Preparation of conophylline affinity nano-beads and identification of a target protein

Conophylline, a vinca alkaloid extracted from the tropical plant Ervatamia microphylla, has been shown to induce the differentiation of insulin-producing β-cells in cultured cells and in animals. However, its mechanism of action and the molecular target have remained unclear. Therefore, we prepared a fishing probe with conophylline to identify the target protein by using latex nano-beads, which are newly innovated tools for affinity-purification. With these conophylline-linked nano-beads, we found that conophylline directly interacted with ARL6IP. ARL6IP may thus be involved in the mechanism of cellular differentiation of β-cells, and this probe should be useful to find other target proteins.     

Kinetic aspects of hemoglobin.haptoglobin-receptor interaction in rat liver plasma membranes, isolated liver cells, and liver cells in primary culture

125I-Hemoglobin.haptoglobin injected intravenously into rats was incorporated into liver parenchymal cells as evidenced by a cell separation technique. A mixture of freshly isolated liver parenchymal and nonparenchymal cells failed to internalize and degrade the 125I-hemoglobin.haptoglobin added, although it retained the ability to bind the molecule. The liver parenchymal cells in primary culture also lacked the ability to degrade 125I-hemoglobin.haptoglobin, although they bound the molecule more extensively as compared with the freshly isolated liver cells. It was confirmed that the 125I-hemoglobin.haptoglobin which was bound to the freshly isolated liver parenchymal cells localized on the outer surface of liver plasma membranes. Scatchard plots revealed the existence of two binding sites for 125I-hemoglobin-haptoglobin on the isolated liver plasma membrane: an apparent high affinity binding site (Kd = 1.3 X 10(-7) M) and an apparent low affinity binding site (Kd = 4.0 X 10(-6) M) at 37 degrees C. In contrast, freshly isolated liver parenchymal cells had only an apparent low affinity binding site (Kd = 1.4 X 10(-6) M) at 37 degrees C. Impairment of the apparent high affinity binding site during the isolation procedure with collagenase seemed to be related to loss of the ability to internalize and degrade the 125I-hemoglobin.haptoglobin molecules into the freshly isolated liver parenchymal cells or liver parenchymal cells in primary culture.     

Mapping of the human C3G gene coding a guanine nucleotide releasing protein for Ras family to 9q34.3 by fluorescence in situ hybridization

C3G, a human guanine nucleotide releasing protein for Ras protein, was mapped to human chromosome 9q34.3 by fluorescence in situ hybridization with Rbanded chromosomes. C3G was originally identified as one of the CRK-binding proteins, similar to c-ab1 (9q34.1). Our result suggests that the downstream factors of Crk are localized in close proximity on chromosome 9.     

Partial characterization of an unusual 185 kDa protein synthesized by dermal fibroblasts from patients with Marfan syndrome: identification of the protein as type IV collagen

Production of an unusual collagenous protein was observed in culture of dermal fibroblasts from four patients with Marfan syndrome. The apparent molecular weight of the protein was about 185 kDa after reduction with 2-mercaptoethanol and 175 kDa after limited pepsin treatment. The 185 kDa protein was susceptible to the bacterial collagenase but resistant to the animal collagenase. Immunoprecipitation revealed the specific interaction of the pepsin-treated 175 kDa collagenous protein with monoclonal and polyclonal antibodies to human type IV collagen. From the patterns of CNBr peptide mapping the 185 kDa band was identified as alpha 1 (IV) chain. Type IV collagen in the skin is generally considered to be of non-fibroblastic origin. However, in "diseased" condition, dermal fibroblasts might produce type IV collagen. The clinical manifestation in relation to production of type IV collagen by cultured skin fibroblasts from Marfan patients is discussed.     

Complete amino acid sequence of an immunomodulatory protein, ling zhi-8 (LZ-8). An immunomodulator from a fungus, Ganoderma lucidium, having similarity to immunoglobulin variable regions

The complete amino acid sequence of a novel immunomodulatory protein, ling zhi-8 (LZ-8), isolated from a fungus, Ganoderma lucidium (Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., Shimizu, K., and Tsunoo, H. (1989) J. Biol. Chem. 264, 472-478), was determined by protein sequencing. The polypeptide consists of 110 amino acid residues with an acetylated amino end and has a molecular mass of 12,420 Da including an amino-end blocking group. There is no attachment site for an Asn-linked oligosaccharide chain, consistent with the very low carbohydrate content of LZ-8. These results indicate that the native form of LZ-8 with a molecular mass of 24 kDa is a homodimer of the LZ-8 polypeptide whose sequence is described here. Furthermore, the LZ-8 chain shows considerable similarity to the variable region of immunoglobulin heavy chain both in its sequence and in its predicted secondary structure. The interesting possibility that LZ-8 is related to an ancestral protein of the immunoglobulin superfamily is also discussed.     

