Effect of individualized occupational therapy on cognition among patients with schizophrenia: A randomized controlled trial.

This study aimed to evaluate the feasibility and efficacy of individualized occupational therapy (IOT) plus group occupational therapy (GOT) as standard care for cognition compared to GOT alone, and to determine which IOT component has the greatest effect on cognitive outcome in patients with schizophrenia. This study was conducted at 14 clinical sites across Japan and enrolled recently hospitalized patients with schizophrenia. The IOT consisted of motivational interview, self-monitoring, individualized visits, craft activities, individualized psychoeducation, and discharge planning. Among the 68 patients who were randomized to the GOT + IOT group (n = 34) and GOT alone group (n = 34), 67 completed the trial (GOT + IOT group, n = 34; GOT alone group, n = 33). There were significant improvements in change from baseline to post-treatment between the groups in verbal memory, working memory, verbal fluency, attention, executive function domains, and the composite score of the Brief Assessment of Cognition in Schizophrenia (BACS). The BACS composite score was significantly associated with the number of craft activity sessions. The addition of IOT to GOT has a favorable feasibility profile and efficacy for cognition in schizophrenia. Craft activity is the most effective IOT component in improving cognition.

Predicting drug release and degradation kinetics of long-acting microsphere formulations of tacrolimus for subcutaneous injection.

Today, tacrolimus represents a cornerstone of immunosuppressive therapy for liver and kidney transplants and remains subject of preclinical and clinical investigations, aiming at the development of long-acting depot formulations for subcutaneous injection. One major challenge arises from establishing in vitro-in vivo correlations due to the absence of meaningful in vitro methods predictive for the in vivo situation, together with a strong impact of multiple kinetic processes on the plasma concentration-time profile. In the present approach, two microsphere formulations were compared with regards to their in vitro release and degradation characteristics. A novel biorelevant medium provided the physiological ion and protein background. Release was measured using the dispersion releaser technology under accelerated conditions. A release of 100% of the drug from the carrier was achieved within 7 days. The capability of the in vitro performance assay was verified by the level A in vitro-in vivo correlation analysis. The contributions of in vitro drug release, drug degradation, diffusion rate and lymphatic transport to the absorption process were quantitatively investigated by means of a mechanistic modelling approach. The degradation rate, together with release and diffusion characteristics provides an estimate of the bioavailability and therefore can be a guide to future formulation development.

Effect of BCAA supplement timing on exercise-induced muscle soreness and damage: a pilot placebo-controlled double-blind study.

BACKGROUND: The aim of the present study was to compare the effects of branched-chain amino acid (BCAA) supplementation taken before or after exercise on delayed onset muscle soreness (DOMS) and exercise-induced muscle damage (EIMD). METHODS: Fifteen young men (aged 21.5±0.4 years) were given either BCAA (9.6 g·day-1) or placebo before and after exercise (and for 3 days prior to and following the exercise day) in three independent groups: the control group (placebo before and after exercise), the PRE group (BCAA before exercise and placebo after exercise), and the POST group (placebo before exercise and BCAA after exercise). Participants performed 30 repetitions of eccentric exercise with the non-dominant arm. DOMS, upper arm circumference (CIR), elbow range of motion (ROM), serum creatine kinase (CK), lactate dehydrogenase (LDH), and aldolase, BCAA, and ß-hydroxy-ß-methylbutyrate (3HMB) were measured immediately before and after the exercise and on the following 4 days. RESULTS: Serum BCAA and 3HMB concentrations increased significantly in the PRE group immediately after the exercise, recovering to baseline over the following days. In the days following the exercise day, DOMS, CIR, and ROM were significantly improved in the PRE group compared to the control group, with weaker effects in the POST group. Serum activities of CK, LDH, and aldolase in the days following the exercise day were significantly suppressed in the PRE group compared to control group. CONCLUSIONS: The present study confirmed that repeated BCAA supplementation before exercise had a more beneficial effect in attenuating DOMS and EIMD induced by eccentric exercise than repeated supplementation after exercise.

