Molecular basis for the p phenotype. Identification of distinct and multiple mutations in the alpha 1,4-galactosyltransferase gene in Swedish and Japanese individuals

p phenotype individuals lack both P(k) (Gb3) and P (Gb4) glycolipid antigens of the P blood group system. To explore the molecular basis for this phenotype, DNA sequences of Gb3 synthase (alpha1, 4-galactosyltransferase; alpha1,4Gal-T) in six p phenotype individuals from Japan and Sweden were analyzed. A missense mutation P251L and a nonsense mutation W261stop in three and one Japanese indivuiduals, respectively, and missense mutations M183K and G187D in one each of two Swedish p individuals were found, indicating that p individuals from Japan and Sweden have distinct and multiple homozygous point mutations in the coding region. In the function analysis of the mutated alpha1,4Gal-Ts by the transfection of the expression vectors, P251L and M183K mutations showed complete loss of enzyme function, and W261stop and G187D mutations resulted in the marginal activity. BLAST analysis of homologous sequences of alpha1, 4Gal-T revealed that three residues, Met(183), Gly(187), and Pro(251), at which missense mutations were found, were highly conserved among all species examined, suggesting their importance for the function of alpha1,4Gal-T.     

Possible involvement of the p57(Kip2) gene in bone metabolism

We previously uncovered that growth stimulation of rat primary osteoblasts by transforming growth factor-beta1 (TGF-beta1) resulted in a dramatic decrease in p57(Kip2), a member of cyclin-dependent kinase (CDK) inhibitors, through the proteasomal degradation pathway (Urano et al., J. Biol. Chem. 274, 12197-12200, 1999). Here we demonstrated that the amount of p57 protein increases markedly, when rat calvarial primary osteoblasts treated with 1,25-dihydroxyvitamin D3 transit from proliferation toward differentiation. Next, we have analyzed the association of four amino acids deletion polymorphism of p57 and bone mineral density (BMD). The p57 genotype was determined in 154 postmenopausal Japanese women. When we separated the subjects into two groups, one having one or two copies of deletion polymorphism and the other without the deletion, the former subjects had higher BMD (Z score of total body, 0.67 +/- 0.93 vs 0. 23 +/- 0.90, mean +/- standard deviation; P = 0.021). Taken together, these findings suggest that the p57 regulated in the osteoblast proliferation and differentiation may play a role in determination of bone mineral density and pathogenesis of osteoporosis.     

Protein farnesylation is critical for maintaining normal cell morphology and canavanine resistance in Schizosaccharomyces pombe

Protein farnesyltransferase (FTase) plays important roles in the growth and differentiation of eukaryotic cells. In this paper, we report the identification of the Schizosaccharomyces pombe gene cpp1(+) encoding the beta-subunit of FTase. The predicted amino acid sequence of the cpp1(+) gene product shares significant similarity with FTase beta-subunits from a variety of organisms. S. pombe FTase purified from E. coli exhibits high enzymatic activity toward the CAAX farnesylation motif substrates (where C represents cysteine, A represents aliphatic amino acid, and X is preferentially methionine, cysteine, serine, alanine, or glutamine) while showing little preference for CAAL geranylgeranylation motif substrates (where L represents leucine or phenylalanine). cpp1(+) is not essential for growth as shown by gene disruption; however, mutant cells exhibit rounded or irregular cell morphology. Expression of a geranylgeranylated mutant form, Ras1-CVIL, which can bypass farnesylation, rescues these morphological defects. We also identify a novel phenotype of cpp1(-) mutants, hypersensitivity to canavanine. This appears to be due to a 3-4-fold increase in the rate of arginine uptake as compared with wild-type cells. Expression of the geranylgeranylated mutant form of a novel farnesylated small GTPase, SpRheb, is able to suppress the elevated arginine uptake rate. These results demonstrate that protein farnesylation is critical for maintaining normal cell morphology through Ras1 and canavanine resistance through SpRheb.     

