration of the final stage of the film thinning process. The obtained results demonstrate that the various boosters affect different characteristics of the foaming solution, and which of the additives is appropriate for a particular application depends strongly on the time-scale of interest.

Abstract: We report on five patients with dermatomyositis (DM) and cutaneous necrosis. Patients presented with classic DM skin eruptions, mild myositis, and a high incidence (4/5) of interstitial pneumonia. Cutaneous necrosis developed independently of steroid therapy, with the majority of lesions being cured following several months of sterilization treatment. In addition, one patient with accompanying cancer presented with multiple necrotic lesions. Topical treatment using gentiana violet against local infection was considered to have been essential in accelerating healing.

Betaines (a particular class of amphoteric surfactants) are commonly used as foam boosters in various products to improve their foamability and foam stability. Foaming media often contain dispersed drops of silicone or hydrocarbon oil, which act as foam destruction agents (antifoams). A complementary set of experiments on foams and foam films stabilized by an anionic surfactant, sodium dodecyl-polyoxyethylene-3-sulfate (SDP3S), or by mixtures of SDP3S and Betaine, is performed in the present study to clarify the mechanisms of:  (1) foam destruction by silicone oil drops, and (2) foam boosting effect of betaine in the presence of oil. The experiments show that foams stabilized by SDP3S are very stable in the absence of oil, while they are unstable and decay with time in the presence of oil (antifoam effect of the oil). The introduction of 40 molar % betaine in the mixture leads to complete foam stabilization in both cases&sbd;with and without oil (foam boosting effect). Notably, the size of the oil droplets has a significant effect on the foam stability&sbd;a substantial amount of silicone oil can be introduced without deteriorating the foam stability, if the drop diameter is below ca. 5 μm. Optical observations of the process of foam film thinning show that the oil drops leave the films without destroying them in all of the studied systems (stable and unstable) relatively soon after foam formation&sbd;typically, within less than a minute. The foam destruction occurs at a later stage of the foam evolution, when the oil drops are compressed by the walls of the narrowing Plateau channels as a result of liquid drainage from the foam. Surface and interfacial tension measurements show that variations in the values of entry, E, spreading, S, and bridging, B, coefficients cannot be used to explain the observed foam boosting effect of betaine. On the other hand, direct measurements of the critical capillary pressure leading to drop entry demonstrate that the barrier to drop entry is much higher in the presence of betaine. The data unambiguously show that the main role of betaine as a foam booster in the studied systems is to increase the barrier to drop entry, which leads to suppressed activity of the silicone oil as an antifoam. The obtained results provide deeper insight into the foam boosting effect and suggest some clues about the properties which an efficient booster should possess.

Accurate detection of dew condensation implies thermal control of sensor's surface to match the thermal behavior of target's one. Herein, thermal behavior of our moisture sensor was controlled by attaching heat sink to its backside while preserving thermal insulation. Temperature change of the sensor's surface was linearly related to the heat capacity of the heat sink and successfully relaxed against that of environmental target. A physical model based on Newton's law of cooling explains that the thermal insulation for the heat sink is another dominant factor to control the thermal behavior. Such combination enhanced the accurate detection of dew condensation.

The epithelium of the pharynx contributes to the formation of gills in hemichordates, urochordates, cephalochordates and primitive vertebrates, and is therefore a key structure for understanding developmental mechanisms underlying the establishment of chordate body plans. Pax1- and Pax9-related genes encode transcription factors which are expressed in the pharyngeal region of cephalochordates as well as in the vertebrate pharyngeal pouch epithelium that forms the thymus and parathyroid glands. To explore the molecular basis underlying the occurrence and modifications of the pharyngeal epithelium during evolution, we isolated cDNA clones for Pax1- and Pax9-related genes of urochordates (HrPax1/9 of Halocynthia roretzi and CiPax1/9 of Ciona intestinalis) and a hemichordate (PfPax1/9 of Ptychodera flava) from gill cDNA libraries. Each gene is present as a single copy per haploid genome. All of the cDNAs encode typical paired domains and octapeptides but not a homeodomain, as is also true of other Pax1- and Pax9-related genes. Molecular phylogenetic analysis based on comparison of the paired domain amino-acid sequences suggests that HrPax1/9, CiPax1/9 and PfPax1/9 belong to the Pax1/9 subfamily, and that they are descendants of a single precursor of Pax1/Pax9. Screening of HrPax1/9 cDNA clones yielded six different types of transcripts which were generated by alternative splicing. Northern blot, RT-PCR/Southern and in situ hybridization analyses revealed that HrPax1/9, CiPax1/9 and PfPax1/9 are not expressed during early embryogenesis but are expressed in the epithelia of differentiating gills, suggesting that these genes encode gill-specific transcription factors. The Pax1/9 genes therefore might provide the first developmental genetic corroboration of hypotheses of organ-level homology that unifies hemichordates, urochordates and cephalochordates.

The critical micelle concentration (CMC), micelle ionization degree (α), Krafft point (KP), and chemical stability in water of sodium and calcium salts of the title surfactants, CmH2m+1NHCO(CH2)nOSO3Me, abbreviated as m-n-Me (Me = Na, 0.5Ca), have been investigated as a function of the numbers m and n of the hydrocarbon chain length and the spacer methylene chain length, respectively. For the m-n-Na series, the CMC was reduced with increasing both the spacer methylene chain length (n = 1, 3, 5) and the hydrocarbon chain length (m = 12, 14, 16). The value of the ratio of reduction in log CMC for m to n was found to be just 4:1, suggesting that the spacer methylene chain is less hydrophobic than in the alkyl chain. Inclusion of an amide linkage near the headgroup in alkyl sulfates leads to a significant increase in the micelle ionization degree and to an increase in Krafft temperature. Furthermore, we found a striking and interesting exception in the Krafft temperature for the sodium and calcium salt pair of m-1-Me homologues. The KP of the 12-1-0.5Ca surfactant was found to be below 0 °C and was lower than that of the corresponding sodium salt (25.6 °C). This phenomenon is believed to be the first observation that the KP of the divalent salt is lower than that of the monovalent salt having the same surface active ion.