Mesoscale structures from magnetocapillary self-assembly.

When identical soft ferromagnetic particles are suspended at some water-air interface, capillary attraction is balanced by magnetic repulsion induced by a vertical magnetic field. By adjusting the magnetic field strength, the equilibrium interdistance between particles can be tuned. The aim of this paper is to study the ordering of particles for large assemblies. We have found an upper size limit above which the assembly collapses due to capillary effects. Before reaching this critical number of particles, defects are always present and limit the perfect ordering expected for that system. This is due to the curvature of the interface induced by the weight of the self-assembly.

Four-year study of lamivudine and adefovir combination therapy in lamivudine-resistant hepatitis B patients: influence of hepatitis B virus genotype and resistance mutation pattern.

To investigate the efficacy of long-term lamivudine (3TC) and adefovir dipivoxil (ADV) combination therapy in 3TC-resistant chronic hepatitis B virus (HBV) infected patients, we analysed 28 3TC-resistant patients treated with the combination therapy during 47 months (range, 9-75). At 12, 24, 36, and 48 months, the rates of virological response with undetectable HBV DNA (≤ 2.6 log copies/mL) were 56, 80, 86, and 92%, respectively. Among 17 hepatitis B e antigen (HBeAg)-positive patients, HBeAg disappeared in 24% at 12 months, 25% at 24 months, 62% at 36 months, and 88% at 48 months. When HBV genotypes were compared, patients with genotype B achieved virological response significantly more rapidly than those with genotype C (P=0.0496). One patient developed virological breakthrough after 54 months, and sequence analysis of HBV obtained from the patient was performed. An rtA200V mutation was present in the majority of HBV clones, in addition to the 3TC-resistant mutations of rtL180M+M204V. The rtN236T ADV-resistant mutation was observed in only 25% clones. In vitro analysis showed that the rtA200V mutation recovered the impaired replication capacity of the clone with the rtL180M+M204V mutations and induced resistance to ADV. Moreover, rtT184S and rtS202C, which are known entecavir-resistant mutations, emerged in some rtL180M+M204V clones without rtA200V or rtN236T. In conclusion, 3TC+ADV combination therapy was effective for most 3TC-resistant patients, especially with genotype B HBV, but the risk of emergence of multiple drug-resistant strains with long-term therapy should be considered. The mutation rtA200V with rtL180M+M204V may be sufficient for failure of 3TC+ADV therapy.

A phase I/II trial of intrabone marrow cord blood transplantation and comparison of the hematological recovery with the Japanese nationwide database.

Intrabone marrow cord blood transplantation (IB-CBT) was proposed as a promising treatment modality to improve hematological recovery. However, clinical advantages of IB-CBT over conventional IV CBT have been unclear. We conducted a prospective single-center trial of IB-CBT to evaluate its safety and superiority in terms of hematological recovery. Fifteen adults with hematological malignancies were enrolled. A thawed and unwashed single cord blood unit was injected into the bilateral superior-posterior iliac crests under local anesthesia. Engraftments of neutrophils and platelets were achieved in 13 cases, with medians of 17 and 45 days, respectively. For the control, we extracted data from the Japanese nationwide database and compared the hematological recovery of contemporaneously transplanted 1135 CBT cases. Multivariate analysis revealed that IB-CBT enhanced platelet recovery (hazard ratio, 2.13; P=0.007), but neutrophil recovery did not differ significantly (hazard ratio, 1.70; P=0.19). Better donor chimerism was seen in the bone marrow of the ilium than of the sternum on day 14, suggesting that the local hematopoiesis at the injected site was established earlier than that at the remote bone marrow site. Collectively, IB-CBT was well tolerated and may enhance local engraftment, which promotes prompter platelet recovery than does IV-CBT.

Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Plasma GH responses to GHRH and insulin-induced hypoglycemia in man.

Changes in plasma GH levels in response to an intravenous bolus injection of 200 micrograms GHRH-44 or 0.1 U/kg body weight regular insulin were examined in normal men who were pre-treated with 200 micrograms GHRH-44 or 0.1 U/kg body weight regular insulin 120 min in advance. The prior bolus injection of GHRH-44 inhibited the plasma GH response to the subsequent administration of GHRH-44 whereas the plasma GH response to the subsequent injection of insulin was not influenced by the prior administration of GHRH-44. The prior administration of insulin attenuated the plasma GH response to the subsequently given GHRH-44. These results suggest that desensitization of GHRH receptors in somatotrophs and/or somatostatin hypersecretion induced by increase in plasma GH levels following the prior GHRH-44 administration may be involved in the mechanism by which the prior GHRH-44 administration or insulin-induced hypoglycemia suppressed plasma GH responses to the following GHRH-44 administration. Sudden suppression of somatostatin secretion, which causes rebound of GH secretion, may occur in insulin-induced hypoglycemia.