The relationship between the violet pigment PP-V production and intracellular ammonium level in Penicillium purpurogenum.

Penicillium purpurogenum is the fungus that produces an azaphilone pigment. However, details about the pigment biosynthesis pathway are unknown. The violet pigment PP-V is the one of the main pigments biosynthesized by this fungus. This pigment contains an amino group in a pyran ring as its core structure. We focused on this pigment and examined the relationship between intracellular ammonium concentration and pigment production using glutamine as a nitrogen source. The intracellular ammonium level decreased about 1.5-fold in conditions favoring PP-V production. Moreover, P. purpurogenum was transferred to medium in which it commonly produces the related pigment PP-O after cultivating it in the presence or absence of glutamine to investigate whether this fungus biosynthesizes PP-V using surplus ammonium in cells. Only mycelia cultured in medium containing 10 mM glutamine produced the violet pigment, and simultaneously intracellular ammonium levels decreased under this condition. From comparisons of the amount of PP-V that was secreted with quantity of surplus intracellular ammonium, it is suggested that P. purpurogenum maintains ammonium homeostasis by excreting waste ammonium as PP-V.

PP-O and PP-V, Monascus pigment homologues, production, and phylogenetic analysis in Penicillium purpurogenum.

The production of pigments as secondary metabolites by microbes is known to vary by species and by physiological conditions within a single strain. The fungus strain Penicillium purpurogenum IAM15392 has been found to produce violet pigment (PP-V) and orange pigment (PP-O),Monascus azaphilone pigment homologues, when grown under specific culture conditions. In this study, we analysed PP-V and PP-O production capability in seven strains of P. purpurogenum in addition to strain IAM15392 under specific culture conditions. The pigment production pattern of five strains cultivated in PP-V production medium was similar to that of strain IAM15392, and all violet pigments produced by these five strains were confirmed to be PP-V. Strains that did not produce pigment were also identified. In addition, two strains cultivated in PP-O production medium produced a violet pigment identified as PP-V. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region sequences from the eight P. purpurogenum strains were sequenced and used to construct a neighbor-joining phylogenetic tree. PP-O and PP-V production of P. purpurogenum was shown to be related to phylogenetic placement based on rDNA ITS sequence. Based on these results, two hypotheses for the alteration of pigment production of P. purpurogenum in evolution were proposed.

Release mechanisms of tacrolimus-loaded PLGA and PLA microspheres and immunosuppressive effects of the microspheres in a rat heart transplantation model.

The objective of this study was to elucidate the release and absorption mechanisms of tacrolimus loaded into microspheres composed of poly(lactic-co-glycolic acid) (PLGA) and/or polylactic acid (PLA). Tacrolimus-loaded microspheres were prepared by the o/w emulsion solvent evaporation method. The entrapment efficiency correlated with the molecular weight of PLGA, and the glass transition temperature of PLGA microspheres was not decreased by the addition of tacrolimus. These results indicate that intermolecular interaction between tacrolimus and the polymer would affect the entrapment of tacrolimus in the microspheres. Tacrolimus was released with weight loss of the microspheres, and the dominant release mechanism of tacrolimus was considered to be erosion of the polymer rather than diffusion of the drug. The whole-blood concentration of tacrolimus in rats was maintained for at least 2 weeks after a single subcutaneous administration of the microspheres. The pharmacokinetic profile of tacrolimus following subcutaneous administration was similar to that following intramuscular administration, suggesting that the release and dissolution of tacrolimus, rather than the absorption of the dissolved tacrolimus, were rate-limiting steps. Graft-survival time in a heart transplantation rat model was prolonged by the administration of tacrolimus-loaded microspheres. The microsphere formulation of tacrolimus would be expected to precisely control the blood concentration while maintaining the immunosuppressive effect of the drug.

Construction of transformation system in Penicillium purpurogenum.

