Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells

Maintenance and differentiation of human pluripotent stem cells (hPSCs) usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth—Holm—Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp) motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs.     

Beyond simple adaptation: Incorporating other evolutionary processes and concepts into eco-evolutionary dynamics

Studies of eco-evolutionary dynamics have integrated evolution with ecological processes at multiple scales (populations, communities and ecosystems) and with multiple interspecific interactions (antagonistic, mutualistic and competitive). However, evolution has often been conceptualised as a simple process: short-term directional adaptation that increases population growth. Here we argue that diverse other evolutionary processes, well studied in population genetics and evolutionary ecology, should also be considered to explore the full spectrum of feedback between ecological and evolutionary processes. Relevant but underappreciated processes include (1) drift and mutation, (2) disruptive selection causing lineage diversification or speciation reversal and (3) evolution driven by relative fitness differences that may decrease population growth. Because eco-evolutionary dynamics have often been studied by population and community ecologists, it will be important to incorporate a variety of concepts in population genetics and evolutionary ecology to better understand and predict eco-evolutionary dynamics in nature.     

MASCOT: multiple alignment system for protein sequences based on three-way dynamic programming

A multiple alignment methodology that can produce high-qualityalignment is extremely important for predicting the structureof unknown proteins. Nearly all the methodologies developedso far have employed two-way alignment only. Although thesemethods are fast, the alignments they produce lose reliabilityas the similarity of sequences reduces. We developed the MASCOTmultiple alignment system. MASCOT can sustain the reliabilityof alignment even when the similarity of sequences is low. MASCOTachieves high-quality alignment by employing three-way alignmentin addition to two-way alignment. The resultant alignments arerefined by simulated annealing to higher quality. We also usea cluster analysis of sequences to produce highly reliable alignments.     

The molecular defect in propionic acidemia: Exon skipping caused by an 8-bp deletion from an intron in the PCCB allele

Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl CoA carboxylase (PCC) activity. To investigate the genetic basis of propionic acidemia, we isolated a cDNA encoding the precursor of the subunit of human PCC ( PCC). The cloned cDNA sequence was 1,832 bp long and the open reading frame of 1,617 nucleotides encoded a polypeptide of 539 amino acids with a molecular mass of 58,202 Da. The human PCC sequence shared a high degree of homology (91%) with the full-length cDNA of rat PCC at the amino acid level; there were only 47 differences among 539 amino acid residues compared. Polymerase chain reaction amplification and sequencing of cDNA from a subunit-deficient Japanese patient revealed a deletion of 101 nucleotides consisting of one exon from mature mRNA. This deletion resulted in a frameshift and a stop codon in the new frame. Analysis of the genomic DNA revealed a homozygous 8-bp deletion from bp3 to bp10 of the intron just downstream of the deleted exon. This deletion disrupted the consensus 5 splice signal and led to exon skipping.     

The possible involvement of protein phosphatase 1 in thrombin-induced Ca2+ influx of human platelets

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327–334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca²⁺ influx. In the presence of 1 mM Ca²⁺, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca²⁺ (EGTA 1 mM). Furthermore, thrombin-induced Mn²⁺ influx but not intracellular Ca²⁺ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca²⁺ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca²⁺ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca²⁺ channel of human platelets.     

Improvement of long-term prognosis in patients with ovarian cancers by adjuvant sizofiran immunotherapy: a prospective randomized controlled study

The effect of immunotherapy using sizofiran (SPG) on the prognosis of patients with ovarian cancers was prospectively studied in a total of 68 patients, who were randomly assigned to either a cisplatin, adriamycin and cyclophosphamide (PAC) therapy group or a PAC plus SPG combination therapy group.The survival rate was significantly higher in patients with stage Ic, II or III cancers treated with the PAC plus SPG combination, compared with the patients treated with PAC alone. In the SPG-receiving patients with stage Ic or more advanced cancers who were treated with four cycles or more of PAC, the outcome was improved (Cox-Mantel, p=0.074; generalized Kruskal-Wallis, p=0.032). Similar improvement was also observed in the patients with non-serous adenocarcinomas (Cox-Mantel, p-0.076; generalized Krukal-Wallis, p=0.045). No side effects attributable to SPG were recorded.The present results suggest that the use of SPG in combination with long-term chemotherapy improves the postoperative prognosis in ovarian cancer patients.Abbreviations SPG sizofiran     

