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Background:: Obesity and metabolic syndrome are the major risk factors for cardiovascular disease. Obesity is caused by increased food intake and/or decreased energy expenditure. Leptin potently inhibits food intake and promotes energy expenditure. These effects of leptin involve the activation of proopiomelanocortin (POMC) neurons in the hypothalamus arcuate nucleus (ARC). Disruption of leptin signaling in POMC neuron is considered one of the major causes for obesity. Aims:: The present study aimed to examine whether overexpression of interleukin-10 (IL-10) could substitute for the leptin action and ameliorate obesity in leptin-deficient Lepob/obmice. Design:: Adeno-associated virus (AAV) expressing murine IL-10 (AAV-mIL-10) was injected into the skeletal muscle to overexpress IL-10 in mice. These mice were subsequently subjected to analysis of body weight, food intake, glucose metabolism and underlying mechanisms. Results:: In Lepob/obmice, AAV-IL-10 ameliorated hyperphagia, obesity, glucose intolerance and insulin resistance, as well as attenuated tumor necrosis factor-a expression. The IL-10 treatment also improved glucose-induced insulin release. Furthermore, IL-10 treatment increased POMC mRNA expression in ARC and phosphorylation of signal transducer and activator of transcription-3 (STAT3) in ARC and white adipose tissue (WAT). In neuron-specific STAT3-null mice that exhibited obesity and hyperphagia, AAV-mIL-10 administration failed to affect food intake, body weight and phosphorylation of STAT3 in WAT. Conclusions:: These results demonstrate that peripheral overexpression of IL-10 induces STAT3 phosphorylation in ARC POMC neurons, and thereby ameliorates hyperphagia and obesity caused by leptin deficiency. IL-10 gene transfer may provide an effective approach for preventing progression of metabolic syndrome due to leptin resistance.

Methodologies for generating functional neuronal cells directly from human fibroblasts [induced neuronal (iN) cells] have been recently developed, but the research so far has only focused on technical refinements or recapitulation of known pathological phenotypes. A critical question is whether this novel technology will contribute to elucidation of novel disease mechanisms or evaluation of therapeutic strategies. Here we have addressed this question by studying Tay-Sachs disease, a representative lysosomal storage disease, and Dravet syndrome, a form of severe myoclonic epilepsy in infancy, using human iN cells with feature of immature postmitotic glutamatergic neuronal cells. In Tay-Sachs disease, we have successfully characterized canonical neuronal pathology, massive accumulation of GM2 ganglioside, and demonstrated the suitability of this novel cell culture for future drug screening. In Dravet syndrome, we have identified a novel functional phenotype that was not suggested by studies of classical mouse models and human autopsied brains. Taken together, the present study demonstrates that human iN cells are useful for translational neuroscience research to explore novel disease mechanisms and evaluate therapeutic compounds. In the future, research using human iN cells with well-characterized genomic landscape can be integrated into multidisciplinary patient-oriented research on neuropsychiatric disorders to address novel disease mechanisms and evaluate therapeutic strategies.

The role of calmodulin (CaM) during mating in Saccharomyces cerevisiae was examined by using a set of Phe-to-Ala substitutions. We identified ten CaM mutants that exhibited significantly reduced mating efficiencies when crossed to a strain of the opposite mating type harboring the same CaM mutation. Most of the mating-defective CaM mutants were bilateral, i.e., they also exhibited mating defects, albeit minor ones, when crossed to the wild type. When strains carrying different bilateral CaM mutations were mated, the mating efficiencies recovered dramatically. We termed this phenomenon "intragenic mating complementation", and classified the mating-defective CaM mutations into two intragenic mating complementation groups. Two mutant alleles belonging to different groups showed minor defects in cell adhesion and cell fusion, but exhibited severe defects in karyogamy. CaM is known to bind to the essential spindle pole body component Nuf1p. This binding appears to be important for karyogamy because the nuf1 C911R mutation, which impairs CaM-Nuf1p binding, resulted in a severe defect in karyogamy. Indeed, the two mating-defective CaM mutations were found to compromise formation of the CaM/Nuf1p complex, and the mating defects of these two CaM mutants were suppressible by a dominant, CaM-independent, mutation in NUF1. Taken together, these results suggest that loss of CaM binding to Nuf1p causes a defect in karyogamy, thereby inhibiting productive mating.

