Serum cholesterol-decreasing effect of heat-moisture-treated high-amylose cornstarch in cholesterol-loaded rats

Rats were fed on a diet containing cholesterol (Chol) at a level corresponding to the standard Chol intake in humans, and the influence of heat-moisture-treated high-amylose cornstarch (HHA) on their serum Chol level was investigated. HHA decreased the serum level of Chol in rats fed on the diet containing 0.1% Chol, which corresponds to a Chol intake in humans of 800 mg/d, although the liver levels of Chol increased in these rats. HHA did not influence the fecal excretion of Chol/bile acids. It is possible that the decrease in serum Chol level in the rats fed on the high-Chol diet can be attributed to the promotion of Chol uptake in the liver.     

Crystal structures of N-myristoylated lipopeptide-bound HLA class I complexes indicate reorganization of B-pocket architecture upon ligand binding

Rhesus monkeys have evolved MHC-encoded class I allomorphs such as Mamu-B∗098 that are capable of binding N-myristoylated short lipopeptides rather than conventional long peptides; however, it remains unknown whether such antigen-binding molecules exist in other species, including humans. We herein demonstrate that human leukocyte antigen (HLA)-A∗24:02 and HLA-C∗14:02 proteins, which are known to bind conventional long peptides, also have the potential to bind N-myristoylated short lipopeptides. These HLA class I molecules shared a serine at position 9 (Ser9) with Mamu-B∗098, in contrast to most MHC class I molecules that harbor a larger amino acid residue, such as tyrosine, at this position. High resolution X-ray crystallographic analyses of lipopeptide-bound HLA-A∗24:02 and HLA-C∗14:02 complexes indicated that Ser9 was at the bottom of the B pocket with its small hydroxymethyl side chain directed away from the B-pocket cavity, thereby contributing to the formation of a deep hydrophobic cavity suitable for accommodating the long-chain fatty acid moiety of lipopeptide ligands. Upon peptide binding, however, we found the hydrogen-bond network involving Ser9 was reorganized, and the remodeled B pocket was able to capture the second amino acid residue (P2) of peptide ligands. Apart from the B pocket, virtually no marked alterations were observed for the A and F pockets upon peptide and lipopeptide binding. Thus, we concluded that the structural flexibility of the large B pocket of HLA-A∗2402 and HLA-C∗1402 primarily accounted for their previously unrecognized capacity to bind such chemically distinct ligands as conventional peptides and N-myristoylated lipopeptides.     

Release of Cell Body Agglutinins into the Medium from Gametes of Chlamydomonas reinhardtii upon Treatment with Proteases

Brief treatment of mt⁺ gametes of Chlamydomonas reinhardtiiwith trypsin or -chymotrypsin resulted in complete loss of flagellaragglutinability, without loss of cell motility, and concomitantrelease of cell body agglutinins (CBAs) into the culture medium.Release of CBAs also occurred when walled gametes without flagellawere treated with proteases. ¹Present address: Department of Biology, Washington UniversitySt. Louis, MO 63130, U.S.A.     

Enhancement of antitumor effect by peptide vaccine therapy in combination with anti-CD4 antibody: Study in a murine model

PurposeThe clinical efficacy of cancer peptide vaccine therapy is insufficient. To enhance the anti-tumor effect of peptide vaccine therapy, we combined this therapy with an anti-CD4 mAb (GK1.5), which is known to deplete CD4⁺ cells, including regulatory T cells (Tregs).MethodsTo determine the treatment schedule, the number of lymphocyte subsets in the peripheral blood of mice was traced by flow cytometry after administration of anti-CD4 mAb. The ovalbumin (OVA)_257–264 peptide vaccine was injected intradermally and anti-CD4 mAb was administered intraperitoneally into C57BL/6 mice at different schedules. We evaluated the enhancement of OVA peptide-specific cytotoxic T lymphocyte (CTL) induction in the combination therapy using the ELISPOT assay, CD107a assay, and cytokine assay. We then examined the in vivo metastasis inhibitory effect by OVA peptide vaccine therapy in combination with anti-CD4 mAb against OVA-expressing thymoma (EG7) in a murine liver metastatic model.ResultsWe showed that peptide-specific CTL induction was enhanced by the peptide vaccine in combination with anti-CD4 mAb and that the optimized treatment schedule had the strongest induction effect of peptide-specific CTLs using an IFN-γ ELISPOT assay. We also confirmed that the CD107a⁺ cells secreted perforin and granzyme B and the amount of IL-2 and TNF produced by these CTLs increased when the peptide vaccine was combined with anti-CD4 mAb. Furthermore, metastasis was inhibited by peptide vaccines in combination with anti-CD4 mAb compared to peptide vaccine alone in a murine liver metastatic model.ConclusionThe use of anti-CD4 mAb in combination with the OVA peptide vaccine therapy increased the number of peptide-specific CTLs and showed a higher therapeutic effect against OVA-expressing tumors. The combination with anti-CD4 mAb may provide a new cancer vaccine strategy.     