Characterization of Two Distinct Monoclonal Antibodies to Paired Helical Filaments: Further Evidence for Fetal-Type Phosphorylation of the τ in Paired Helical Filaments

Abstract: Two monoclonal antibodies C5 and M4 raised against Sarkosyl-insoluble paired helical filaments (PHF) specifically labeled fetal τ, but hardly labeled normal adult τ. C5 immunoreactivity was eliminated by alkaline phosphatase treatment at 37°C, whereas M4 reactivity could be removed only by the treatment at 67°C. Epitope analysis showed that C5 and M4 recognition sites are in residues 386–406 and 198–250, respectively, according to the numbering of the longest human τ isoform. Thus, the phosphorylation sites are located in the amino- and carboxyl-terminal portions of the microtubule-binding region. These two well-characterized monoclonals should be valuable in the identification of a protein kinase(s) that converts normal τ into PHF-τ.     

Loss of tubulin during cold treatment of cultured carrot cells

When carrot cells, Daucus carota L. cv. Kintoki, in suspension culture were chilled on ice, more than 90% of the cortical microtubules disappeared within an hour. Colchicine-binding activity in the soluble extract decreased gradually during the prolonged cold treatment and after 4 days reached a minimum level. In SDS-polyacrylamide gel electrophoretograms of the cold-treated cell extracts, the bands corresponding to tubulin subunits disappeared, whereas most of the other protein bands remained. The level of tubulin was restored within a day after the cells were returned to 267deg;C. The relative ratio of the two major β-tubulin isoforms was reversed during the cold treatment, indicating that the cold sensitivity of tubulin differs according to its molecular species. When the cells were returned to 26°C, the ratio was restored to the original state within 24 h.     

Detection of Coxiella burnetii specific DNA in blood samples from Japanese patients with chronic nonspecific symptoms by nested polymerase chain reaction

Sprague Dawley rats, previously infected with Phase-I Bordetella pertussis , developed more severe abnormal respiratory sounds than normal animals, but not coughing, when exposed to aerosolized capsaicin, one of several cough-inducing agents tested. Stethoscope examination suggested that greater production of pulmonary mucus might be occurring after capsaicin challenge of the infected animals, compared to the uninfected controls. Rats of three other strains gave characteristically different responses from the Sprague Dawleys. The administration of capsaicin to B. pertussis -infected rats may provide useful insights into the pathophysiology of excess mucus secretion in human pertussis.     

ANALYSIS OF THE EXPRESSION OF GENES FOR P-TYPE ATPASE IN THE HALOTOLERANT ALGA DUNALIELLA SALINA (CHLOROPHYCEAE)1

A partial complementary DNA (cDNA) (DSA8) for a P-type ATPase was obtained from the halotolerant alga Dunaliella salina (Dunal) Teod. (Chlorophyceae). The cDNA exhibited greater than 90% homology to the cDNA for a H⁺-ATPase in D. bioculata Butcher. The expression of the gene that corresponded to DSA8 was decreased strongly by increases in NaCl concentration. The expression of a gene that corresponded to another ATPase (DSA1; possibly for a Ca²⁺-ATPase) from D. salina did not show the same decrease as did the DSA8. However, increased osmotic pressure due to glycerol resulted in the same decrease in the DSA8 gene. Under salt or osmotic stress, the activity of a H⁺-ATPase from microsomes of this alga also decreased. We suggest that expression of the gene for the plasma membrane H⁺-ATPase of D. salina is regulated by osmotic pressure rather than by the concentration of NaCl.