The tradeoff between the speed and quality of the solutions obtained by various construction and local search algorithms for the elementary bin packing problem (BPP) are analyzed to obtain useful information for designing algorithms for real-world problems that can be modeled as BPPs. On the basis of intensive computational experiments, we observe that the framework of a solution (i.e., a part of a solution consisting of large items or items with tight constraints) should be constructed in the early stages of a local search. New local search algorithms are proposed as empirical support for the observation.

Mouse thymic lymphomas are one of the classic models of radiation-induced malignancies. However, little genetic study has been performed, although the mouse systems offer a number of useful features for genetic and physical mapping. We have carried out large-scale mapping toward the isolation of the genes involved in lymphoma development. Two different types of genes are chosen as targets for positional cloning. One is the tumor suppressor gene and the other is the susceptibility or resistance-giving gene, which predispose to the lymphoma development. One susceptibility locus was localized near D4Mit12 on chromosome 12 by an association study for backcross and congenic mice, and three loci, probably harboring a tumor suppressor gene, were localized by allelic loss mapping on physical maps that were covered by BAC clones. The maps are invaluable to facilitate the identification of candidate tumor suppressor genes. Also, success in identification of Ikaros as a tumor suppressor gene is described.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I–VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.

Intercellular signaling through the cell-surface receptor Notch plays important roles in a variety of developmental processes as well as in pathogenesis of some human cancers and genetic disorders. However, the mechanisms by which Notch signals are transduced into cells still remain elusive. Here we investigated the signaling mechanisms for Notch in the cell fate control of neural progenitor cells. We show that Deltex-1 (DTX1), a mammalian homolog of Drosophila Deltex, mediates a Notch signal to block differentiation of neural progenitor cells. We found that a significant fraction of DTX1 proteins were localized in the nucleus and physically interacted with the transcriptional coactivator p300. Through its binding to p300, DTX1 inhibited transcriptional activation by the neural-specific helix-loop-helix-type transcription factor MASH1, and this mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. Our results further suggest that DTX1 regulates transcription independently of the previously characterized Notch signaling pathway involving RBP-J and HES1/HES5. Thus, DTX1 serves as an important signaling component downstream of Notch that regulates transcription in the nucleus.

In situ detection of neural progenitor cells including stem-like cells is essential for studying the basic mechanisms of the generation of cellular diversity in the CNS, upon which therapeutic treatments for CNS injuries, degenerative diseases, and brain tumors may be based. We have generated rat monoclonal antibodies (Mab 14H1 and 14B8) that recognize an RNA-binding protein Musashi1, but not a Musashi1-related protein, Musashi2. The amino acid sequences at the epitope sites of these anti-Musashi1 Mabs are remarkably conserved among the human, mouse, and Xenopusproteins. Spatiotemporal patterns of Musashi1 immunoreactivity in the developing and/or adult CNS tissues of frogs, birds, rodents, and humans indicated that our anti-Musashi1 Mabs reacted with undifferentiated, proliferative cells in the CNS of all the vertebrates tested. Double or triple immunostaining of embryonic mouse brain cells in monolayer cultures demonstrated strong Musashi1 expression in Nestin(+)/RC2(+) cells. The relative number of Musashi1(+)/Nestin(+)/RC2(+) cells increased fivefold when embryonic forebrain cells were cultured to form ‘neurospheres’ in which stem-like cells are known to be enriched through their self-renewing mode of growth. Nestin(+)/RC2(–) cells, which included Tα1-GFP(+) neuronal progenitor cells and GLAST(+) astroglial precursor cells, were also Musashi1(+), as were GFAP(+) astrocytes. Young neurons showed a trace of Musashi1 expression. Cells committed to the oligodendroglial lineage were Musashi(–). Musashi1 was localized to the perikarya of CNS stem-like cells and non-oligodendroglial progenitor cells without shifting to cell processes or endfeet, and is therefore advantageous for identifying each cell and counting cells in situ.

