The neural cell adhesion molecule, NCAM, is known to be expressed in chicken muscle as at least three principal molecular forms (molecular masses of 155 kDa, 145 kDa, and 120 kDa). They are generated from a single gene by alternative splicing. To distinguish these molecular species and to investigate their expressions in muscle differentiation during chicken embryonic development, we prepared antipeptide antibodies against three different domains of the NCAM. The antibody named MSD<+> was designed to detect musclespecific domain (MSD) which was inserted into a muscle-specific NCAM by alternative splicing. The locus encompassing the MSD insertion site was detected with the antibody named MSD<->, and cytoplasmic sites near the transmembrane region were detected with the antibody named CYT. Immunoblot analysis utilizing the peptide antibodies prepared here revealed that, of three NCAMs, two isoforms, 155 kDa and 120 kDa, were recognized with the antibody MSD<+> and the same 155-kDa and the other 145-kDa molecule were recognized with the antibody CYT. The antibody MSD<-> was capable of detecting all three isoforms of the NCAM.Expression of the 120-kDa form with MSD but lacking a cytoplasmic tail increased but that of the 145-kDa form with cytoplasmic tail but lacking MSD declined during embryonic day 5 to day 18. The 155-kDa form NCAM with both MSD and cytoplasmic tail was expressed specifically and transiently during embryonic day 11 to day 14 in chick muscle; this period coincides with the period of extensive myotube formation. Thus, this largest isotype seems to play an important role in muscle differentiation.

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Photodamage of insulin-releasing pancreatic β-cells during measurements of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) with fura-2 was studied. Keeping the fluorescence intensities at low levels, regular oscillations of [Ca²⁺]_i with a frequency of 0.2-0.5 min^-1 could be recorded for more than 60 min in glucose-stimulated cells. However, after a ten fold increase of the excitation energies, oscillations disappeared in most cells, the glucose response being transformed into an elevated concentration of [Ca²⁺]_i with irregular fluctuations. Under corresponding conditions, the loss of fluorescence due to photobleaching during the initial 10 min increased from 7 to 16% in cell-sized fura-2-containing droplets. In further attempts to investigate the effects of photodamage, cells were irradiated by pulses (< 1 ms) of intense UV light (300-400 nm). A single flash of 4-7 mJ/mm² perturbed the oscillatory pattern with maintenance of the glucose response in terms of raised [Ca²⁺]_i. More severe lesions obtained by repeated pulses of 13-15 mJ/mm² involved an excessive and uncontrolled rise of [Ca²⁺]_i. It is concluded that there is a significant potential risk of photodamage when using the fura-2 technique. In the pancreatic β-cells, exposure to intense excitation light may even result in a selective suppression of the oscillatory response to glucose.

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The in vitro simian virus 40 (SV40) replication system has been developed as a model system of cellular DNA replication, because the replication initiated from the replication origin of SV40 and replication fork proceeds bidirectionally. In this system, SV40 T-antigen (TAg) is the only factor provided by viral genes, while all other factors are supplied by the host cells. A suppression of replication has been observed in the linear template containing SV40 replication origin, compared with the closed circular template in the SV40 in vitro replication system using a crude extract of HeLa cells. However in the in vitro replication system reconstituted from partially purified factors, less preference was observed for the replication of the closed circular DNA over the linear DNA.
In a mono-polymerase system supplemented by crude extracts, a suppression of replication in a linear template was also observed, when compared with a closed circular template. This suppression effect of crude extract was abolished by heat treatment, suggesting that the suppression was induced by some protein factors. A crude extract of HeLa cells was fractionated by stepwise elution with buffers containing 0.2 M, 0.4 M, 0.6 M and 1M NaCl on a phosphocellulose column, and characterization of factors that suppress linear DNA replication has been done. Both fractions that were eluted at 0.4 M and 0.6 M from phosphocellulose were necessary to suppress linear DNA replication efficiently. The factors in the 0.6 M fraction that suppressed linear DNA replication synergistically with the 0.4 M fraction were partially purified by successive chromatography with heparin-sepharose and dsDNA-cellulose followed by glycerol gradient centrifugation. These results suggested that multiple factors are required to suppress DNA replication of the linear template.

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A protein phosphatase inhibitor, tautomycin induces blebs on the surface of human myeloid leukemia K562 cells within 10 min. In this paper we examined the cytoskeleton of tautomycin-treated cells. In the presence of tautomycin, cells with blebs turned into segmented forms with smooth surfaces after 15 min and into smooth round shapes without microprotuberance after 60 min. Observation with fluorescence microscopy showed that F-actin detached from the plasma membraneat the site of the blebs. Further treatment with tautomycin induced the accumulation of F-actin at the segmentation centers. Under electron microscopy, an electron dense ring-structure was detected at the segmentation center. Tautomycin did not induce major changes of the microtubule network although F-actin accumulated near the microtubule organizing center. The amount of F-actin increased in tautomycin-treated cells. These results indicate that the morphological changes are induced by reorganization of actinfilaments.