Studies on the response of growth hormone (GH) secretion to GH-releasing hormone, thyrotropin-releasing hormone, gonadotropin-releasing hormone, and somatostatin in acromegaly.

The plasma GH response to GH-releasing hormone (GHRH), TRH, or GnRH administration was examined in 25 acromegalic patients. Plasma GH levels increased in 21 patients after GHRH, in 19 after TRH, and in 4 after GnRH. The four GHRH nonresponders had had acromegaly longer than had the GHRH responders. No specific combination of GH responsiveness to these 3 releasing hormones was found among the patients. Infusion of 1 mg GHRH for 150 min gradually increased plasma GH levels, with some fluctuations, from the beginning to the end of infusion in normal subjects and in 7 patients who were GHRH responders, but a bolus injection of 100 micrograms GHRH at the end of the infusion did not further elevate plasma GH levels. These results suggest that desensitization to GHRH occurred in the normal subjects and acromegalic patients. However, in 5 acromegalic patients who responded to both GHRH and TRH, a bolus injection of 500 micrograms TRH given at the end of the 150-min infusion of 1 mg GHRH evoked a further plasma GH rise. In 5 normal subjects and 2 patients who were responders to GHRH but not TRH, a bolus injection of 500 micrograms TRH did not cause plasma GH elevation at the end of 150-min infusion of 1 mg GHRH. These results imply that TRH and GnRH stimulate GH secretion from the adenoma cells in vivo through receptors different from those for GHRH. In vitro studies using cultured pituitary adenoma cells from 2 patients revealed that the responses of GH secretion to GHRH were similar to those in vivo. These data, therefore, suggest that the responsiveness of GH secretion to stimuli is determined by the specificity of the receptors on adenoma cells. The action of somatostatin-28 was more potent than that of somatostatin-14 in the suppression of GH secretion from adenoma cells.

Response to skin grafts exchanged among siblings of larval and adult gynogenetic diploids in Xenopus laevis.

Gynogenetic diploid individuals were produced in an anuran amphibian, Xenopus laevis, and their response to skin grafts exchanged among siblings was studied. All skin grafts exchanged among nongynogenetic sibling froglets, as well as those from genetically unrelated donors, were rejected within 30 days. More than half (57%) of the gynogens that received grafts from sibling partners exhibited a prolonged survival (over 30 days), including long-term survival of over 120 days in 13%. The skin grafted from genetically unrelated froglets onto Nieuwkoop and Faber stage 42-56 larvae and onto perimetamorphic stage 58-65 animals was rejected within 30 days. Similarly, most (96%) of the skin grafts from outbred sibling froglets onto larvae at these stages were rejected acutely or subacutely (12-39 days). However, the skin grafted from sibling froglets to gynogens at larval stage 42-56 and perimetamorphic stage 58-61 enjoyed a long-term survival significantly more frequently (81%) than that in the final metamorphic (stage 64-65) counterparts (57%). These results support the view that in the adult Xenopus allograft responses are reactions to a single MHC as well as to cumulative, multiple minor H-locus barriers. The results also suggest that in larval stages the responses against minor H-locus barriers are generated only mildly.

Autoradiographic study on the distribution of thymus-derived and thymus-independent lymphocytes in the spleen of Xenopus laevis.

Non-thymectomized and early-thymectomized Xenopus laevis were injected with 3H-thymidine, and labelled lymphocytes from thymus, spleen, and peripheral blood were transferred to histocompatible recipients to study their distribution by autoradiography. An extremely high proportion of labelled thymocytes was localized in the splenic red pulp of both non-thymectomized and thymectomized recipients after transfer. Labelled splenic lymphocytes were localized in a significantly higher density in the splenic white- than red pulp, particularly 24 hr after cell transfer. This preference of labelled lymphocytes in the white pulp was more evident when early-thymectomized toads were used as lymphocyte donors. These results strongly suggest that in Xenopus, thymus-independent lymphocytes preferentially localize in the splenic white pulp, and thymus-derived lymphocytes possibly in the red pulp.

In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor.

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.

Derivative (1;7)(q10;p10) in a patient with de novo acute erythroblastic leukemia (AML-M6).