Novel Hydrophobic Surface Binding Protein, HsbA, Produced by Aspergillus oryzae

Hydrophobic surface binding protein A (HsbA) is a secreted protein (14.5 kDa) isolated from the culture broth of Aspergillus oryzae RIB40 grown in a medium containing polybutylene succinate-co-adipate (PBSA) as a sole carbon source. We purified HsbA from the culture broth and determined its N-terminal amino acid sequence. We found a DNA sequence encoding a protein whose N terminus matched that of purified HsbA in the A. ozyzae genomic sequence. We cloned the hsbA genomic DNA and cDNA from A. oryzae and constructed a recombinant A. oryzae strain highly expressing hsbA. Orthologues of HsbA were present in animal pathogenic and entomopathogenic fungi. Heterologously synthesized HsbA was purified and biochemically characterized. Although the HsbA amino acid sequence suggests that HsbA may be hydrophilic, HsbA adsorbed to hydrophobic PBSA surfaces in the presence of NaCl or CaCl₂. When HsbA was adsorbed on the hydrophobic PBSA surfaces, it promoted PBSA degradation via the CutL1 polyesterase. CutL1 interacts directly with HsbA attached to the hydrophobic QCM electrode surface. These results suggest that when HsbA is adsorbed onto the PBSA surface, it recruits CutL1, and that when CutL1 is accumulated on the PBSA surface, it stimulates PBSA degradation. We previously reported that when the A. oryzae hydrophobin RolA is bound to PBSA surfaces, it too specifically recruits CutL1. Since HsbA is not a hydrophobin, A. oryzae may use several types of proteins to recruit lytic enzymes to the surface of hydrophobic solid materials and promote their degradation.     

Clinical application of an enzyme immunoassay for cholecystokinin‐like immunoreactive substance for determination of the human plasma levels: the effect of metoclopramide on gastrointestinal peptides and stress‐related hormones

Metoclopramide, a prokinetic drug, is widely used to treat vomiting and nausea. Delayed gastric emptying and continual stress are considered important factors, among others, that induce nausea and vomiting. One gastrointestinal motility regulatory factor has been assumed to be the induction of changes in the levels of peptides such as gastrin, somatostatin, motilin, and cholecystokinin (CCK) in plasma. In contrast, adrenocorticotropic hormone (ACTH) and cortisol are used as indicators of stress. Here, we studied the effects of metoclopramide on human plasma gastrin‐, somatostatin‐, motilin‐, and CCK‐like immunoreactive substances (ISs) and ACTH‐IS and cortisol under stress conditions using repetitive blood sampling in healthy subjects. Metoclopramide hydrochloride at a dose of 30 mg or placebo was orally administered to five healthy male volunteers. Blood samples were taken before and 20, 40, 60, 90, 120, 180, and 240 min after administration, subject to extracting procedures, and submitted to a highly sensitive enzyme immunoassay system. A single administration of metoclopramide caused significant increases in plasma somatostatin‐IS levels compared with the placebo. Metoclopramide significantly decreased plasma gastrin‐ and suppressed ACTH‐IS and cortisol levels compared with the placebo. We hypothesize that metoclopramide might have an accelerating gastric‐emptying effect and a modulatory effect on the hypothalamo‐pituitary‐adrenal (HPA) axis and the autonomic nervous function. These effects might be beneficial in stress‐related diseases, which suggest that this medicine has clinicopharmacological activities. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

Four-Dimensional Spatial Nanometry of Single Particles in Living Cells Using Polarized Quantum Rods

Single particle tracking is widely used to study protein movement with high spatiotemporal resolution both in vitro and in cells. Quantum dots, which are semiconductor nanoparticles, have recently been employed in single particle tracking because of their intense and stable fluorescence. Although single particles inside cells have been tracked in three spatial dimensions (X, Y, Z), measurement of the angular orientation of a molecule being tracked would significantly enhance our understanding of the molecule’s function. In this study, we synthesized highly polarized, rod-shaped quantum dots (Qrods) and developed a coating method that optimizes the Qrods for biological imaging. We describe a Qrod-based single particle tracking technique that blends optical nanometry with nanomaterial science to simultaneously measure the three-dimensional and angular movements of molecules. Using Qrods, we spatially tracked a membrane receptor in living cells in four dimensions with precision close to the single-digit range in nanometers and degrees.     

The Lutheran/Basal Cell Adhesion Molecule Promotes Tumor Cell Migration by Modulating Integrin-mediated Cell Attachment to Laminin-511 Protein

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.     

Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.