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The tumor suppressor adenomatous polyposis coli (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. APC forms a complex with beta-catenin, Axin, and glycogen synthase kinase-3beta and induces the degradation of beta-catenin. In the present study, we examined whether APC association with Axin is required for degradation of beta-catenin. We found that a fragment of APC that induces beta-catenin degradation was rendered inactive by disruption of its Axin-binding sites. Also, overexpression of an Axin fragment spanning the regulator of the G-protein signaling domain inhibited APC-mediated beta-catenin degradation. An APC fragment with mutated beta-catenin-binding sites but intact Axin-binding sites also failed to induce degradation of beta-catenin. These results suggest that APC requires interaction with Axin and beta-catenin to down-regulate beta-catenin.

We have previously isolated and cloned a novel eosinophil chemoattractant (ECA) from a human T-cell-derived expression library. This ECA, termed ecalectin, is a variant of human galectin-9, a member of a beta-galactoside binding animal lectin family, which contains two conserved carbohydrate recognition domains (CRDs). In the present study, we addressed whether carbohydrate binding activity is required for the ECA activity of ecalectin and whether both CRDs are essential for this activity. Recombinant full-length wild-type ecalectin (ecalectin-WT) and N-terminal and C-terminal CRD (ecalectin-NT and -CT, respectively) were generated. All of these recombinant proteins exhibited affinity for lactose, a property shared by galectins, but ecalectin-WT exhibited substantially higher hemagglutination activities than ecalectin-NT and -CT. Furthermore, ecalectin-WT showed over 100-fold higher ECA activity than ecalectin-NT and -CT; combination of recombinant domain fragments did not reconstitute the ECA and hemagglutination activities of the full-length protein. ECA activity of ecalectin-WT was inhibited by lactose in a dose-dependent manner. Site-directed mutation of positions Arg(65) of ecalectin-NT and Arg(239) of ecalectin-CT to an aspartic acid residue resulted in the loss of both lactose-binding and ECA activities. We conclude that divalent galactoside-binding activity is required for eosinophil chemoattraction by ecalectin.

We have identified a mouse PDZ protein that interacts with the activin type IIA receptor (ActRIIA), which we named activin receptor-interacting protein 1 (ARIP1). By using yeast two-hybrid screening, we isolated a cDNA clone of ARIP1 from a mouse brain cDNA library. We detected two forms of ARIP1, ARIP1-long and ARIP1-short, which may be produced by alternative splicing. ARIP1-long had one guanylate kinase domain in the NH(2)-terminal region, followed by two WW domains and five PDZ domains (PDZ1-5). ARIP1-short had a deletion in the NH(2)-terminal region and lacked the guanylate kinase domain. Both forms interacted with ActRIIA through PDZ5. The COOH-terminal residues of ActRIIA (ESSL) agree with a PDZ-binding consensus motif, and ARIP1 recognized the consensus sequence. ARIP1 interacts specifically with ActRIIA among the receptors for the transforming growth factor beta family. Interestingly, ARIP1 also interacted with Smad3, which is an activin/transforming growth factor beta intracellular signaling molecule. The mRNA of ARIP1 was more abundant in the brain than in other tissues. Overexpression of ARIP1 controls activin-induced and Smad3-induced transcription in activin-responsive cell lines. These findings suggest that ARIP1 has a significant role in assembling activin signaling molecules at specific subcellular sites and in regulating signal transduction in neuronal cells.