Paraneoplastic cerebellar degeneration (PCD) is a disorder in which breast or ovarian tumors express an onconeural antigen termed cdr2, which normally is expressed in cerebellar Purkinje neurons. This leads to an immune response to cdr2 that is associated with tumor immunity and autoimmune cerebellar degeneration. We have found that cdr2, a cytoplasmic protein harboring a helix-leucine zipper (HLZ) motif, interacts specifically with the HLZ motif of c-Myc. Both proteins colocalize in the cytoplasm of adult cerebellar Purkinje neurons, and coimmunoprecipitate from tumor cell lines and cerebellar extracts. cdr2 down-regulates c-Myc-dependent transcription in cotransfection assays, and redistributes Myc protein in the cytoplasm. Disease antisera from six of six PCD patients specifically blocked the interaction between cdr2 and c-Myc in vitro. These data indicate that cdr2 normally sequesters c-Myc in the neuronal cytoplasm, thereby down-regulating c-Myc activity, and suggest a mechanism whereby inhibition of cdr2 function by autoantibodies in PCD may contribute to Purkinje neuronal death.

Analysis of double mutant mice of thep53andscidgenes, which have a combination of cell cycle checkpoint/apoptosis and DNA repair defects, shows that the latter defect synergistically enhances lymphoma development with loss of the former function. These mice lack the ability to eliminate lymphocytes predisposed to neoplastic transformation resulting from faulty antigen receptor gene rearrangement. Here we examine the cooperativity in double heterozygotes ofp53andscidin which normal development of lymphocytes is not impaired. MSM mice carrying a p53-knockout allele were crossed with BALB/c mice heterozygous for thescidlocus and 129 offspring were obtained. They were subjected to γ-ray irradiation, 84 thymic lymphomas being generated. The tumors and host mice were genotyped ofp53andscid.Among 42 mice developing p53-deficient lymphomas,scid/+ and +/+ genotypes did not provide difference in onset and latency. Besides, allelic loss of theScidgene occurred at a high frequency in those lymphomas but the loss exhibited no allelic bias. The results suggest that thescid/+ genotype is not a modifier of loss of p53 function in the double heterozygotes.

Abstract: Patients with constipation may have various pelvic complaints of difficult evacuation coexisting with infrequent evacuation and other abdominal complaints, and the overlapping of symptoms makes it difficult to select appropriate therapy based on clinical history and routine office examination alone. Cinedefecography is an objective method for examining patients who have complex subjective symptoms. This study assessed the value of cinedefecography for evaluating patients with constipation with multiple complaints. We divided 185 patients with constipation into two those with (group 1) or without (group 2) cinedefecographic evidence of difficult evacuation. These groups were compared relative to complaints, manometric results, cinedefecography findings, and the success of biofeedback treatment. Group 1 patients were further evaluated according to the type of abnormal findings: sigmoidocele, rectocele, intussusception, and perineal descent. We found no significant differences in patient complaints between the groups. However, there were more patients in group 2 with intussusception than in group 1; there were also significant differences between the groups in mean resting pressure, maximum resting pressure, and maximum squeeze pressure. In group 1 patients with rectocele complained more frequently of excessive straining, and those with intussusception complained more frequently of incomplete evacuation. Sensory threshold and maximal tolerable capacity were significantly higher in patients with intussusception. Rectocele was predominant in women, and biofeedback treatment was extremely advantageous (86%) for patients in group 1 with a rectocele in association with other pathology. Whether the intussusception or the descent causes decreased mean resting and mean and maximum squeeze pressures is unknown but is an additional and potentially important finding which needs further elucidation for it to have therapeutic significance.