Penicillium purpurogenum attracts attention in the food industry and biomass degradation. We expressed green fluorescent protein (GFP) with pBPE, a novel vector, and constructed a transformation system for P. purpurogenum. The accumulation of GFP was confirmed by fluorescence microscopy. In future, this system may prove useful for the genetic modification of P. purpurogenum.

Importance of the ammonia assimilation by Penicillium purpurogenum in amino derivative Monascus pigment, PP-V, production.

A fungal strain, Penicillium purpurogenum IAM 15392, produced the azaphilone Monascus pigment homolog when cultured in a medium composed of soluble starch, ammonium nitrate, yeast extract, and citrate buffer, pH 5.0. One of the typical features of violet pigment PP-V [(10Z)-12- carboxyl-monascorubramine] is that pyranoid oxygen is replaced with nitrogen. In this study, we found that glutamine synthetase (glnB) and glutamate dehydrogenase (gdh1) genes were expressed in the culture conditions conducive to PP-V production. Gln and Glu both support PP-V biosynthesis, but PP-V biosynthesis was much more efficient with Gln. We determined that synthesis of Gln by glutamine synthetase from ammonium is important for PP-V production.

Effects on vesicular transport pathways at the late endosome in cells with limited very long-chain fatty acids.

Very long-chain fatty acids (VLCFAs), fatty acids with chain-length greater than 20 carbons, possess a wide range of biological functions. However, their roles at the molecular level remain largely unknown. In the present study, we screened for multicopy suppressors that rescued temperature-sensitive growth of VLCFA-limited yeast cells, and we identified the VPS21 gene, encoding a Rab GTPase, as such a suppressor. When the vps21Δ mutation was introduced into a deletion mutant of the SUR4 gene, which encodes a VLCFA elongase, a synthetic growth defect was observed. Endosome-mediated vesicular trafficking pathways, including endocytosis and the carboxypeptidase Y (CPY) pathway, were severely impaired in sur4Δ vps21Δ double mutants, while the AP-3 pathway that bypasses the endosome was unaffected. In addition, the sur4Δ mutant also exhibited a synthetic growth defect when combined with the deletion of VPS3, which encodes a subunit of the class C core vacuole/endosome tethering (CORVET) complex that tethers transport vesicles to the late endosome/multivesicular body (MVB). These results suggest that, of all the intracellular trafficking pathways, requirement of VLCFAs is especially high in the endosomal pathways.

Development of a sandwich ELISA for the 5.9-kDa fibrinogen alpha C chain fragment detected by serum proteome analysis.

PURPOSE: We previously identified novel biomarker candidates in heavy consumers of alcohol using serum proteome analysis. Among several candidates, a 5.9 kDa peptide identified as a fragment of the fibrinogen alpha C chain (FIC5.9) was the most promising. To move FIC5.9 toward potential diagnostic use, we developed an enzyme immunoassay that enables measurement of serum FIC5.9 levels. EXPERIMENTAL DESIGN: Two monoclonal antibodies specific to the N and C-termini of the 5.9-kDa peptide were used to develop a FIC5.9 sandwich ELISA. The assay was evaluated by comparing the results with those obtained by the stable isotope-labeled dilution mass spectrometry (SID-MS) using the ClinProt™ system. RESULTS: The ELISA results correlated with the SID-MS findings (slope=0.795, intercept=-0.011, r(2) =0.908) and the performance of the ELISA was satisfactory in terms of recovery (98.5-103.0%) and within-run (1.4-4.7%) and between-day (2.8-8.4%) reproducibility. The assay was capable of detecting changes in FIC5.9 during abstinence from drinking in patients with alcohol dependency (p<0.0001). CONCLUSIONS AND CLINICAL RELEVANCE: The sandwich ELISA developed in this study will be useful for validation of the diagnostic significance of serum FIC5.9 levels in various pathological conditions, including alcoholism.

Adsorption of urinary proteins on the conventionally used urine collection tubes: possible effects on urinary proteome analysis and prevention of the adsorption by polymer coating.

