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A specific binding site for somatotropin was solubilized by 1% (v/v) Triton X-100 from a crude particulate membrane fraction of pregnant rabbit liver, partially purified and characterized. The solubilized binding site retained many of the characteristics observed in the original particulate fraction, indicating that extraction of the binding site with Triton X-100 does not cause any major changes in its properties. The binding of human 125I-labelled-somatotropin to the solubilized binding site is a saturable and reversible process, depending on temperature, incubation time, pH and ionic environment. Analysis of the kinetic data revealed a finite number of binding sites with an affinity constant of 0.32 × 10(10)M-1. The binding activity for human 125I-labelled-somatotropin was adsorbed to a concanavalin-A-Sepharose column and was dissociated from the column with alpha-methyl-D-glucoside, suggesting that the binding protein may be a glycoprotein. Using affinity chromatography on concanavalin-A-Sepharose, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B, the binding protein was purified 1000-4000-fold from the original liver homogenate. When the partially purified preparation was chromatographed on Sepharose 6B, the binding protein eluted as a molecule with an apparent molecular weight of 200000, with a Stokes' radius of 4.9 nm. Sucrose-density-gradient centrifugation of the preparation showed that the sedimentation coefficient of the binding protein was 7.2S. Isoelectric focusing experiments revealed that a major part of the protein has an acidic pI (4.2-4.5). Exposure of the protein to trypsin decreased the binding activity for human 125I-labelled-somatotropin or bovine 125I-labelled-somatotropin, whereas ribonuclease, deoxyribonuclease, phospholipase C or neuraminidase had little or no effect.

When neuroblasts divide, prospero protein and mRNA segregate asymmetrically into the daughter neuroblast and sibling ganglion mother cell. miranda is known to localize prospero protein to the basal cell cortex of neuroblasts while the staufen RNA-binding protein mediates prospero mRNA localization. Here we show that miranda is required for asymmetric staufen localization in neuroblasts. Analyses using miranda mutants reveal that prospero and staufen interact with miranda under the same cell-cycle-dependent control. miranda thus acts to partition both prospero protein and mRNA. Furthermore, miranda localizes prospero and staufen to the basolateral cortex in dividing epithelial cells, which express the three proteins prior to neurogenesis. Our observations suggest that the epithelial cell and neuroblast (both of epithelial origin) share the same molecular machinery for creating cellular asymmetry.

Like other tissues and organs in vertebrates, multipotential stem cells serve as the origin of diverse cell types during genesis of the mammalian central nervous system (CNS). During early development, stem cells self-renew and increase their total cell numbers without overt differentiation. At later stages, the cells withdraw from this self-renewal mode, and are fated to differentiate into neurons and glia in a spatially and temporally regulated manner. However, the molecular mechanisms underlying this important step in cell differentiation remain poorly understood. In this study, we present evidence that the expression and function of the neural-specific transcription factors Mash-1 and Prox-1 are involved in this process. In vivo, Mash-1- and Prox-1-expressing cells were defined as a transient proliferating population that was molecularly distinct from self-renewing stem cells. By taking advantage of in vitro culture systems, we showed that induction of Mash-1 and Prox-1 coincided with an initial step of differentiation of stem cells. Furthermore, forced expression of Mash-1 led to the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells, and up-regulation of Prox-1, suggesting that Mash-1 positively regulates cell differentiation. In support of these observations in vitro, we found specific defects in cellular differentiation and loss of expression of Prox-1 in the developing brain of Mash-1 mutant mice in vivo. Thus, we propose that induction of Mash-1 and Prox-1 is one of the critical molecular events that control early development of the CNS.

Morphogen gradients of secreted molecules play critical roles in the establishment of the spatial pattern of gene expression. During midgut development in Drosophila, secreted molecules of Decapentaplegic (Dpp) and Wingless (Wg) establish unique transcriptional regulation within target cells to specify the resultant cell types. Here we report the identification of a novel homeobox gene, defective proventriculus (dve), which is required for the midgut specification under the control of Dpp and Wg. In dve mutants, two distinct parts of the midgut, the proventriculus and middle midgut, are abnormally organized. The Wg signal regulates dve expression during proventriculus development. On the other hand, dve is a downstream target of Dpp in the middle midgut and defines the functional specificity of copper cells along with another Dpp target gene, labial. Thus, the dve gene acts under the two distinct extracellular signals at distant parts of the midgut primordia.