We previously reported the purification and characterization of a novel type of alkaline ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368-14373). Here, we report the molecular cloning, sequencing, and expression of the gene encoding the ceramidase of this strain. Specific oligonucleotide primers were synthesized using the peptide sequences of the purified ceramidase obtained by digestion with lysylendopeptidase and used for polymerase chain reaction. DNA fragments thus amplified were used as probes to clone the gene encoding the ceramidase from a genomic library of strain AN17. The open reading frame of 2,010 nucleotides encoded a polypeptide of 670 amino acids including a signal sequence of 24 residues, 64 residues of which matched the amino acid sequence determined for the purified enzyme. The molecular weight of the mature enzyme was estimated to be 70,767 from the deduced amino acid sequence. Expression of the ceramidase gene in Escherichia coli, resulted in production of a soluble enzyme with the identical N-terminal amino acid sequence. Recombinant ceramidase was purified to homogeneity from the lysate of E. coli cells and confirmed to be identical to the Pseudomonas enzyme in its specificity and other enzymatic properties. No significant sequence similarities were found in other known functional proteins including human acid ceramidase. However, we found a sequence homologous to the ceramidase in hypothetical proteins encoded in Mycobacterium tuberculosis, Dictyostelium discoideum, and Arabidopsis thaliana. The homologue of the ceramidase gene was thus cloned from an M. tuberculosis cosmid and expressed in E. coli, and the gene was demonstrated to encode an alkaline ceramidase. This is the first report for the cloning of an alkaline ceramidase.

During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, nonspecific flip-flop of phospholipids play a role. As previous studies in the erythrocyte ghost have shown that polyamines can alter flip-flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoing apoptosis would affect PS appearance, either by altering aminophospholipid translocase activity or phospholipid flip-flop. Cells of the human leukemic cell line, HL-60, were incubated with or without the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apoptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profound depletion of putrescine and spermidine (but not spermine), it had no effect on caspase activity, DNA fragmentation, or plasma membrane vesiculation, typical characteristics of apoptosis. Notably, DFMO treatment prior to ultraviolet irradiation did not alter the decline in PS inward movement by the aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS detected in the flow cytometer. Conversely, the appearance of endogenous PS in the plasma membrane outer leaflet detected with fluorescein isothiocyanate-labeled annexin V and enhanced phospholipid flip-flop detected by the uptake of 1-palmitoyl-1-[6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosphocholine (NBD-PC) seen during apoptosis were significantly inhibited by prior DFMO treatment. Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restored both enhanced phospholipid flip-flop and appearance of PS during apoptosis. Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay. Taken together, these data show that intracellular polyamines can modulate PS appearance resulting from nonspecific flip-flop of phospholipids across the plasma membrane during apoptosis.

We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries and neural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1-5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis by in situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling.

Brain-enriched human FC96 protein shows a close sequence similarity to the Dictyostelium actin-binding protein coronin, which has been implicated in cell motility, cytokinesis, and phagocytosis. A phylogenetic tree analysis revealed that FC96 and two other mammalian molecules (p57 and IR10) form a new protein family, the coronin-like protein (Clipin) family; thus hereafter we refer to FC96 as ClipinC. A WD domain and a succeeding alpha-helical region are conserved among coronin and Clipin family members. ClipinC is predominantly expressed in the brain, and discrete areas in the mouse brain were intensely labeled with anti-ClipinC antibodies. ClipinC was also shown to bind directly to F-actin in vitro. Immunocytochemical analysis revealed that ClipinC accumulated at focal adhesions as well as at neurite tips and stress fibers. Furthermore, ClipinC was associated with vinculin, which is a major component of focal contacts. These results indicate that ClipinC is also a component part of the cross-bridge between the actin cytoskeleton and the plasma membrane. These findings and the previously reported function of coronin suggest that ClipinC may play specific roles in the reorganization of neuronal actin structure, a change that has been implicated in both cell motility and growth cone advance.