The focal adhesion kinase (FAK) protein-tyrosine kinase plays important roles in cell adhesion in vertebrates. Using polymerase chain reaction-based cloning strategy, we cloned a Drosophila gene that is homologous to the vertebrate FAK family of protein-tyrosine kinases. We designated this gene Dfak56 and characterized its gene product. The overall protein structure and deduced amino acid sequence of Dfak56 show significant similarity to those of FAK and PYK2. Dfak56 has in vitro autophosphorylation activity at tyrosine residues. Expression of the Dfak56 mRNA and the protein was observed in the central nervous system and the muscle-epidermis attachment site in the embryo, where Drosophila position-specific integrins are localized. The results suggest that like FAK in vertebrates, Dfak56 functions downstream of integrins. Dfak56 was tyrosine-phosphorylated upon integrin-dependent attachment of the cell to the extracellular matrix. We conclude that the Dfak56 tyrosine kinase is involved in integrin-mediated cell adhesion signaling and thus is a functional homolog of vertebrate FAK.

Oligodendrocytes are myelin-forming cells in the mammalian central nervous system. About 50% of oligodendrocytes undergo cell death in normal development. In addition, massive oligodendrocyte cell death has been observed in multiple sclerosis. Tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of oligodendrocytes in multiple sclerosis. The addition of TNF-α to primary cultures of oligodendrocytes significantly decreased the number of live cells in 72 h. DNA fragmentation was detected in TNF-treated oligodendrocytes at 36 h by TUNEL assay. Chemical inhibitors Ac-YVAD-CHO (a specific inhibitor of caspase-1 [ICE]-like proteases) as well as Ac-DEVDCHO (a specific inhibitor of caspase-3[CPP32]-like proteases) enhanced the survival of oligodendrocytes treated with TNF-α, indicating that caspase-1- and the caspase-3-mediated cell-death pathways are activated in TNF-induced oligodendrocyte cell death. Caspase-11 is involved in activation of caspase-1. Oligodendrocytes fromCASP-11-deficient mice are partially resistant to TNF-induced oligodendrocyte cell death. Our results suggest that the inhibition of caspases may be a novel approach to treat multiple sclerosis.

The myelin deficient shimld mouse is an autosomal recessive mutant, characterized by hypomyelination in the central nervous system. The expression of the myelin basic protein (MBP) gene is inhibited transcriptionally. The MBP gene is duplicated tandemly in mld, and exons 3 to 7 of the upstream copy is inverted. In the present studies, we determined the approximate position of the 5′ boundary and the nucleotide sequence surrounding the 3′ boundary of the inversion and found a number of sequences homologous to the switching regions of mouse immunoglobulin heavy chain gene and J regions of human T cell receptor genes. Antisense RNA complementary to exons 3 and 7, which correspond to the inverted segment, was detected by RNase protection studies. This abnormal transcript was also shown to elongate through the inverted segment to reach the transcription initiation site of the downstream gene.

A hereditary dysmyelinating mutation, named myelin deficient (shi(mld)), is characterized by reduced expression of myelin basic protein (MBP). In shi(mld), the MBP gene is duplicated and its reduced expression is mainly determined by the level of mRNA. We have characterized the structure and function of the promoter regions of the duplicated MBP genes in shi(mld). Among the lambda clones containing promoter regions of the duplicated MBP genes in shi(mld), one (gene 1) had the same restriction enzyme pattern as that in control mice, but another (gene 2) had a rearrangement on a distal part of the promoter. A 712‐bp nucleotide sequence upstream of the first exons of both of the duplicated MBP genes of shi(mld) was completely consistent with that of the control. Promoter activities of 1.3‐kb 5′‐flanking regions from respective genes of shi(mld) measured by in vitro run‐off assay using HeLa whole‐cell extracts were indistinguishable from that of the control MPB gene. Chromosomal mapping by in situ hybridization suggested that the duplicated MBP genes were located closely to each other at the distal part of chromosome 18. A recombinational event including the inversion seemed to have occurred within gene 1 and its possible relationship to the reduced expression of MBP is discussed.