Two high molecular weight heat shock proteins, HSP90 (Mr, 90,000) and HSP100 (Mr, 100,000), were separately purified from extracts of cultured cells of a mouse lymphoma cell line, L5178Y. Both of the HSPs exist in homodimeric form under physiological conditions. Their physicochemical properties are quite similar to each other. Each of the purified HSPs was shown to coprecipitate with rabbit skeletal muscle actin under actin-polymerizing conditions. Both HSP90 and HSP100 increased the low-shear viscosity of filamentous actin solutions in a dose-dependent manner, which suggests that these HSPs cross-link actin filaments. Although some molecular properties and the effects described above on actin solution of HSP90 and HSP100 resemble those of alpha-actinin, the HSPs were distinguished from alpha-actinin by various means, including visualization of molecular shapes by electron microscopy with the aid of the low-angle rotary shadowing technique. Immunofluorescence staining by specific antisera against HSP90 revealed that HSP90 was localized in ruffling membranes in addition to the cytoplasmic space.

beta-tubulin of budding yeast Saccharomyces cerevisiae is a polypeptide of 457 amino acids encoded by the unique gene TUB2. We investigated the function of the carboxy-terminal part of yeast beta-tubulin corresponding to the carboxy-terminal variable domain of mammalian and avian beta-tubulins. The GAA codon for Glu-431 of TUB2 was altered to TAA termination codon by using in vitro site-directed mutagenesis so that the 27-amino acid residues of the carboxyl terminus was truncated when expressed. The mutagenized TUB2 gene (tub2(T430)) was introduced into a haploid strain in which the original TUB2 gene had been disrupted. The tub2(T430) haploid strain grows normally less than 30 but not at 37 degrees C. The truncation of the carboxyl terminus caused hypersensitivity to antimitotic drugs and low spore viability at the permissive temperature for vegetative growth. Immunofluorescence labeling with antitubulin antibody and DNA staining with 4',6'-diamidino-2-phenylindole showed that in these cells at 37 degrees C, formation of spindle microtubules and nuclear division was inhibited and cytoplasmic microtubule distribution was aberrant. These results suggest that functions of the carboxy-terminal domain of yeast beta-tubulin are necessary for cells growing under suboptimal growth conditions although it is not essential for growth under the optimal growth conditions. Cells bearing tub2(411), a tub2 gene in which the GAA codon for Glu-412 was altered to TAA were no more viable at any temperature. In addition, a haploid strain carrying two functional beta-tubulin genes is not viable.

Thyrotropin Binding Inhibiting Immunoglobulins (TBII) were measured in sera of 240 patients with Graves’ disease who were followed 0–25 yr as a cross-sectioned study (21 untreated, 189 under therapy and 30 T3-suppressible and drug-discontinued patients) by using solubilized porcine thyroid TSH receptor. Assays were performed by using 50 μl of serum. All untreated 21 patients showed positive TBII. Frequency of positive patients decreased yearly with treatment although 36% of patients remained positive after 6 yr of therapy. After that time TBII were positive in 61 % of follow-up patients and in 16 positive patients who have been treated for more than 10 yr, drug therapy could not be stopped because of recurrence. TBII were positive in 6 of 30 T3-suppressible patients. As a longitudinal study changes in TBII were studied in 10 patients at the time of relapse or exacerbation. TBII increased in parallel with increases in thyroid hormone concentrations in 3 of 10 patients. Six of the others showed earlier or later TBII increases than those in thyroid hormones. One patient did not show any change in TBII, albeit thyroid hormone concentrations were found to be increased. Our observations suggest that abnormal IgGs detected as TBII in sera of patients with Graves’ disease by the present method do not explain the occurrence of hyperthyroidism.