TheChlamydomonas plus and minus agglutinin: difference in sensitivity to dithiothreitol

The effect of disulfide-reducing agent dithiothreitol (DTT) on the plus and minus agglutinins ofChlamydomonas reinhardtii gametes was studied. Live gametes of mating-type plus (mt ⁺) lost their flagellar agglutinability by DTT treatment without any loss of cell motility and concurrently released into the medium agglutinin in an inactive form. DTT treated cells also lost completely their cell body-agglutinin. By contrast, the mating-type minus (mt ^-) gametes neither lost their agglutinability nor released agglutinin into the medium by DTT even at very high concentrations. In vitro experiments showed that plus agglutinin in solution is just as sensitive as that in vivo to DTT, whereas minus agglutinin is totally insensitive, and the sulfhydryl-oxidizing agent diamide restores the plus agglutinin activity immediately and completely. Isolated flagella from themt ⁺ gametes were also inactivated by DTT, but they retained the inactivated agglutinin on the surfaces. The results indicate that plus agglutinin, but not minus agglutinin, possesses disulfide bonds which are essential for the recognition/adhesion activity.Abbreviations mt ^+/- mating-type plus or minus - DTT dithiothreitol     

Control of Gametic Differentiation and Activity by Light in Chlamydomonas reinhardtii

Laboratory strains of Chlamydomonas reinhardtii, which are descendantsof a 1945 isolate by G.M. Smith (Harris 1989), were dividedinto two groups depending upon whether the vegetative cellsrequire light to differentiate into gametes under ammonium ion-starvedconditions. Light-dependent (LD) strains were unable to becomegametes in the dark, while light-independent (LI) strains coulddo so. All the wild-type strains isolated recently from thefield showed light-dependency, suggesting that the LD-phenotypeis the wild-type. The LD-cells failed to acquire flagellar agglutinability,to accumulate cell body agglutinins, or to form mating structuresin the dark, but did so rapidly after transfer to light. Moreover,the light-induced LD-gametes, but not the Li-gametes, lost theirmating ability, cell body agglutinins, and mating structuresafter transfer to darkness, indicating that the LD-cells requirelight not only for gametic differentiation but also for maintenanceof gametic activity. (Received July 4, 1997; Accepted October 17, 1997)     

Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions

We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/microm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of mutants was analyzed using the multiplex polymerase chain reaction (PCR). The dose-response curves increased steeply up to 0.5 Gy and leveled off or decreased between 0.5 and 1.0 Gy, compared to the response to (137)Cs gamma-rays. The mutation frequency increased up to 105 keV/microm and then there was a downward trend with increasing LET values. The deletion pattern of exons was non-specific. About 75-100% of the mutants produced using LETs ranging from 63 to 335 keV/mum showed all or partial deletions of exons, while among gamma-ray-induced mutants 30% showed no deletions, 30% partial deletions and 40% complete deletions. These results suggested that the dose-response curves of neon-ion-induced mutations were dependent upon LET values, but the deletion pattern of DNA was not.     

Adiponectin promotes migration activities of endothelial progenitor cells via Cdc42/Rac1

Adiponectin has anti-atherosclerotic effects through its direct actions on vascular cells. The present study investigates the molecular mechanisms of adiponectin in the migration of endothelial progenitor cells (EPCs) which play an important role in neovascularization and re-endothelization. The phosphorylation of Akt and the activations of Cdc42 and Rac1 were significantly increased by adiponectin. Adiponectin increased the migration activity of EPCs, which was completely inhibited by a PI3-kinase inhibitor. siRNA of Cdc42 or Rac1 completely inhibited the adiponectin-induced migration, but siRNA of Akt had no effects, indicating that adiponectin promotes the migration activities of EPCs mainly through PI3-kinase/Cdc42/Rac1.

Structured summary

MINT-7217629: PAK1 (uniprotkb:Q13153) physically interacts (MI:0914) with CDC42 (uniprotkb:P60953) by pull down (MI:0096)MINT-7217644: PAK1 (uniprotkb:Q13153) physically interacts (MI:0914) with Rac1 (uniprotkb:P63000) by pull down (MI:0096)     

Comparative Genetics of the Poly-Q Tract of Ataxin-1 and Its Binding Protein PQBP-1

Human PQBP-1 is known to interact with triplet repeat disease gene products such as ataxin and huntingtin through their poly-glutamine (poly-Q) tracts. The poly-Q tracts show extensive variation in both the number and the configuration of repeats among species. A surface plasmon resonance assay showed clear interaction between human PQBP-1 and Q(11), representative of the poly-Q tract of the ataxin-1 of Old World monkeys. No response was observed using Q(2)PQ(2)P(4)Q(2), representative of the poly-Q tract of the ataxin-1 of New World monkeys. This implies that the interaction of human PQBP-1 with ataxin-1 is limited to humans and closely related species. Comparison of the human and mouse PQBP-1 sequences showed an elevated amino acid substitution rate in the polar amino acid-rich domain of PQBP-1 that is responsible for binding to poly-Q tracts. This could have been advantageous to the new biological function of human PQBP-1 through poly-Q tracts.