KMOE -2/05 cells, derived from a patient with acute erythremia, became benzidine-positive after the addition of cytosine arabinoside (CA). Radioimmunoassays using antihuman hemoglobin antibodies revealed an elevated amount of hemoglobin in the CA-exposed cells over that in the control cells (without CA). Isoelectric focusing of the CA-exposed cell lysate formed benzidine-positive foci in the positions of human adult Hb (HbA) and human fetal Hb (HbF). To determine the types of globin synthesized in the CA-exposed cells, globin chains internally labeled with [3H] leucine were purified by carboxy-methyl (CM)-Sephadex column chromatography, immunoadsorption by Sepharose-coupled antihuman Hb antibodies and Sephadex G-100. The labeled globin chains were finally separated by CM-cellulose chromatography in urea. Two distinct peaks of radioactivity were shown in exactly the same fractions as carrier human globin alpha- and beta-chains. These observations indicate that these KMOE -2/05 cells synthesize HbA.

UT-7 is a human leukemic cell line capable of growing in interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo) (Komatsu et al, Cancer Res 51:341, 1991). To study the effect of Epo on proliferation and differentiation of UT-7, we maintained the UT-7 cell culture for more than 6 months in the presence of Epo. As a result, a subline, UT-7/Epo, was established. The growth of UT-7/Epo could be supported by Epo but not by GM-CSF or IL-3. UT- 7/Epo showed a greater level of heme content and ratio of benzidine- positive staining cells than did UT-7. Butyric acid promoted the synthesis of hemoglobin in UT-7/Epo, but not UT-7. Further, the mRNA concentrations of the c-myb oncogene and GM-CSF receptor beta-subunit were decreased substantially in UT-7/Epo cells. These findings showed that UT-7/Epo cells had progressed further in erythroid development than UT-7 cells, and suggested that long-term culture in Epo had promoted this differentiation. Whereas availability of the Epo receptor (Epo-R) for binding of Epo was reduced in UT-7/Epo cells compared with UT-7 cells, the Epo-R showed a similar affinity for Epo. This observation suggested that change(s) in postreceptor signaling step might be involved in the establishment and maintenance of the UT-7/Epo phenotype.

Biopsies of 18 patients with lung cancer and cellular specimens of 21 lung cancer patients were analyzed with human monoclonal antibody HB4C5-clone 3 using avidin-biotin-peroxidase techniques. Analyses with the biopsies showed that HB4C5-clone 3 reacted with 16 of 18 biopsy specimens at a high rate of about 90% and reacted with all specimens of five adenocarcinoma tissues, five of six squamous cell carcinomas, two of three large cell carcinomas, both specimens of small cell carcinomas, and both of the other types of carcinomas. In all the reactive cases, the monoclonal antibody was reactive with greater than 50% of cancer cells. With cellular specimens of lung cancer patients, HB4C5-clone 3 reacted with 7 of 13 adenocarcinoma specimens, two of six squamous cell carcinomas, and one of two small cell carcinomas. The immunoreaction with HB4C5-clone 3 was observed in sites of the cytoplasm of cancer cells. From these data, HB4C5-clone 3 is considered to be of potential use in diagnoses of tissues and cellular specimens of lung cancer.

