Calpastatin domain L is a partial agonist of the calmodulin-binding site for channel activation in Cav1.2 Ca2+ channels

Cav1.2 Ca(2+) channel activity diminishes in inside-out patches (run-down). Previously, we have found that with ATP, calpastatin domain L (CSL) and calmodulin (CaM) recover channel activity from the run-down in guinea pig cardiac myocytes. Because the potency of the CSL repriming effect was smaller than that of CaM, we hypothesized that CSL might act as a partial agonist of CaM in the channel-repriming effect. To examine this hypothesis, we investigated the effect of the competitions between CSL and CaM on channel activity and on binding in the channel. We found that CSL suppressed the channel-activating effect of CaM in a reversible and concentration-dependent manner. The channel-inactivating effect of CaM seen at high concentrations of CaM, however, did not seem to be affected by CSL. In the GST pull-down assay, CSL suppressed binding of CaM to GST fusion peptides derived from C-terminal regions in a competitive manner. The inhibition of CaM binding by CSL was observed with the IQ peptide but not the PreIQ peptide, which is the CaM-binding domain in the C terminus. The results are consistent with the hypothesis that CSL competes with CaM as a partial agonist for the site in the IQ domain in the C-terminal region of the Cav1.2 channel, which may be involved in activation of the channel.     

Discovery of potent CCR4 antagonists: Synthesis and structure-activity relationship study of 2,4-diaminoquinazolines

A new series of quinazolines that function as CCR4 antagonists were discovered during the screening of our corporate compound libraries. Subsequent compound optimization elucidated the structure-activity relationships and led the identification of 2-(1,4'-bipiperidine-1'-yl)-N-cycloheptyl-6,7-dimethoxyquinazolin-4-amine 14a, which showed potent inhibition in the [(35)S]GTPgammaS-binding assay (IC(50)=18nM). This compound also inhibited the chemotaxis of human and mouse CCR4-expressing cells (IC(50)=140nM, 39nM).     

Maximal lactate steady-state prediction through quadratic modeling of selected stages of the lactate minimum test

In this study, we compared the maximal lactate steady state (MLSS) with lactate minimal (LM) intensities determined visually and through a quadratic polynomial function of selected stages of LM test. Eleven male recreational cyclists (27.7 +/- 4.5 years, 175.7 +/- 5.6 cm, 69.5 +/- 10.8 kg, and 12.0 +/- 5.5% body fat) performed one LM test under previous induction of hyperlactaemia with an initial intensity of 75 W with 30-W increments every 3 minutes with blood lactate concentration (HLa) and rating of perceived exertion (RPE) measurements. The LM intensity was determined visually (VLM) and by modeling the lactate response through polynomial function by using: 1) all stages (LMP); 2) the first stage, the stage corresponding to RPE-13 and the last stage/exhaustion (LMP3max); 3) the three lowest lactate concentration stages (LMP3adj); and 4) the initial, RPE-13, and RPE-16 stages (LMP3sub). The MLSS was determined as the highest intensity at a variation not greater than 0.05 mmol.l.min of HLa during the last 20 minutes of a 30-minute exercise session. The MLSS (204.0 +/- 16.0 W), VLM (198.6 +/- 15.2 W), LMP3adj (190.4 +/- 12.9 W), and LMP3sub (192.1 +/- 27.2 W) were not different, well correlated, and in agreement to each other. In conclusion, the polynomial modeling of HLa response to three submaximal stages produced exercise intensities that did not differ from MLSS.     

Trial of integrated laboratory practice

In most laboratory practices for students in medical schools, a laboratory guidebook is given to the students, in which the procedures are precisely described. The students merely follow the guidebook without thinking deeply, which spoils the students and does not entice them to think creatively. Problem-based learning (PBL) could be one means for the students themselves to actively learn, find problems, and resolve them. Such a learning attitude nurtures medical students with lifelong learning as healthcare professionals. We merged PBL and laboratory practices to promote deep thinking habits and developed an integrated laboratory practice. We gave a case sheet to groups of students from several schools. The students raised hypotheses after vivid discussion, designed experimental protocols, and performed the experiments. If the results did not support or disproved the hypothesis, the students set up another hypothesis followed by experiments, lasting for 4 or 5 consecutive days. These procedures are quite similar to those of professional researchers. The main impact achieved was the fact that the students developed the experimental design by themselves, for the first time in their college lives. All students enjoyed the laboratory practice, which they had never experienced before. This is an antidote to the guidebook-navigated traditional laboratory practice, which disappoints many students. As educators in basic medical sciences stand on the edge in terms of educating the next generation, there is a need to provide a strong foundation for medical students to design and perform scientific experiments. The integrated laboratory practice may provide the solution.     