alpha2beta1 integrin, CD36, and GP VI have all been implicated in platelet-collagen adhesive interactions. We have investigated the role of these glycoproteins on activation of the GP IIb-IIIa complex induced by platelet adhesion to type I fibrillar and monomeric collagen under static conditions. In the presence of Mg2+, platelet adhesion to fibrillar collagen induced activation of the GP IIb-IIIa complex and complete spreading. Anti-alpha2beta1 integrin and anti-GP VI antibodies inhibited the activation of the GP IIb-IIIa complex by about 40 and 50%, respectively, at 60 min although minimal inhibitory effects on adhesion were seen. Platelet spreading was markedly reduced by anti-alpha2beta1 integrin antibody. The combination of anti-alpha2beta1 integrin with anti-GP VI antibody completely inhibited both platelet adhesion and activation of the GP IIb-IIIa complex. Anti-CD36 antibody had no significant effects on platelet adhesion, spreading, and the activation of the GP IIb-IIIa complex at 60 min. Aspirin and the thromboxane A2 receptor antagonist SQ29548 inhibited activation of the GP IIb-IIIa complex about 30% but had minimal inhibitory effect on adhesion. In the absence of Mg2+, there was significant activation of the GP IIb-IIIa complex but minimal spreading was observed. Anti-GP VI antibody completely inhibited adhesion whereas no effect was observed with anti-alpha2beta1 integrin antibody. Anti-CD36 antibody partially inhibited both adhesion and the activation of the GP IIb-IIIa complex. Platelet adhesion to monomeric collagen, which requires Mg2+ and is exclusively mediated by alpha2beta1 integrin, resulted in partial activation of the GPIIb-IIIa complex and spreading. No significant effects were observed by anti-CD36 and anti-GP VI antibodies. These results suggest that both alpha2beta1 integrin and GP VI are involved in inside-out signaling leading to activation of the GP IIb-IIIa complex after platelet adhesion to collagen and generation of thromboxane A2 may further enhance expression of activated GP IIb-IIIa complexes.

Externally applied Ni(2+), which apparently competes with Ca(2+) in all three isoforms of Na(+)/Ca(2+) exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li(+) is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423-C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni(2+) or Li(+) by measuring intracellular Na(+)-dependent (45)Ca(2+) uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal alpha-1 and alpha-2 repeats are individually responsible for the alteration of Ni(2+) sensitivity, both together accounting for approximately 80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the alpha-2 repeat fully accounts for the differential Li(+) sensitivity between the isoforms. The Ni(2+) sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the alpha-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the alpha-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li(+) sensitivity was mimicked by double substitution mutation in the alpha-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn(125) and Val(820) of NCX1 caused significant alterations in the interactions of the exchanger with Ca(2+) and Ni(2+), and Ni(2+) and Li(+), respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the alpha repeats, these regions might form an ion interaction domain in the exchanger.

A functional element of an enzyme can be defined as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. In order to elucidate the distribution of functional elements in an active site flexible loop (the M20 loop) of Escherichia coli dihydrofolate reductase, systematic cleavage of main chain connectivity was performed using circular permutation. Our analysis is based on the assumption that a permutation within a functional element would significantly reduce enzyme function, whereas ones outside or at the boundaries of the elements would affect the function only slightly. Thus, a functional element would be assigned as the minimum peptide chain between the identified boundaries. Comparison of the activities of the circularly permuted variants revealed that the peptide chain around the M20 loop could be divided into four regions (regions 1-4). Region 1 was found to play an important role in overall tertiary fold because most variants permuted at region 1 did not accumulate in E. coli cells stably. A distinction between region 2 and region 3 was in agreement with the extent of movements calculated from the coordinates of alpha carbons, supporting the idea that the movement of peptide backbone is a key feature of enzyme function. The boundary between region 3 and region 4 coincided with that between the M20 loop and the following alpha helix. From equilibrium binding studies, region 2 was found to be involved in the binding of nicotinamide substrates, whereas region 4 appeared to be very important for the binding of pterin substrates.

We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.