Cytochrome cfrom various sources, such as Candida krusei, yeast, horse, and cattle, was found to be recognized by human monoclonal antibody HB4C5 specific to lung cancer. Therefore, the cytochrome cwas applied to the measurement of antibody amount in patient sera with a similar reactivity to the antibody HB4C5 for serodiagnosis of cancer. The cytochrome cfrom Candida kruseiwas most valuable for the serodiagnosis of various cancers, and the yeast cytochrome cwas also useful. However, horse and bovine cytochrome cdid not react with antibody of the cancer patients. By using Candidacytochrome c, lung, bile duct, esophagus, and liver cancers were detected at high rates of more than 50%. In the case of lung cancer, the detection rates of small-cell, squamous, large-cell and adenocarcinoma were 78%, 63%, 100%, and 34%, respectively. The rate for small-cell carcinoma was higher than that with the currently used NSE assay system, and the rate for squamous carcinoma was comparable to that with the SCC assay system, although the system using cytochrome cdid not show similar reactivity to that with the SCC system. Furthermore, lung cancer was detected at early stages by using cytochrome c, and even in the case of adenocarcinoma, the rate at early stages with the cytochrome csystem was higher than that with the CEA assay system. On the other hand, false positive rates of benign diseases and normal were low–8% and 2%, respectively.

We investigated the effect of orally administered trimebutine maleate on gastric and gallbladder emptying and on the release of gut peptide, pancreatic polypeptide (PP), and gastrin in humans for 120 min after ingestion of a solid meal. Gastric emptying was measured by a radionuclide technique. Gallbladder emptying was estimated by real-time ultrasonography. The oral administration of 200 mg of trimebutine maleate significantly shortened the lag time in starting gastric emptying (P<0.05). Considering gallbladder emptying, trimebutine significantly inhibited the fasting emptying induced by neural reflex. Postprandially, there was a tendency toward an accelerated gallbladder emptying in the early phase. Neither the maximal percentage of gallbladder emptying nor the time of peak gallbladder emptying were affected. Trimebutine significantly blunted the postprandial PP response in the cephalic and gastric phases, reflecting a vagal-cholinergic activity (P<0.05). The PP response in the intestinal phase was also blunted. Gastrin release was significantly augmented only during the period of fasting after drug administration (P<0.05). The major effect of trimebutine maleate appears to be a shortening of the lag time at the start of gastric emptying probably via its anticholinergic activity.

Studies have been made of poly- and single Si etching induced by excimer-laser irradiation of the silicon surfaces in halogenated gases. Etching was investigated for different conduction types, impurity concentrations and crystallographic planes. Chlorine atoms accept electrons generated in photoexcited, undoped p-type Si, thus becoming negative ions which are pulled into the Si. However, the n+-type Si is etched spontaneously by Cl- as a result of the availability of conduction electrons. Fluorine atoms, with the highest electronegativity, take in electrons independent of whether the material is n- or p-type. And thus, the easy F- ion penetration into Si causes spontaneous etching in both types. New anisotropic etching for n+ poly-Si is investigated because of its importance to microfabrication technology. Methyl methacrylate (MMA) gas, which reacts with Cl atoms, produces a deposition film on the n+ poly-Si surface. The surface, from which the film is removed by KrF (5 eV) laser irradiation, is etched by Cl atoms, while the film remains on the side wall to protect undercutting. However, with the higher photon energy for the ArF (6.4 eV) laser, the Si-OH bonds are broken and electron traps are formed. These electrontrapping centers are easily annealed out in comparison to the plasma-induced centers. Pattern transfer etching for n+ poly-Si has been realized using reflective optics. The problems involved in obtaining finer resolution etching are discussed.

ced-9, a member of the bcl-2 gene family in Caenorhabditis elegans plays a central roles in preventing cell death in worms. Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether ced-9 can regulate cell death when expressed in other organisms. We demonstrated that the CED-9 protein is co-localized with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to specific cytoplasmic compartments. Overexpression of ced-9 only poorly protected cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduced the cell death induced by ced-3 in Drosophila SL2 cells. Furthermore, apoptosis of SL2 cells that was induced by a Drosophila cell-death gene, reaper, was shown to be partially prevented by ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of bcl-2 family members, including ced-9, is conserved in Drosophila cells. In addition, SL2 cells provide a unique systems for dissecting the main machinery of cell death.