A rare association of der(1;7)(q10;p10) with de novo acute erythroblastic leukemia (AML-M6) in a 63-year-old male is reported. While this unbalanced 1;7 translocation, der(1;7), has been reported often in therapy-related myelodysplastic syndrome (t-MDS) or therapy-related acute myeloid leukemia (t-AML), its associations with de novo AML-FAB-M6 have rarely been reported. Although der(1;7) has been reported as a cytogenetic factor for poor prognosis in t-MDS/AML, our patient showed a good response to chemotherapy and obtained complete remission, although longer observation is required to evaluate the prognosis.

Bulbectomy of neonatal mice induces migration of basal cells from the olfactory epithelium.

Bulbectomy of neonatal mice induced cell migration from the olfactory epithelium in the nasal septum. We examined cell types of migrating clusters by immunohistochemistry using anti-keratin and anti-BrdU antibodies, and by electron microscopy. At 1-2 days after unilateral bulbectomy of P1 mice, cells migrated from the olfactory epithelium to the lamina propria of the septal olfactory mucosa. Horizontal basal cells that reacted specifically with anti-keratin antibody, and globose basal cells characterized by a round shape and poor content of organellae in their cytoplasm, were contained in the cluster. At 1 week, migrated clusters that contained keratin-positive horizontal basal cells were observed in both the lamina propria and olfactory bulb on the unoperated side. At 1 month, not only basal cells but also olfactory cells and presumed supporting cells were involved in the clusters in the lamina propria and olfactory bulb, suggesting that migrated cells do not transform to other phenotypes.

Expression of the neural cell adhesion molecule (NCAM) during second- and third-molar development in the mouse.

Distribution of the neural cell adhesion molecule (NCAM) during the development of the mandibular second- and third-molars of the mouse was studied by indirect immunofluorescence techniques. At the initial stage, NCAM was intensely expressed by the mesenchymal cells surrounding the dental lamina, and by the cap stage NCAM expression by the mesenchymal cells became restricted to the dental follicle. After that, in addition to the follicular mesenchyme, some cells in the basal part of the dental papilla showed NCAM-immunoreactivity for a while after the hard tissue formation had started. During root formation, the follicular cells lost NCAM first from the level of the cervical root and later from the coronal part, while an additional NCAM positive area appeared deep in the dental papilla. Even after the teeth had erupted, NCAM was expressed in the tissue surrounding the apical root and in the pulp core. During the initial and bud stages, the pattern of NCAM expression in the second and third molars was different from that in the first molar, where NCAM was found only after the late bud stage; while from the cap stage onward, it changed in the same sequence as in the first molar. The different pattern of NCAM expression implies that there is a difference in developmental events between the early stages of the first and the other two molars. On the other hand, the common sequence of NCAM expression in the tooth germs later than the cap stage suggests that NCAM plays an essential role in the formation of the basic structure of the teeth and periodontal tissues.

Expression of neural cell adhesion molecule (NCAM) during the first molar development in the mouse.

NCAM, the neural cell adhesion molecule, was immunolocalized in the mandibular first molar tooth germ of the mouse. NCAM was first detected in the tooth germ of the late bud stage, where only the cells in the outer part of the condensed mesenchyme (primitive dental follicle) exhibited faint immunoreactivity. The entire dental follicle was intensely immunostained for NCAM from cap stage to the stage when root formation started. During root formation, NCAM disappeared from the follicular tissue surrounding the cervical root as well as from the part covering the crown top. This loss of NCAM proceeded in the direction of the root apex, but even after the tooth had achieved functional occlusion, NCAM was still expressed by the mesenchymal cells adjacent to the root apex. On the other hand, NCAM was negative in the dental papilla until birth. After birth, NCAM-immunoreactivity appeared in the basal portion of the dental papilla, but this NCAM-positive area gradually diminished in width during the root elongation. Instead, another NCAM-positive zone appeared in the core of the pulp during root formation. Even in the tooth that had already erupted, the pulp core contained cells that were strongly positive for NCAM immunostaining. In addition to its expression in the above two mesenchymal cell lineages, NCAM was transiently expressed by epithelial components of the tooth germ, some of the cells of the dental lamina and the enamel organ. The results suggest that NCAM participates in several processes of tooth development.

Distribution of the neural cell adhesion molecule (NCAM) during pre- and postnatal development of mouse incisors.