Cell adhesion molecules (CAMs) are cell surface glycoproteins that play important roles in morphogenesis and histogenesis, particularly in defining discrete borders between cell populations. Previous studies have suggested that the cytoplasmic domains of CAMs play a significant role in their adhesion properties. These domains may also be involved in regulating other cellular interactions, such as those involved in the sorting-out of cells to form tissues. In the present studies, we have compared the effects of replacing the cytoplasmic domain of one CAM with that of another CAM of different homophilic binding specificity on cell adhesion and cell sorting-out. The molecules studied were liver CAM (L-CAM) and the neural CAM (N-CAM) sd polypeptide. One cDNA was constructed that encodes a chimeric molecule composed of the extracellular domain of L-CAM and the cytoplasmic plus transmembrane domains of the sd polypeptide of chicken N-CAM (called L/N-CAM). Another was constructed encoding a truncated L-CAM missing the last 50 residues of the cytoplasmic domain. Permanently transfected lines of mouse L cells were obtained expressing the truncated L-CAM ("L-L-50 cells") or the chimeric L/N-CAM ("L-L/N cells") and were compared with cells expressing intact L-CAM ("L-L cells"). Immunoblotting and ELISA analyses demonstrated that these various cell lines expressed similar amounts of CAMs at the cell surface. Aggregation of L-L and L-L/N cells occurred at similar rates in short-term aggregation assays and was inhibited by antibodies to the extracellular L-CAM binding domain. In contrast, L-L-50 cells did not aggregate. Incubation of transfected cells with cytochalasin D, which disrupts microfilaments, markedly inhibited aggregation of L-L cells but had no effect on L-L/N cell aggregation. Mixed L-L and L-L/N cells co-aggregated in short-term assays; in the longer-term sorting-out assays, however, they behaved differently: L-L cells sorted out from both L-L/N and untransfected cells, whereas L-L/N cells did not sort out from untransfected cells. These studies not only suggest that interactions of cytoplasmic domains of different CAMs with the cytoskeleton can modulate cell adhesion but also suggest that specific interactions with certain cytoskeletal components are required for events such as cell sorting and cell patterning.

Pleiomorphic mouse sarcoma S180 cells were transfected with cDNAs for the liver cell adhesion molecule (L-CAM), the neural cell adhesion molecule (N-CAM), or both CAMs. Transfected cells expressed the appropriate CAMs at their surface and those expressing L-CAM (S180L cells) changed from adjoining spindle or round shapes to a closely linked "epithelioid" sheet when grown to confluence. Cells transfected with cDNA for N-CAM (S180N cells) also expressed this CAM on the cell surfaces and bound brain vesicles containing N-CAM but showed no phenotypic change to an epithelioid state. In S180L cells and doubly transfected (S180L/N) cells, L-CAM was concentrated at regions of cell contact and was codistributed with cortical actin. In S180N cells, N-CAM was uniformly distributed on the cell surface. When S180L cells were cocultured with S180L/N cells, N-CAM was not concentrated at boundaries between the S180L and S180L/N cells but was concentrated at boundaries between pairs of S180L/N cells. Fab' fragments of anti-L-CAM dissociated the epithelioid sheets of S180L or S180L/N cells into cells with shapes resembling those of untransfected cells. Cells in epithelioid sheets were polygonal in shape but, unlike cells in true epithelia, had no basement membrane or polar structure; they also lacked tight junctions and desmosomes. Ultrastructural examination showed that, in contrast to the untransfected phenotype, cells in epithelioid sheets had large increases in adherens junctions and gap junctions. Dye coupling experiments indicated that the gap junctions were functional. The frequency of expression of both kinds of junctions was sharply decreased by treatment with anti-L-CAM Fab' fragments. These experiments provide support for the precedence hypothesis, which proposes that the linkage of cells by means of CAMs is a necessary event for the extensive expression of junctional structures.

Cofilin is a widely distributed, pH-sensitive, actin-modulating protein with an apparent molecular mass of 21 kDa, which forms intranuclear and/or cytoplasmic actin/cofilin rods in cultured fibroblastic cells under specific conditions. In this study, a cDNA library from porcine brain mRNA was constructed, and full-length brain cofilin cDNA clones were isolated by screening with oligonucleotide probes. The deduced amino acid sequence of cofilin is 166 residues long and contains a sequence of Lys-Lys-Arg-Lys-Lys which is very similar to the nuclear transport signal sequence (Pro-Lys-Lys-Lys-Arg-Lys-Val) of SV40 large T antigen. The sequence may act as a signal capable of inducing nuclear accumulation of cofilin in cells exposed to heat shock or dimethyl sulfoxide. The cofilin sequence contains a hexapeptide (Asp-Ala-Ile-Lys-Lys-Lys) identical to the amino-terminal sequence (residues 2-7) of muscle and nonmuscle tropomyosin. Cofilin also has in the carboxyl-terminal portion a region homologous to the sequence shared by gelsolin, fragmin, and Acanthamoeba profilin. Furthermore, the overall amino acid sequence of cofilin shows weak homology with the rod portion of myosin and suggests a high alpha-helical content.