Orexin-A and -B are recently identified potent orexigenic peptides that are derived from the same precursor peptide and are highly specifically localized in neurons located in the lateral hypothalamic area, a region classically implicated in feeding behavior. We cloned the whole length of the human prepro-orexin gene and corresponding cDNA. The human prepro-orexin mRNA was predicted to encode a 131-residue precursor peptide (prepro-orexin). The human prepro-orexin gene consists of two exons and one intron distributed over 1432 base pairs. The 143-base pair first exon includes the 5'-untranslated region and a small part of the coding region that encodes the first seven residues of the secretory signal sequence. The second exon contains the remaining portion of the open reading frame and 3'-untranslated region. The 3.2 kilobase pairs of the 5'-upstream region from a cloned human prepro-orexin gene promoter is sufficient to direct the expression of the Escherichia coli beta-galactosidase (lacZ) gene in transgenic mice to neurons in the lateral hypothalamic area and adjacent regions. The lacZ-positive neurons were positively stained with anti-orexin antibody but not with anti-melanin-concentrating hormone antibody. These findings suggest that this genomic fragment contains all the necessary elements for appropriate expression of the gene and will be useful for the targeted expression of the exogenous gene in orexin-containing neurons. These mice might also be useful for examining the molecular mechanisms by which orexin gene expression is regulated.

An endo-beta-glucuronidase acting on chondroitin sulfate was isolated from rabbit liver and purified about 550-fold, using a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephacryl S-300, affinity chromatography through heparin-Sepharose CL-6B and preparative polyacrylamide gel electrophoresis. The pH optimum of this enzyme was 4.0 and the Km value 7 X 10(-3) M for chondroitin sulfate (Mr 40,000). The isoelectric point of the enzyme was found to be at pH 5.4. The molecular weight, estimated by gel filtration through Sephacryl S-200 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 35,000. This enzyme, which was found in the liver, kidney, spleen, and lung, hydrolyzed the glucuronyl galactose linkage of the linkage region of chondroitin sulfate possessing a very small peptide segment. The enzyme did not hydrolyze proteoglycan. It was concluded that an endo-beta-glucuronidase is involved in the catabolism of proteoglycan chondroitin sulfates.

Three glycoproteins (GPs), namely GPIa-IIa, GPVI, and GPIV, have been recently implicated in platelet-collagen adhesive interactions. We have employed antibodies to these GPs to investigate further their role in platelet adhesion to immobilized monomeric and polymeric fibrillar collagen under static conditions in the presence and the absence of Mg2+. In the presence of Mg2+, each antibody inhibited platelet adhesion to fibrillar collagen from 70 to 85%, especially during the early phase (<15 min), but the inhibitory effects diminished dramatically to 25% or less by 60 min. Combination of anti-GPVI with anti-GPIa-IIa antibodies completely inhibited platelet adhesion at 60 min. Anti-GPIV and anti-GPIa-IIa or anti-GPVI antibodies in combinations were more effective in inhibiting adhesion than was anti-GPIa-IIa or anti-GPVI alone. In the absence of Mg2+, anti-GPVI completely inhibited adhesion at 60 min, while anti-GPIV antibody inhibited adhesion by about 50% and minimal effects were seen with anti-GPIa-IIa, suggesting that GPIa-IIa does not play a significant role in the divalent cation-independent platelet adhesion to immobilized fibrillar collagen. Under either divalent cation-dependent or -independent conditions, platelets adhered to fibrillar collagen were able to secrete contents of both alpha-granules and dense granules and generate thromboxane A2 (TXA2), but platelets adhering to acid soluble monomeric collagen neither secreted their granular contents nor generated TXA2. Although anti-GPVI antibodies were not able to inhibit Mg2+-dependent adhesion, they completely inhibited TXA2 generation under both divalent cation-dependent and -independent conditions. With the other antibodies, TXA2 generation corresponded with the amount of adhesion observed. These results suggest that GPVI is directly associated with the TXA2 generating system during platelet-collagen interaction.