Developmental changes in the distribution of the neural cell adhesion molecule (NCAM) were investigated in mouse incisors by means of the indirect immunofluorescence method. During the prenatal stages of development, NCAM was predominantly found in the dental follicle, but not in the dental papilla; the results were analogous to the distribution of NCAM during molar development. After birth, the expression of NCAM continued in the tissue between the enamel organ and the alveolar bone on the labial aspect. In contrast, the follicular tissue covering the lingual aspect of the incisor gradually lost NCAM immunoreactivity from its outer zone as it differentiated into the highly organized periodontal ligament. The intermediate zone of the ligament continued to express NCAM-immunoreactivity even in mice of 6 weeks of age. This pattern of NCAM expression was different from that found in molar teeth, where the organized peridontal ligament was NCAM-negative. The dental pulp, in which we previously reported that an NCAM-positive area appeared at later stages of molar tooth development, did not express NCAM immunoreactivity even at the latest stage of development covered in this study. These differences in the distribution of NCAM between the incisors and the molars might be related to the fact that rodent incisors continue to grow throughout the life of the animal.

Effect of denervation on lymphocytes and dendritic cells in the rat circumvallate and foliate papillae.

The distribution of dendritic (Langerhans) cells and lymphocytes in rat circumvallate and foliate papillae was examined by immunohistochemistry and electron microscopy. Light microscopic immunohistochemistry using anti-OX62 antibody, which recognizes both gammadelta T lymphocytes and dendritic cells, showed that many OX62-immunoreactive cells had invaded into the trench wall epithelium from the connective tissue at 6 days after sectioning of the glossopharyngeal nerves. The presence of OX62-immunoreactive cells in the epithelium was observed up to 17 days after the denervation, by which time the taste buds had disappeared from the trench wall. The OX62-positive cells were again observed in the connective tissue at 24 and 40 days when taste buds regenerated. The local circulation of OX62-positive cells between the epithelium and connective tissue is suggested. Most of the OX62-positive-cells in the epithelium of circumvallate and foliate papillae were suggested to be gammadelta T cells, since they were round or spindle-shaped. Electron micrographs of OX62-positive cells also indicated that they were lymphocytes. Furthermore, they expressed CD3 but lacked CD4 and CD8 surface markers. A few dendritic cells, which reacted with anti-OX6 antibody, were observed in the circumvallate and foliate papillae in the control and denervated animals, and they were irregular in shape with long cytoplasmic processes. Electron micrographs taken at 6 days showed that the dendritic cells, which were characterized by the presence of Birbeck granules in the cytoplasm, were in contact with lymphocytes. The finding suggests that gammadelta T lymphocytes and dendritic cells in the rat circumvallate and foliate papillae interact with each other to respond to changes such as the presence or absence of taste buds in the epithelium.

A comparative study of an olfactory epithelial area lacking olfactory neurons and a nearby presence of TGF-alpha-like immunoreactive olfactory neurons.

Our previous study has shown that ddY mice have special patches of nasal epithelium in the posterior roof of the nasal cavity that exclusively consists of olfactory supporting cells and horizontal basal cells. Here, we extend this finding to Balb/c and DBA/2 mice, Wistar and Sprague-Dawley rats, hamsters, and guinea pigs. In the mice, rats, and hamsters studied, the patches lacked olfactory cells and their precursor, globose basal cells. In rats and hamsters, the supporting cells were arranged in a single layer, in mice as three or four layers. Horizontal basal cells were located in a single layer in these species. In the guinea pigs, the specialized roof structure was less clear and could be seen at the level of ultrastructure as an olfactory neuron-lacking area. Distinct populations of transforming growth factor (TGF)-alpha-like immunoreactive olfactory cells occupied an area close to the epithelial patches. In this region, the TGF-alpha-like immunoreactive neurons were negative for the usual olfactory markers, either OMP or protein gene product (PGP) 9.5 or beta-tubulin. These cells are suggested to project to the so-called 'necklace glomeruli' and use a different cGMP-driven, transduction pathway. Three-dimensional analysis of double-labeled (TGF-alpha, PGP9.5) serial sections revealed a unique relation among the epithelial patches, TGF-alpha-like immunoreactive neurons and olfactory epithelium.

Expression of E- and P-cadherin during tooth morphogenesis and cytodifferentiation of ameloblasts.