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Liver UDP-glucuronosyltransferase (UDPGT) is localized in the endoplasmic reticulum (ER), with its catalytic domain exposed to the lumenal side of the membrane structure. The proteins expressed from the transfected UDPGT cDNA in cultured cells were found to be localized in the ER membrane. Its enzyme activity was in a latent state and was fully expressed in an in vitro assay system when the membrane integrity was disrupted by a detergent, Triton X-100, suggesting that the orientation of the expressed enzyme in the membrane was the same as that of the liver enzyme.
To investigate how the expressed UDPGT was retained in the ER, we constructed chimeric plasmids of cDNAs of UDPGT and ErbB2 which is a receptor protein localized in the cell membranes. Analysis of chimeric proteins expressed in the stable transformants of the cultured cells transfected with these plasmids to reveal that the cytoplasmic tail of UDPGT is responsible for the ER retention of the expressed proteins. Deletion and mutation analysis in the cytoplasmic tail of the enzyme demonstrated that the two lysine residues positioned at 3 and 5 from the C-terminus of the molecule are important for conferring the ER residency. Furthermore, the distance of the ER retention signal composed of the two lysine residues from the transmembrane domain may be influential for the efficiency of the ER retention activity.

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Type V collagen inhibits the cell-substratum adhesion of many types of cells. In this study, inhibitory effects of type V collagen on the adhesion of mouse melanoma B16-F10 cells to fibronectin, laminin and vitronectin were investigated. When the culture dishes were coated with a mixture of fibronectin and type V collagen, adhesion of the cells was inhibited by 50% at a fibronectin/collagen molar ratio of 10/1. At a similar molar ratio, adhesion of the cells to laminin was inhibited moderately, but that to vitronectin was not significantly affected. Type V collagen added into culture medium was less effective in inhibiting cell adhesion. The antiadhesive activity of type V collagen was partially retained in the 1(V) chain of heat-denatured collagen. The α1 (V) chain was split into two large fragments, 90 kDa and 60 kDa, by limited digestion with Staphylococcus aureus V8 proteinase. The 90-kDa fragment, which was derived from the C-terminal half of the α1 (V) chain, inhibited the cell adhesion more profoundly than α1(V). However, little fibronectin bound to the 90-kDa fragment, while fibronectin bound to the 60-kDa fragment, which was less antiadhesive than the 90-kDa fragment, with the same extent as α1(V). Wetherefore concluded that the antiadhesive effect of type V collagen was not due to its specific binding to the fibronectin molecule.

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We have previously shown that mouse L?P3 cells secrete fibronectin and a novel protein, gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). Here, we screened and characterized cell-adhesive proteins in the conditioned media (CM's) of a series of cell lines growing in a protein- and lipidfree synthetic medium (P3 cell lines). Although cell-attachment activity of the CM's ranged from undetectable to highly significant, fractionation with affinity columns revealed the presence of significant cell-attachment activity in all CM's. Using cell-attachment assay and immunoassay on blotted filters, various cell-adhesive proteins were detected in the CM's, and most of them were identified as fibronectin, laminin, vitronectin, and collagen. GNCP-like proteins were detected in the CM's of HeLa?P3, JTC-16?P3, L?P3, and JTC-12?P3. There was no relationship between the origin of the cell lines and the cell-adhesive proteins secreted. GNCP purified from L?P3-CM was separated into 120-, 140-, 150-, and 160-kDa proteins on SDS-PAGE, which were judged to be a type of mouse type I procollagen from the following results: (1) they were digested by collagenase, (2) pepsin treatment converted the 150- and 160-kDa proteins into 120- and 140-kDa proteins, (3) they were recognized by anti-type I collagen antiserum, and (4) amino-terminal sequence of the pepsin-digested 140-kDa protein had significant homology with type I collagen. GNCP showed half-maximum activity of cell attachment at 0.03 μg/ml, indicating that GNCP was a cell-adhesive protein with an extremely high specific activity compared to other known cell-adhesive proteins.

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Primary hepatocytes were cultured on collagen gel in serum-free, α-modified Eagle's minimum essential medium containing 0.1 μMinsulin, 0.1 μM dexamethasone, 10 mM pyruvate and supplements such as glucagon, epinephrine or growth hormone. The activities of alkaline phosphatase, 5' -nucleotidase and γ-glutamyltransferase were assayed in cell extracts prepared from the cultures. All three enzyme activities were induced by glucagon, epinephrine or dibutyryl cAMP.The maximally effective concentration of glucagon was 5-10 nM for both alkaline phosphatase and 5' -nucleotidase and 100 nMfor γ-glutamyltransferase. Only alkaline phosphatase activity was suppressed by growth hormone, which caused marked suppression at about 1 μU (0.25 ng)/ml. Taurocholate also induced both alkaline phosphatase and γ-glutamyltransferase activities at 1 mM.

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Ankyrin is an important key protein transferring the signal between the inside and outside of eukaryotic cells, because of its ability to bind both to ionic channels of the plasma membranes and to cytoskeletal proteins. In this study, we investigated new ankyrin binding proteins in rat cerebral membrane. Five main proteins in the extract of the demyelinated membranes were bound to erythrocyte ankyrin as examined by affinity chromatography. One of the proteins had a molecular weight of 97 kD that was almost identical with that of the alpha-subunit of Na⁺, K⁺-ATPase. ¹²⁵I-labeled erythrocyte ankyrin was bound to the alpha-subunit of cerebral Na⁺, K⁺-ATPase, which contains both alpha and alpha(+) subunits. The binding experiment also showed that 70% of the total erythrocyte ankyrin bound to cerebral Na⁺, K⁺-ATPase. On the other hand, erythrocyte ankyrin binds less to Na⁺, K⁺-ATPase prepared from rat brain stem axolemma which contained only alpha(+) subunit. These results suggest that erythrocyte ankyrin may bind with high affinity to a cerebral Na⁺, K⁺-ATPase isoform with alpha subunit, but with much lower affinity to axonal Na⁺, K⁺-ATPase containing alpha(+) subunit.

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