We have examined the regulation of apolipoprotein A-I (apoA-I) gene expression in response to glucose and insulin. In Hep G2 cells, endogenous apoA-I mRNA was suppressed by one-half or induced 2-fold following 48 h of exposure to high concentrations of glucose (22.4 mM) or insulin (100 microunits/ml), respectively, compared with control. Transcriptional activity of the rat apoA-I promoter (-474 to -7) in Hep G2 cells paralleled endogenous mRNA expression, and this activity was dependent on the dose of glucose or insulin. Deletional analysis showed that a 50-base pair fragment spanning -425 to -376 of the promoter mediated the effects of both insulin and glucose. Within this DNA fragment there is a motif (-411 to -404) that is homologous to a previously identified insulin response core element (IRCE). Mutation of this motif abolished not only the induction of the promoter by insulin but also abrogated its suppression by glucose. Electrophoretic mobility shift assay analysis of nuclear extracts from Hep G2 cells revealed IRCE binding activity that formed a duplex with radiolabeled probe. The IRCE binding activity correlated with insulin induction of apoA-I expression. In summary, our data show that glucose decreases and insulin increases apoA-I promoter activity. This effect appears to be mediated by a single cis-acting element.

We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rac1 small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1(val12) (Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 23363-23367). Here we identified an IQGAP1-interacting molecule with molecular mass of 43 kDa (p43) from bovine brain cytosol, using glutathione S-transferase (GST)-IQGAP1 affinity column chromatography. The amino acid sequencing of the protein revealed that p43 was identical to beta- and gamma-actin. IQGAP1 was cosedimentated with filamentous actin (F-actin). The amino-terminal domain (amino acids 1-216) of IQGAP1 was responsible for the interaction with F-actin. Falling ball viscometry assay revealed that IQGAP1 cross-linked the F-actin. This IQGAP1 activity was further enhanced by guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).GST-Cdc42 but not by GDP.GST-Cdc42. The gel filtration analysis of IQGAP1 revealed that IQGAP1 appeared as oligomers and that GTPgammaS.GST-Cdc42 but not GDP.GST-Cdc42 enhanced the oligomerization of IQGAP1. These results strongly suggest that IQGAP1, acting downstream of Cdc42, can cross-link the actin filament through its oligomerization.

We have identified and characterized a cDNA encoding a novel Ca2+-binding protein named calumenin from mouse heart by the signal sequence trap method. The deduced amino acid sequence (315 residues) of calumenin contains an amino-terminal signal sequence and six Ca2+-binding (EF-hand) motifs and shows homology with reticulocalbin, Erc-55, and Cab45. These proteins seem to form a new subset of the EF-hand protein family expressed in the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Purified calumenin had Ca2+-binding ability. The carboxyl-terminal tetrapeptide His-Asp-Glu-Phe was shown to be responsible for retention of calumenin in ER by the retention assay, immunostaining with a confocal laser microscope, and the deglycosylation assay. This is the first report indicating that the Phe residue is included in the ER retention signal. Calumenin is expressed most strongly in heart of adult and 18.5-day embryos. The calumenin gene (Calu) was mapped at the proximal portion of mouse chromosome 7.

We have isolated cDNA clones encoding novel proteins belonging to the cadherin family. These novel proteins are encoded by two distinct mRNA species generated by alternative splicing from a single gene, and based on preferential expression in the pituitary gland and brain, we named it PB-cadherin. One mRNA species encodes long type PB-cadherin composed of 803 amino acid residues with a longer cytoplasmic domain, whereas the other species encodes short-type PB-cadherin composed of 694 amino acid residues with a shorter cytoplasmic domain. Both long and short type PB-cadherin contain five repeats of a cadherin motif in the extracellular domain, the transmembrane domain, and the cytoplasmic domain, and the deduced amino acid sequences have a 30% homology to those of E-, N-, and P-cadherins. Although the primary structure of N-terminal amino acids is identical between long and short type PB-cadherin, the following structures in the cytoplasmic regions are completely different. The long type PB-cadherin but not the short type contains the putative catenin-binding domain. When these two distinct forms of PB-cadherins were stably expressed in L cells, L cells expressing long type PB-cadherin or short type PB-cadherin both acquired a Ca2+-dependent cell adhesion property, thereby indicating that both types of PB-cadherin are responsible for Ca2+-dependent cell adhesion. Persistent expression of PB-cadherin mRNA was found in the brain of rat embryos at least from embryonic day 15 to the postnatal period. In situ localization of PB-cadherin mRNA in the adult rat brain indicated that PB-cadherin mRNA is expressed in the inner granular layer of the olfactory bulb, Purkinje cell layer of the cerebellum, and in the pineal gland. PB-cadherin may play an important role in morphogenesis and tissue formation in neural and non-neural cells for the development and maintenance of the brain and neuroendocrine organs by regulating cell-cell adhesion.