Cell-cell adhesion is fundamental in morphogenesis and is known to be mediated by several groups of cell adhesion molecules. Cadherins are a group of such molecules involved in the Ca2+-dependent cell-cell adhesion mechanism and are found in most kinds of tissue. In this study using indirect immunofluorescence microscopy, we analyzed the distribution of two kinds of cadherins, E- and P-cadherin, in developing tooth germs. In the molar tooth germs at the early bud stage, marginal cells of the epithelial tooth bud expressed both E- and P-cadherin, whereas central cells expressed only E-cadherin. At the cap stage, in addition to the cells of the inner and outer enamel epithelium, which outline the enamal organ, cells of the enamel knot, which is thought to control tooth morphogenesis, strongly expressed P-cadherin. The expression of P-cadherin was prominent in the inner enamel epithelium during the early to mid bell stage, and was also evident in the non-dividing cell masses at future cusp tips, which are the so-called secondary enamel knots. In the tooth germ at the late bell stage when the cells of the inner enamel epithelium began to polarize to differentiate into ameloblasts, the polarizing ameloblasts lost P-cadherin and strongly expressed E-cadherin. However, E-cadherin was also lost from polarized ameloblasts at later stages. The stratum intermedium and the stellate reticulum were E-cadherin positive from the bell stage onward even at the stages when the ameloblasts became E-cadherin negative again. These results suggest that the differential expression of E- and P-cadherin during morphogenetic stages plays a role in the regulation of tooth morphogenesis, whereas alteration of E-cadherin expression during later stages of tooth development is related to differentiation and function of the ameloblasts and other cells supporting amelogenesis.

Colchicine-induced cell death and proliferation in the olfactory epithelium and vomeronasal organ of the mouse.

The cytotoxic agent colchicine induced apoptotic cell death and subsequent regeneration in the mouse olfactory epithelium and vomeronasal organ. The TUNEL method revealed the presence of many apoptotic bodies in the middle to basal region of the septal olfactory epithelium and vomeronasal organ near the boundary of the respiratory epithelium at 1 day after a single i.p. injection of colchicine (4 mg/kg b.w.). In some regions of the third and the fourth nasal turbinates, massive apoptosis was observed in the olfactory epithelium. Electron micrographs of the septum showed that immature olfactory cells and globose basal cells were killed by the colchicine and had been phagocytized by the supporting cells and macrophages. In the vomeronasal organ, immature sensory cells and precursors died in response to the colchicine. In response to cell death, active proliferation of precursor cells (globose basal cells) and subsequent regeneration of olfactory cells occurred in the olfactory epithelium and vomeronasal organ. Incorporation of the mitotic tracer BrdU by precursor cells reached its peak at 4 days after colchicine treatment in the vomeronasal organ, and at 6 to 7 days in the olfactory epithelium; however, in some regions in the third and the fourth nasal turbinates, where many olfactory cells and globose basal cells had died by colchicine effect, the regeneration did not occur even in 1 month, forming the epithelium of only supporting cells and horizontal basal cells. In the next month, these regions became normal olfactory epithelium. This suggests that the globose basal cells in the surrounding normal olfactory epithelium might invade these regions to give rise to the olfactory cells.

Effects of lithium and purinergic compounds on the behavioral and physiological aspects of restraint stress in rats.

This study investigates the effects of lithium and caffeine on psychomotor activities, defecation, and gastric lesions induced by restraint stress. Rats exposed to restraint stress typically exhibited a biphasic response consisting of an initial hypermotility (such as tail-flipping, body-rolling, jaw movement, and vocalization) accompanied by defecation, and followed by hypomotility (decrease in motility) accompanied by gastric ulceration. Lithium chloride (150 micrograms, ICV; 50 and 100 mg/kg, IP) significantly attenuated these responses while N6-cyclohexyl adenosine (CHA; 1.5 micrograms, ICV; 0.3 mg/kg, IP), a potent adenosine A1 receptor agonist, attenuated the behavioral effects but potentiated the gastric ulceration. Caffeine (3 micrograms, ICV; 1.0 mg/kg, IP), an adenosine receptor antagonist, inhibited the effects of CHA in animals exposed to 3 h of stress, but aggravated the effects in animals exposed to 6-12 h of stress. These results suggest that caffeine consumption may produce supersensitivity of adenosine receptors, which potentiate the actions of adenosine or CHA. Lithium may modulate the effects of stress by indirectly inhibiting central adenosine receptor activity.

Immunofluorescence detection of cadherins in mouse tooth germs during root development.

The distribution of two cell-adhesion molecules, E- and P-cadherin, was studied in relation to morphological changes in Hertwig's epithelial root sheath Before root dentinogenesis had started, the root sheath expressed both cadherins. As dentinogenesis proceeded, the sheath fragmented and lost P-cadherin rapidly and E-cadherin slowly, whereas the intact sheath at the apical end continued to express both. These results suggest that the two cadherins play a part in root as well as in crown development, and indicate that the decrease in the amount of these molecules and the fragmentation of the epithelial root sheath are interrelated.