The mouse myelin basic protein promoter was transcribed in brain nuclear extracts. The distal promoter region from -253 to -54 directed preferential transcription in brain extracts, whereas the same region repressed transcription activity in liver extracts. Stimulation of transcription was observed when the distal region was located only in a native orientation. The proximal region downstream from -53 alone still directed preferential transcription. It is suggested that cooperative function by the two promoter regions may be required for higher specificity.

The chordotonal (Ch) organ, an internal stretch receptor located in the subepidermal layer, is one of the major sensory organs in the peripheral nervous system of Drosophila melanogaster. Although the cell lineage of the Ch organ has been well characterized in many studies, the determination machinery of Ch organ precursor cells (COPs) remains largely unresolved. Here we report that the rhomboid (rho) gene and the activity of the Drosophila EGF receptor (DER) signaling pathway are necessary to induce specifically three of the eight COPs in an embryonic abdominal hemisegment. The cell-lineage analysis of COPs using the yeast flpase (flp/FRT) method indicated that each of the eight COPs originated from an individual undifferentiated ectodermal cell. The eight COPs in each abdominal hemisegment seemed to be determined by a two-phase induction: first, five COPs are determined by the action of the proneural gene atonal and neurogenic genes. Subsequently, these five COPs start to express the rho gene, and rho activates the DER-signaling pathway in neighboring cells and induces argos expression. Three of these argos-expressing cells differentiate into the three remaining COPs and they prevent neighboring cells from becoming extra COPs.

The myelin deficient shimld mouse is an autosomal recessive mutant, characterized by hypomyelination in the central nervous system. The expression of the myelin basic protein (MBP) gene is inhibited transcriptionally. The MBP gene is duplicated tandemly in mld, and exons 3 to 7 of the upstream copy is inverted. In the present studies, we determined the approximate position of the 5′ boundary and the nucleotide sequence surrounding the 3′ boundary of the inversion and found a number of sequences homologous to the switching regions of mouse immunoglobulin heavy chain gene and J regions of human T cell receptor genes. Antisense RNA complementary to exons 3 and 7, which correspond to the inverted segment, was detected by RNase protection studies. This abnormal transcript was also shown to elongate through the inverted segment to reach the transcription initiation site of the downstream gene.

Recent genetic studies of yeast calmodulin (yCaM) have shown that alterations of different sets of Phe residues result in distinct functional defects (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). To examine the importance of Phe residues for target binding and activation, we purified mutant yCaMs containing single or double Phe to Ala substitutions and determined their ability to bind and activate two target proteins, calcineurin and CaM-dependent protein kinase (CaMK). Binding assays using the gel overlay technique and quantitative analyses using surface plasmon resonance measurements indicated that the binding of yCaM to calcineurin is impaired by either double mutations of F16A/F19A or a single mutation of F140A, while binding to CaMK is impaired by F89A, F92A, or F140A. These same mutant yCaMs fail to activate calcineurin and CaMK, respectively, in vitro. In addition, F19A exhibited a severe defect in activation of both enzymes. F12A activated calcineurin to only 50% of the level achieved by wild-type calmodulin but fully activated CaMK. These results suggest that each target protein requires a specific and distinct subset of Phe residues in yCaM for target binding and activation.

Additional subtypes of the inositol 1,4,5-trisphosphate (InsP3) receptor are expressed in a tissue-specific and developmentally specific manner. They differ from the InsP3 receptor structure previously reported in two small variably spliced segments. One segment (SI) is located within the InsP3 binding site, whereas another segment (SII) is located near putative sites for phosphorylation and ATP binding to modulate InsP3 action on Ca2+ flux. Therefore, we speculate that selective use of InsP3 receptor subtypes permits a tissue-specific and developmentally specific expression of functionally distinct channels.