Characteristics of hepatitis B virus isolates of genotype G and their phylogenetic differences from the other six genotypes (A through F)

Eight hepatitis B virus (HBV) isolates of genotype G were recovered from patients and sequenced over the entire genome. Six of them had a genomic length of 3,248 bp and two had genomic lengths of 3,239 bp (USG15) and 3,113 bp (USG18) due to deletions. The 10 HBV/G isolates, including the 8 sequenced isolates as well as the original isolate (AF160501) and another isolate (B1-89), had a close sequence homology of 99.3 to 99.8% among themselves (excluding USG18 with a long deletion) but of <88.7% to any of the 68 HBV isolates of the other six genotypes with the full-length sequence known. The eight HBV/G isolates possessed an insertion of 36 bp in the core gene and two stop codons in the precore region, as did the AF160501 and B1-89 isolates. The 10 HBV/G isolates clustered on a branch separate from those bearing the other six genotypes (A through F [A-F]) in the phylogenetic tree constructed from full-length sequences of 78 HBV isolates as well as in those constructed from the core, polymerase, X, and envelope genes. Despite two stop codons in the precore region that prohibited the translation of the HBV e antigen (HBeAg), all of the eight patients with HBV/G infection possessed the HBeAg in serum. By restriction fragment length polymorphism of the surface gene, all of the eight patients were found to be coinfected with HBV of genotype A (HBV/A), which would be responsible for the expression of HBeAg in them. It is worthy of examination to determine how coinfection occurs and whether HBV/G needs HBV/A for replication.     

Fungal melanin inhibitor and related compounds from Penicillium decumbens

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.     

A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels

Calpastatin, an endogenous inhibitor of calpain, is composed of domain L and four repetitive homologous domains 1-4. Domains 1-4 inhibit calpain, whereas domain L partially reprimes L-type Ca2+ channels for voltage-gated activation. In the present study, the effects on Ca2+ channel activity of four isoforms and a series of fragments of calpastatin domain L were investigated in guinea-pig ventricular myocytes with the patch-clamp method. With one exception, all the isoforms and fragment peptides that contained amino acid residues 54-64 of domain L reprimed the Ca2+ channels to comparable levels (9-15% of control activity) to those observed previously with a full-length form of calpastatin. These results suggest that the region containing amino acid residues 54-64 (EGKPKEHTEPK) is responsible for the Ca2+ channel repriming function of calpastatin domain L.     

Pepstatin A, an aspartic proteinase inhibitor, suppresses RANKL-induced osteoclast differentiation

Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. Except for its role as a proteinase inhibitor, however, the pharmacological action of pepstatin A upon cells remain unclear. In this study, we found that pepstatin A suppressed receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Pepstatin A suppressed the formation of multinuclear osteoclasts dose-dependently. This inhibition of the formation only affected osteoclast cells, i.e., not osteoblast-like cells. Furthermore, pepstatin A also suppressed differentiation from pre-osteoclast cells to mononuclear osteoclast cells dose-dependently. This inhibition seems to be independent of the activities of proteinases such as cathepsin D, because the formation of osteoclasts was not suppressed with the concentration that inhibited the activity of cathepsin D. Cell signaling analysis indicated that the phosphorylation of ERK was inhibited in pepstatin A-treated cells, while the phosphorylation of IkappaB and Akt showed almost no change. Furthermore, pepstatin A decreased the expression of nuclear factor of activated T cells c1 (NFATc1). These results suggest that pepstatin A suppresses the differentiation of osteoclasts through the blockade of ERK signaling and the inhibition of NFATc1 expression.     

Receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4

Perturbations to the well-being of tissues in plants and invertebrates generate fragments of endogenous molecules that are recognized by innate immune receptors. Vertebrates have homologous receptors on specialized cells such as dendritic cells, but whether these receptors respond to fragments of endogenous molecules is not known. We tested the idea that Toll-like receptors on dendritic cells might recognize polysaccharide fragments of heparan sulfate proteoglycan. Dendritic cells were found to mature in response to heparan sulfate as measured by costimulatory protein expression, morphology, and T lymphocyte stimulation, but this maturation was absent when Toll-like receptor 4 was mutated or inhibited. These findings suggest that Toll-like receptors in vertebrates may monitor tissue well-being by recognizing fragments of endogenous macromolecules.