Coexpression of the CUG-binding protein reduces DM protein kinase expression in COS cells.

Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy. Patients have a large CTG repeat expansion in the 3' untranslated region of the DMPK gene, which encodes DM protein kinase. RNA trans-dominant models, which hypothesize that the expanded CUG trinucleotide repeat on DMPK mRNA sequesters a factor or disrupts the RNA metabolism of the DMPK mRNA itself and other mRNAs in a trans dominant manner, have been proposed. A candidate for the sequestered factor, termed CUG-binding protein (CUG-BP), exists in several alternatively spliced isoforms. We found a human isoform with a twelve base insertion (deduced amino acids Leu-Tyr-Leu-Gln) and an isoform with a three base insertion (deduced amino acid Ala) insertion. In order to elucidate the effects of CUG-BP on DMPK expression, we introduced CUG-BP and DMPK cDNA transiently into COS-7 cells. Cotransfection of CUG-BP did not significantly affect the expression of either wild type or mutant DMPK at the mRNA level. On the other hand, cotransfection of CUG-BP significantly affected the expression of both the wild type and mutant DMPKs at the protein level. This reduction was remarkable when the mutant DMPK construct was used.     

Passive Hemagglutination Assays for the Detection of Antibodies to Herpes Viruses

A simple and effective method for the detection of antibodies to herpes simplex virus (HSV), human cytomegalovirus (HCMV) and varicella-zoster virus (VZV), has been established using the passive hemagglutination assay (PHA) in combination with viral specific glycoproteins. The results obtained with the PHA were compared with those from neutralization (NT) and complement fixation (CF) tests. The PHA test for each of the herpes viruses appears to compare favorably with the other assays tested. The specificity and sensitivity of HSV PHA to NT were 100%, whereas the specificity and sensitivity of HSV CF test to NT were 98% and 100%, respectively. For HCMV, the specificity and sensitivity of PHA to NT and PHA to CF were 100%. Similarly, the specificity and sensitivity of VZV PHA to NT were 100%. Because of the low sensitivity of the VZV CF, the sensitivity of CF to NT was 83%. Furthermore, the range of antibody titers and their absolute levels obtained in the PHAs were significantly greater than those in the NT and CF tests.     

Immortalization of mutant p53-transfected human fibroblasts by treatment with either 4-nitroquinoline 1-oxide or x-rays

Summary The study of in vitro cell transformation is valuable for understanding the multistep carcinogenesis of human cells. The difficulty in inducing neoplastic transformation of human cells by treatment with chemical or physical agents alone is due to the difficulty in immortalizing normal human cells. Thus, the immortalization step is critical for in vitro neoplastic transformation of human cells. We transfected a mutant p53 gene (mp53: codon 273^Arg-His) into normal human fibroblasts and obtained two G418-resistant mp53-containing clones. These clones showed an extended life span but ultimately senesced. However, when they were treated with either 4-nitroquinoline 1-oxide or X rays, they were immortalized. The immortalized cells showed both numerical and structural chromosome abnormalities, but they were not tumorigenic. The expression of mutant but not wild type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in increased cdk2 and cdc2 kinase activity. However, there was no significant difference between the normal and immortalized human cells in expression of another tumor suppressor gene, p16. These findings indicate that the p53-p21 cascade may play an important role in the immortalization of human cells.     

A cyanophycin synthetase from <Emphasis Type="Italic">Thermosynechococcus elongatus</Emphasis> BP-1 catalyzes primer-independent cyanophycin synthesis

Cyanophycin synthesis is catalyzed by cyanophycin synthetase (CphA). It was believed that CphA requires l-aspartic acid (Asp), l-arginine (Arg), ATP, Mg²⁺, and a primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of a low-molecular mass cyanophycin. Despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear, and already-known CphAs were primer-dependent enzymes. In the present study, we found that recombinant CphA from Thermosynechococcus elongatus BP-1 (Tlr2170 protein) catalyzed in vitro cyanophycin synthesis in the absence of a primer. The Tlr2170 protein showed strict substrate specificity toward Asp and Arg. The optimum pH was 9.0, and Mg²⁺ or Mn²⁺ was essential for cyanophycin synthesis. KCl enhanced the cyanophycin synthesis activity of the Tlr2170 protein; in contrast, dithiothreitol did not. The Tlr2170 protein appeared to be a 400 ± 9 kDa homo-tetramer. The Tlr2170 protein showed thermal stability and retained its 80% activity after a 60-min incubation at 50°C. In addition, we examined cyanophycin synthesis at 30°C, 40°C, 50°C, and 60°C. SDS-PAGE analysis showed that the molecular mass of cyanophycin increased with increased reaction temperature.