Three continuous human cell lines, designated KMOE, derived from a patient with acute erythremia (Di Guglielmo's disease) are reported. The cell lines are the cultures of (1) bone marrow cells, (2) peripheral blood cells, and (3) cells from a tumor developed into an athymic nude mouse after transplantation of the cultured bone marrow cells. Cells of all three lines show morphology of immature erythroblast and have i(17q) marker chromosome. They are negative for both Philadelphia chromosome and Epstein-Barr virus nuclear antigen. Although all KMOE cells in suspension culture are benzidine-negative, benzidine-positive cells are found within colonies formed in semi-solid culture media. The relative number of colonies with benzidine-positive cells is increased when sodium butyrate is added to the culture.

MacMARCKS is a member of the MARCKS family of protein kinase C (PKC) substrates. Biochemical evidence demonstrates that these proteins integrate calcium and PKC-dependent signals to regulate actin structure at the membrane. We report here that deletion of the MacMARCKS gene prevents cranial neural tube closure in the developing brain, resulting in anencephaly. This suggests a central role for MacMARCKS and the PKC signal transduction pathway in the folding of the anterior neural plate during the early phases of brain formation, and supports the hypothesis that actin-based motility directs cranial neural tube closure.

A hereditary dysmyelinating mutation, named myelin deficient (shi(mld)), is characterized by reduced expression of myelin basic protein (MBP). In shi(mld), the MBP gene is duplicated and its reduced expression is mainly determined by the level of mRNA. We have characterized the structure and function of the promoter regions of the duplicated MBP genes in shi(mld). Among the lambda clones containing promoter regions of the duplicated MBP genes in shi(mld), one (gene 1) had the same restriction enzyme pattern as that in control mice, but another (gene 2) had a rearrangement on a distal part of the promoter. A 712‐bp nucleotide sequence upstream of the first exons of both of the duplicated MBP genes of shi(mld) was completely consistent with that of the control. Promoter activities of 1.3‐kb 5′‐flanking regions from respective genes of shi(mld) measured by in vitro run‐off assay using HeLa whole‐cell extracts were indistinguishable from that of the control MPB gene. Chromosomal mapping by in situ hybridization suggested that the duplicated MBP genes were located closely to each other at the distal part of chromosome 18. A recombinational event including the inversion seemed to have occurred within gene 1 and its possible relationship to the reduced expression of MBP is discussed.

Ceramide has emerged as a novel lipid mediator in cell proliferation, differentiation, and apoptosis. In this work, we demonstrate that the levels of c-jun mRNA, c-Jun protein, and DNA binding activity of a nuclear transcription factor AP-1 to 12-o-tetradecanoylphorbol 13-acetate responsive elements all increased following treatment with the cell-permeable ceramide, N-acetylsphingosine in human leukemia HL-60 cells. N-Acetylsphingosine (1-10 microM) increased the levels of c-jun mRNA in a dose-dependent manner, and maximal expression was achieved 1 h after treatment. Increase of c-jun expression treated with 5 microM N-acetyldihydrosphingosine, which could not induce apoptosis, was one third of that with 5 microM N-acetylsphingosine. Ceramide-induced growth inhibition and DNA fragmentation were both prevented by treatment with curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione (an inhibitor of AP-1 activation), or antisense oligonucleotides for c-jun. These results suggest that the transcription factor AP-1 is critical for apoptosis in HL-60 cells and that an intracellular sphingolipid mediator, ceramide, modulates a signal transduction inducing apoptosis through AP-1 activation.

The mouse recessive deafness mutation,shaker-2(sh-2),represents a plausible model for an autosomal recessive form of human non-syndromic genetic deafness,DFNB3.Here we report the use of a positional cloning approach to show that the gene mutated insh-2mice encodes a novel type of unconventional myosin. A G-to-A transition changing cysteine to tyrosine in the conserved actin binding domain is detected insh-2but absent in laboratory strains and wild mice belonging to different mouse subspecies and species. This suggests that the novel myosin gene is a strong candidate forDFNB3.