There are two types of the activin-binding protein follistatin (FS), FS-288 and FS-315. These result from alternative splicing of mRNA. FS-288 exhibits high affinity for cell-surface heparan sulfate proteoglycans, whereas FS-315 shows low affinity. To understand the physiological role of cell-associated FS, we investigated the binding of activin to cell-associated FS and its behavior on the cell surface using primary cultured rat pituitary cells. Affinity cross-linking experiments using 125I-activin A demonstrated that activin bound to rat pituitary cells via FS as well as to their receptors on the cell surface. FS-288 promoted the binding of activin A to the cell surface more markedly than FS-315. When the cells were incubated with 125I-activin A in the presence of FS-288, significant degradation of activin A was observed, and this was dependent on the FS-288 concentration. This activin degradation was abolished by heparan sulfate, chloroquine, and several lysosomal enzyme inhibitors. Moreover, FS-288 stimulated cellular uptake of activin A, whereas chloroquine suppressed lysosomal degradation following internalization, as demonstrated by microscopic autoradiography. These results suggest that cell-associated FS-288 accelerates the uptake of activin A into pituitary cells, leading to increased degradation by lysosomal enzymes, and thus plays a role in the activin clearance system.

We have cloned cDNA for protein tyrosine phosphatase beta2, which had been implicated in erythroid differentiation of mouse erythroleukemia cells. Expression of cDNA constructs, in which beta2 cDNA is placed under the control of mouse metallothionein-I promoter, by ZnCl2 converted a significant portion (20 to 38%) of the cells to erythroid-like cells, which is 25-50% of the erythroid differentiation efficiency observed by conventional erythroid-inducing agents. Furthermore, introduction and expression of altered protein tyrosine phosphatase beta2 cDNA constructs designed to produce the enzyme lacking the phosphatase activity inhibited erythroid differentiation by 100-20%, depending upon the concentration of erythroid-inducing agents employed. These results strongly suggest that protein tyrosine phosphatase beta2 is involved in triggering erythroid differentiation in mouse erythroleukemia cells.

A potent anticoagulant, anti-lipopolysaccharide (LPS) factor, found in limulus hemocytes inhibits the LPS-mediated activation of limulus coagulation cascade and shows an antibacterial action against R-types of Gram-negative bacteria (Morita, T., Ohtsubo, S., Nakamura, T., Tanaka, S., Iwanaga, S., Ohashi, K., and Niwa, M. (1985) J. Biochem. (Tokyo) 97, 1611-1620). The complete amino acid sequence of this substance was determined by sequencing the peptides obtained by selective proteolytic cleavage. The NH2-terminal end of anti-LPS factor was pyroglutamic acid. Anti-LPS factor had two variant residues at position 36 and the COOH-terminal end, respectively. The following sequence was assigned to anti-LPS factor, and it was also confirmed by fast atom bombardment mass spectrometry. less than EGGIWTQLALALVKNLATLWQSGDFQFLGHE (formula; see text) Limulus anti-LPS factor consisted of a single chain of 102 residues with 2 half-cystines in disulfide linkage. Its NH2-terminal region up to 20 residues was highly hydrophobic, and positively charged residues were clustered mainly within the disulfide loop. By searching the homologous sequence in known protein sequences with that of anti-LPS factor, we found a structural homology between anti-LPS factor and alpha-lactalbumin/lysozyme family.

Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+-loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form.