One possible factor determining recovery of trace amount of protein biomarker candidates during proteome analyses could be adsorption on urine tubes. This issue, however, has not been well addressed so far. Recently, a new technical device of surface coating by poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (poly(MPC-co-BMA)) has been developed mainly to prevent the adsorption of plasma proteins. We assessed whether conventionally used urine tubes adsorb trace amount of urinary proteins and, if any, whether the surface coating by poly(MPC-co-BMA) can minimize the adsorption. Proteinuric urine samples were kept in poly(MPC-co-BMA)-coated and noncoated urine tubes for 15 min and possibly adsorbed proteins and/or peptides onto urine tubes were analyzed by SDS-PAGE, 2-DE, and the MALDI-TOF MS. It was found that a number of proteins and/or peptides adsorb on the conventionally used urine tubes and that surface coating by poly(MPC-co-BMA) can minimize the adsorption without any significant effects on routine urinalysis test results. Although it remains to be clarified to what extent the protein adsorption can modify the results of urinary proteome analyses, one has to consider this possible adsorption of urinary proteins when searching for trace amounts of protein biomarkers in urine.

Generation of a monoclonal antibody specific for tissue-nonspecific alkaline phosphatase and its use in a clinical diagnostic study.

Tissue-nonspecific alkaline phosphatase (TNSALP) in serum comprises liver alkaline phosphatase (liver-ALP) and bone alkaline phosphatase (bone-ALP). Liver-ALP is a marker of liver disease; thus a specific method for its measurement would be useful. Measurement of ALP by electrophoresis is difficult, although all of the isozymes can be assessed simultaneously. Total ALP can also be measured by automated analyzer, but it is difficult to determine the cause of a high ALP value because bone-, intestine-, placenta-, and tumor-ALP are measured together. Thus, anti-TNSALP monoclonal antibodies that can resolve these problems are needed. Here we have generated an anti-TNSALP monoclonal antibody, 3-29-3R. This clone has specificity to liver-ALP rather than to bone-ALP. In electrophoresis, 3-29-3R reacted with TNSALP and shifted the bands. The use of 3-29-3R enabled easy interpretation of the results. Furthermore, we tested 3-29-3R by developing an immunocapture enzymatic assay (IEA). Preliminary results of the IEA show that this method is effective for measurement of liver-ALP. Thus, the monoclonal antibody that we have established may be a useful tool for clinical diagnosis.

Development of an ELISA method for detecting immune complexes between tissue-nonspecific alkaline phosphatase and immunoglobulin G.

A convenient method for measuring immune complexes between tissue-nonspecific alkaline phosphatase (TNSALP) and immunoglobulin G (IgG) (i.e., TNSALP-IgG) would be highly useful for routine analyses. Here, we identified a surface-active agent that would dissolve membrane but not dissociate TNSALP-IgG complexes. Next, we developed an enzyme-linked immunosorbent assay (ELISA) method for detecting TNSALP-IgG complexes with two monoclonal antibodies (MoAbs): 3-29-3R was coated on assay plates and captured TNSALP-IgG from a specimen; an horseradish peroxidase (HRP)-conjugated anti-human IgG1 then reacted with captured TNSALP-IgG to form an "immunocomplex sandwich." The immunocomplex was detected via the absorbance of an HRP substrate, resulting in a semiquantitative assay. The mean absorbance of 0.195 (n=5) measured in sera from healthy donors was designated as an arbitrary unit (AU/mL) of TNSALP-IgG concentration. The ELISA values of patient sera known to contain TNSALP-IgG complexes were greater than those of normal sera (normal, 1.86 plusmn; 0.61; patient, 9.30 plusmn; 5.44), and these data were confirmed by electrophoresis. Thus, the ELISA could detect TNSALP-IgG complexes. The intraassay coefficient of variation (CV) was within 7.4% and analytical recovery was excellent. There was no significant interference from hemolytic, lipemic, or icteric serum. In summary, an ELISA using 3-29-3R MoAb and HRP-conjugated anti-human IgG1 constitutes a reliable and convenient method for the semiquantitative detection of TNSALP-IgG complexes in human serum.