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.

The liver cell adhesion molecule (L-CAM) and N-cadherin or adherens junction-specific CAM (A-CAM) are structurally related cell surface glycoproteins that mediate calcium-dependent adhesion in different tissues. We have isolated and characterized a full-length cDNA clone for chicken N-cadherin and used this clone to transfect S180 mouse sarcoma cells that do not normally express N-cadherin. The transfected cells (S180cadN cells) expressed N-cadherin on their surfaces and resembled S180 cells transfected with L-CAM (S180L cells) in that at confluence they formed an epithelioid sheet and displayed a large increase in the number of adherens and gap junctions. In addition, N-cadherin in S180cadN cells, like L-CAM in S180L cells, accumulated at cellular boundaries where it was colocalized with cortical actin. In S180L cells and S180cadN cells, L-CAM and N-cadherin were seen at sites of adherens junctions but were not restricted to these areas. Adhesion mediated by either CAM was inhibited by treatment with cytochalasin D that disrupted the actin network of the transfected cells. Despite their known structural similarities, there was no evidence of interaction between L-CAM and N-cadherin. Doubly transfected cells (S180L/cadN) also formed epithelioid sheets. In these cells, both N-cadherin and L-CAM colocalized at areas of cell contact and the presence of antibodies to both CAMs was required to disrupt the sheets of cells. Studies using divalent antibodies to localize each CAM at the cell surface or to perturb their distributions indicated that in the same cell there were no interactions between L-CAM and N-cadherin molecules. These data suggest that the Ca(++)-dependent CAMs are likely to play a critical role in the maintenance of epithelial structures and support a model for the segregation of CAM mediated binding. They also provide further support for the so-called precedence hypothesis that proposes that expression and homophilic binding of CAMs are necessary for formation of junctional structures in epithelia.

Epidermal growth factor stimulated both [3H]thymidine uptake and proliferation of rat AH66 hepatoma cells. However, the increase in cell number was not accompanied by a proportional increase in the levels of α-fetoprotein of the culture media. The effects of EGF on the cell proliferation were antagonized by N6, O2′-dibutyryl cAMP.

Both 12-O-tetradecanoyl-phorbol-1-acetate and teleocidin B stimulated the secretion of human chorionic gonadotropin by cultured choriocarcinoma cells. These tumor promoters also stimulated production of progesterone in the cells. However, the 2 tumor promoters did not exert a marked effect on the cellular binding of epidermal growth factor that also had a stimulatory effect on production of these hormones.

In mammalian cerebral cortex, neural progenitor cells change their cytogenetic behavior dynamically as development proceeds: while progenitors in early embryonic days mainly generate two progenitor cells in a symmetric manner, those in the middle stage give rise to two different types of daughters in an asymmetric manner (i.e., a stem-like daughter cell and a daughter cell to soon proceed to differentiation). To understand how this stage-dependent cytogenesis is achieved, we sought to identify genes that are expressed by cortical progenitor cells in a temporally regulated manner. We first compared the gene expression profiles of progenitor cells isolated from mouse cortex at E11, E14 and E16 using the single-cell cDNA amplification method followed by microarray analysis. Single-cell gene expression profiles could classify the neocortical progenitor cells into the apical (stem-like) progenitors and the basal (non-stem-like) progenitors at all developmental stages examined. Next, we performed the unsupervised principal component analysis (PCA) of all stages apical progenitor cells, found that the expression pattern of E11 apical progenitors is distinct from that of E14 apical progenitors. This result suggests that apical progenitors may change their property between E11 and E14. Based on these results, we identified a set of genes whose expression increases or decreases during this E11–E14 period. To ask whether these genes are involved in the stage-dependent change of cytogenesis pattern (from symmetric or purely proliferative to asymmetric or more differentiating), we are now doing “heterochronic” in utero erectroporation experiments. For example, a gene that is normally expressed strongly by E14 apical progenitors but not by E11 apical progenitors is artificially expressed in the cortical wall at E10–E11.