When hepatocytes isolated from adult rats were cultured in the presence of 10 mMnicotinamide, insulin- and epidermal growth factor-induced DNA synthesis and cell proliferation were found to be greatly stimulated, and the cells were able to be kept alive for more than one month. In the nicotinamide-treated hepatocytes, albumin and tryptophan 2,3-dioxygenase mRNAs were present at much higher levels than in the untreated control, and the inducibility of tryptophan oxygenase gene expression by dexamethasone and glucagon was also preserved. Without nicotinamide, primary cultured hepatocytes were viable for only 5–7 days and the hepatocyte-specific phenotypes were rapidly lost. The intracellular NAD level was maintained in the nicotinamide-treated hepatocytes at or above the level in intact liver but depleted in hepatocytes without nicotinamide. These results suggest that the maintenance of the intracellular NAD level is essential for the growth and functioning of hepatocytes and that nicotinamide can preserve the NAD level by blocking NAD degradation as well as by acting as a precursor for NAD synthesis.

A cellular receptor for erythroid differentiation factor (EDF) was demonstrated by incubation of 125I-labeled EDF with rat follicular granulosa cell cultures. The specific binding of labeled EDF to the cells showed saturation; Scatchard analysis of binding data indicated a single class of receptors having Kd = 3.4 x 10(-10) M. A large excess of unlabeled EDF reduced labeled EDF binding almost completely, whereas similar doses of inhibin and transforming growth factor type beta, which are quite similar to EDF in protein structure and subunit organization, had no effect; EDF did not share receptors with those factors. EDF receptor (activin A receptor) expression was enhanced in granulosa cells cultured in the presence of follicle-stimulating hormone; follicle-stimulating hormone raised the number of EDF binding sites/cell from 13,000 to 96,000 without altering the binding affinity.

Induction of translatable mRNA for serine dehydratase (SDH) in primary cultured rat hepatocytes requires both dexamethasone and glucagon or cAMP (Noda, C., Tomomura, M., Nakamura, T., and Ichihara, A. (1986) J. Biochem. (Tokyo) 95, 37-45). This unique hormone requirement was studied further with a cDNA probe complementary to SDH mRNA in primary cultured hepatocytes of adult rats. Dot-blot hybridization analysis of RNA showed that SDH mRNA was induced by dexamethasone and glucagon together, but not by either alone. Insulin or epinephrine caused 40% inhibition of this induction of SDH mRNA. Cycloheximide prevented the induction of SDH mRNA by dexamethasone and glucagon, suggesting that ongoing protein synthesis is required for the induction by glucocorticoids and glucagon. In vitro transcription experiments using nuclei isolated from cultured hepatocytes showed that transcription of the SDH gene was not affected by either dexamethasone or glucagon alone, but markedly enhanced by both hormones together, and that this enhancement was inhibited by insulin or epinephrine. These results indicate that the inhibition of SDH induction by epinephrine or insulin was due to effects of these hormones on the transcriptional rate of the SDH gene.

We have found a novel enzyme that exclusively decomposes L-selenocysteine into L-alanine and H2Se in various mammalian tissues, and have named it selenocysteine lyase. The enzyme from pig liver has been purified to homogeneity. It has a molecular weight of approximately 85,000, and contains pyridoxal 5'-phosphate as a coenzyme. Its maximum reactivity is at about pH 9.0. Balance studies showed that 1 mol of selenocysteine is converted to equimolar amounts of alanine and H2Se. The following amino acids are insert: L-cysteine, L-serine, L-cysteine sulfinate, selenocysteamine, Se-ethyl-DL-selenocysteine, and L-selenohomocysteine. L-Cysteine (Ki, 1.0 mM) competes with L-selenocysteine (Km, 0.83 mM) to inhibit the enzyme reaction. The enzyme is the first proven enzyme that specifically acts on selenium compounds.