[The mechanism of TRACP 5b maturation].

Serum tartrate resistant acid phosphatase 5b (TRACP 5b) is an isozyme of osteoclast origin. Indeed, measurement of TRACP 5b activity is used as an index of osteoclast activity. However, the precise mechanism of TRACP 5b maturation is unclear. This study aimed to clarify the mechanism of generation of TRACP 5b. We used a highly sensitive fiber-type DNA chip to investigate the mechanism of generation of TRACP 5b at the genetic level. Genes derived from three related cell types (monocytes, macrophages and osteoclasts) were compared. In addition, at the protein level, posttranscriptional modification was tested by Western blotting using an antiserum specific for the flexible loop region of TRACP 5. Our DNA chip study shows that genes implicated in oligosaccharide construction do not show significant differences in expression levels between the cell types under investigation. Strongly expressed Cathepsin K was observed in osteoclasts. Western blotting demonstrated that TRACP undergoes unique partial degradation during bone resorption, such that serum TRACP 5b lacks the flexible loop found in TRACP 5a. In conclusion, TRACP 5b generated by a specific posttranscriptional modification pathway undergoes partial digestion in resorption lacunae or inside osteoclasts. Serum TRACP 5b lacking the flexible loop differs from TRACP 5a in terms of optimum pH, isoelectric point, sugar chain and antigenicity. The measurement of TRACP 5b could therefore be of great use for monitoring of osteoporosis, rheumatoid arthritis and bone metastasis.

Developmental word grounding through a growing neural network with a humanoid robot.

This paper presents an unsupervised approach of integrating speech and visual information without using any prepared data. The approach enables a humanoid robot, Incremental Knowledge Robot 1 (IKR1), to learn word meanings. The approach is different from most existing approaches in that the robot learns online from audio-visual input, rather than from stationary data provided in advance. In addition, the robot is capable of learning incrementally, which is considered to be indispensable to lifelong learning. A noise-robust self-organized growing neural network is developed to represent the topological structure of unsupervised online data. We are also developing an active-learning mechanism, called "desire for knowledge," to let the robot select the object for which it possesses the least information for subsequent learning. Experimental results show that the approach raises the efficiency of the learning process. Based on audio and visual data, they construct a mental model for the robot, which forms a basis for constructing IKRI's inner world and builds a bridge connecting the learned concepts with current and past scenes.

Development and characterization of novel monoclonal antibodies against tartrate-resistant acid phosphatase 5.

Serum band 5 tartrate-resistant acid phosphatase (TRACP 5; EC 3.1.3.2) is a glycoprotein that exists as two very similar isoforms, TRACP 5a and TRACP 5b. The similarity of these two isoforms has made it difficult to establish monoclonal antibodies specific for either isoform. We report here the development of a monoclonal antibody with high specificity for TRACP 5b. We prepared TRACP 5b antigens from four sources: TRACP 5b purified from human bone, recombinant TRACP 5 from Escherichia coli, recombinant TRACP 5 from insect cells, and a synthetic TRACP 5b peptide. Thirty-seven mice were each immunized with 1 of the 4 different TRACP antigens to generate 473 antibody-producing clones. Three of these clones, Trk27, Trk49, and Trk62, reacted with TRACP 5b. These three clones were all established from mice exposed to native bone TRACP 5b antigen. In fact, none of the other antigens were able to generate anti-TRACP 5b monoclonal antibodies in mice. Furthermore, Trk62 interacted more strongly with TRACP 5b than with TRACP 5a. These results suggested that although recombinant proteins can be effective antigens, the native TRACP 5 protein might be more effective at generating monoclonal antibodies of greater specificity due to its more faithful representation of the native three-dimensional structure of the protein.