Overexpression,purification, and functional analysis of recombinant human tubulin dimer

Microtubules consisting of tubulin dimers play essential roles in various cellular functions. Investigating the structure–function relationship of tubulin dimers requires a method to prepare sufficient quantities of recombinant tubulin. To this end, we simultaneously expressed human α1- and β3-tubulin using a baculovirus-insect cell expression system that enabled the purification of 5 mg recombinant tubulin per litre of cell culture. The purified recombinant human tubulin could be polymerized into microtubules that glide on a kinesin-coated glass surface. The method provides a powerful tool for in vitro functional analyses of microtubules.     

Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.     

Targeted recombination with single-stranded DNA vectors in mammalian cells.

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.     

The Human Prohibitin (PHB) Gene Family and Its Somatic Mutations in Human Tumors

Five cosmid clones, isolated by procedures to screen genomic libraries for homologous variants of the human prohibitin gene (PHB), were analyzed to determine their genomic structures. Four of these (PHBP1-4) were found to be processed pseudogenes, each located on a different chromosome from their counterparts on chromosome 17q21. The DNA sequence of one clone (PHBP1, on chromosome 6q25) shared a 91.3% identity at the nucleotide level with the cDNA of functional prohibitin. A large number of human tumors of the breast, ovary, liver, and lung were examined for somatic mutations in the PHB gene. Although mutations were observed in a few sporadic breast cancers, none were identified in any of the other cancers.     

Cloning of the cDNA encoding neural adhesion molecule F3 from bovine brain

We cloned a bovine cDNA encoding the neural adhesion molecule F3 and analyzed its nucleotide sequence. The coding region consisted of 3054 bp encoding 1018 amino acid (aa) residues. The M_r calculated from the deduced as sequence was 113 383. Bovine F3 had 93, 94 and 77% as identity with the mouse, human and chicken homologs, respectively. Bovine F3, similar to those of chicken and human, was devoid of two as residues (Ile-Thr) in the sixth immunoglobulin type C2-like domain, as compared with the mouse homolog. Parts of bovine F3 protein were overproduced in Escherichia coli. The antibodies raised against the recombinant proteins in rabbits reacted specifically with F3. F3 protein was detected in cerebellum, cerebrum and spinal cord in Western blot analysis.     

Murine monoclonal antibody recognizing human α(1,3/1,4)fucosyltransferase

We prepared a mouse monoclonal antibody, FTA1-16, that specifically recognizes human (1,3/1,4)fucosyltransferase without crossreactivity to any other members of the (1,3)fucosyltransferase family. The specificity was confirmed by both immunofluorescense staining of native antigens in the Golgi apparatus and Western blotting analysis, using stable transformant cells transfected with each gene of the (1,3)fucosyltransferase family. Western blotting analysis on a series of human tumour cell lines from various tissues revealed that some epithelial cancer cell lines from digestive organs expressed an amount of (1,3/1,4)fucosyltransferase in good correlation with expression of sialyl Lewis a antigen. Immunohistochemical staining by FTA1-16 on colon cancer tissues revealed enhanced expression of the enzyme in cancer cells in comparison to normal cells. Finally, the antigenic epitope recognized by FTA1-16 was determined using truncated recombinant peptides which were expressed inE. coli. A minimal length determined was a fragment, amino acid positions 132–153, of the (1,3/1,4)fucosyltransferase.     

Stimulatory effect of hormonal signaling factors on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes: Cross talk with regucalcin

The effect of hormonal signaling factors on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of inositol-glycan (10^–7–10^–5M), dibutyryl cAMP (10^–4 and 10^–3M) or inositol 1,4,5-trisphosphate (IP₃; 10^–6 and 10^–5 M) in the enzyme reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity. These effects were completely inhibited by the presence of vanadate (10^–4 M), an inhibitor of the enzyme phosphorylation, and N-ethylmaleimide (5×10^–3 M), a SH group modifying reagent. Meanwhile, regucalcin, a Ca²⁺-binding protein isolated from rat liver cytosol, increased the enzyme activity by binding to the SH groups of (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes. The presence of regucalcin (0.25 M) with an effective concentration completely inhibited the effect of inositol-glycan (10^–5 M) to increase (Ca²⁺–Mg²⁺)-ATPase activity, while the effect of dibutyryl cAMP (10^–3M) or IP₃ (10^–5M) was not altered. The inositol-glycan effect was not modulated by the presence of dibutyryl cAMP or IP₃. Now, the preincubation of the plasma membranes with regucalcin did not modify the effect of inositol-glycan on the enzyme activity, suggesting that regucalcin competes with inositol-glycan for the binding to the plasma membranes. The present results suggest that there may be a cross talk with regucalcin and hormonal signaling factors in the regulation of (Ca²⁺–Mg²⁺)-ATPase activity in liver plasma membranes.     

Novel Light-Dark Change of Proline Levels in Halophyte (Mesembryanthemum crystallinum L.) and Glycophytes (Hordeum vulgare L. and Triticum aestivum L.) Leaves and Roots under Salt Stress

Proline accumulation was determined in a facultative halophyte,Mesembryanthemum crystallinum and glycophytes, barley (Hordeumvulgare L.) and wheat (Triticum aestivum L.) Proline accumulationpreceded the shift of CAM in M. crystallinum and did not occurin the continuous darkness. The novel light-dark change of prolinelevel (high in the light and low in the dark) was observed inleaves of all three plants. Proline levels of shoots in barleyand wheat also showed the same light-dark change, suggestingthat proline accumulated in the leaves in the light was nottranslocated to other tissues in the dark period. These resultssuggest that proline has a bifunctional role in the acclimationto high salt stress; an osmoregulant role in the light, anda substrate for dark respiration to supply energy to compartmentationof ions into vacuole in the dark. ¹Present address: Kyoto Biological Res. Lab., Bio-Chiba Inc.Watsuka,Soraku, Kyoto, 619-12 Japan ²Present address: Kobayashi Pharmaceutical Co., Ltd. Doshomachi,Chuo-ku, Osaka, 541 Japan