During treatment of primary cultured hepatocytes with either glucagon or isoproterenol for several hours, the stimulations of cAMP formation by these hormones decreased time dependently. Glucagon treatment also reduced the response to isoproterenol, but isoproterenol treatment did not decrease the response to glucagon. Treatment with isoproterenol caused more rapid desensitization than treatment with glucagon. Assays of glucagon and beta-adrenergic receptors showed that the receptor number of only the hormone with which the cells were treated decreased and that dissociation constants of the receptors did not change. Moreover, in glucagon-desensitized cells, the effect of GTP on competition of the bindings of antagonist and agonist for the beta-adrenergic receptor did not change. After treatment with isoproterenol, stimulation of adenylate cyclase activity by the agonist was decreased without any decrease in the stimulations of activity by other effectors. In contrast, glucagon treatment greatly decreased the stimulations of activity by glucagon, isoproterenol, and guanyl-5'-yl imidodiphosphate and slightly decreased that by fluoride. However, after glucagon treatment, the cells showed normal responses to cholera toxin of activation of adenylate cyclase and ADP-ribosylation of guanine nucleotide binding regulatory protein (Ns). The maximal response of glucagon-treated cells to forskolin was about two-thirds that of untreated cells and this treatment also impaired the shift toward a low Kact value for forskolin observed in the presence of either glucagon or isoproterenol. These results indicate that isoproterenol caused homologous desensitization consisting of only a “down regulation” of the beta-adrenergic receptor, whereas glucagon caused heterologous desensitization, mainly by alteration of the Ns component, as well as “down regulation” of glucagon receptor. This altered Ns seems to be coupled normally to the beta-adrenergic receptor, but to have impaired coupling to the catalytic component of adenylate cyclase.

In freshly isolated parenchymal hepatocytes of adult rats, the beta-adrenergic agonist isoproterenol (Ip) did not stimulate cAMP formation, protein kinase activity, or glycogenolysis, although glucagon markedly stimulated all these activities. However, the beta-adrenergic response appeared when rat hepatocytes were cultured as monolayers. This response had already appeared after 2-h culture and increased during further culture. The appearance of the beta-adrenergic response during culture was blocked by cycloheximide, actinomycin D, or alpha-amanitin. Thus adult rat hepatocytes acquired marked ability to respond to Ip during culture through the syntheses of mRNA and protein. Freshly isolated hepatocytes from postnatal rats showed a high beta-adrenergic response that did not increase further during culture. This response gradually decreased during development and had almost disappeared about 60 days after birth. In plasma membranes prepared from freshly isolated cells of adult rats the basal and NaF-stimulated activities of adenylate cyclase (EC 4.6.1.1) were similar to those of cultured cells and the enzyme activity was also stimulated by guanyl-5'-yl imidodiphosphate. However, in plasma membranes of freshly isolated cells Ip scarcely stimulated adenylate cyclase, but glucagon did. The intact cells, whether they were freshly isolated or cultured, accumulated cAMP when exposed to cholera toxin. Moreover, the two subunits of GTP-binding regulatory protein (also named G/F or Ns site) were detected by [32P]ADP ribosylation with cholera toxin and [32P]NAD+ in freshly isolated cells as well as in cultured cells. These results indicate that freshly isolated and cultured hepatocytes of adult rats contain sufficient levels of all the components of the postreceptor-adenylate cyclase system for activity. However, the number of beta-adrenergic receptors measured by binding of [125I]iodocyanopindolol, a potent beta-adrenergic antagonist, was very low in purified plasma membranes of freshly isolated cells (20 fmol/mg of protein), and the number increased about 6-fold without change in the dissociation constant (Kd = 132 pM) when the cells were cultured for 7 h. This increase in beta-adrenergic receptor sites was completely abolished by cycloheximide and alpha-amanitin. Thus it is concluded that the unresponsiveness of adult rat hepatocytes to Ip was due to a very low amount of beta-adrenergic receptor and that the appearance of a beta-adrenergic response during primary culture was due to new synthesis of beta-adrenergic receptor through synthesis of mRNA.