Chondrosarcoma of the urinary bladder and establishment of a human chondrosarcoma cell line (OCUU-6)

Chondrosarcoma is a very rare tumor of the urinary bladder, with only 4 cases reported to date. In this study, we report on a case of a 73-year-old male who presented bladder mass and right hydroureteronephrosis. Radical cystectomy, right nephrectomy and left ureterocutaneoustomy were performed, and histological study disclosed chondrosarcoma of the urinary bladder. As reported in other cases, the tumor was highly aggressive with a short clinical course, and the patient died of carcinomatous pleuritis at one month after surgery. Subsequently, we successfully established a human chondrosarcoma cell line (OCUU-6) from the pleural effusion of the patient.     

Utilization of Escherichia coli outer-membrane endoprotease OmpT variants as processing enzymes for production of peptides from designer fusion proteins

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp(97) is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp(97) were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1'). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg downward arrow motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.     

Protein kinase C alpha requirement in the activation of p38 mitogen-activated protein kinase, which is linked to the induction of tumor necrosis factor alpha in lipopolysaccharide-stimulated microglia

Activated microglia have been suggested to produce a cytotoxic cytokine, tumor necrosis factor alpha (TNF alpha), in many pathological brains. Thus, determining the molecular mechanism of this induction and suppression has been the focus of a great deal of research. Using lipopolysaccharide (LPS) as an experimental inducer of TNF alpha, we investigated the regulatory mechanism by which TNFalpha is induced or suppressed in microglia. We found that LPS-induced TNF alpha is suppressed by pretreatment with the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580. Similar suppression was achieved by pretreatment with specific protein kinase C (PKC) inhibitors, G?6976, myristoylated pseudosubstrate (20-28), and bisindolylmaleimide. These results suggest that PKC alpha activity as well as p38MAPK activity is associated with TNF alpha induction in LPS-stimulated microglia. The requirement of PKC alpha in LPS-dependent TNFalpha induction was verified in PKC alpha-downregulated microglia which could be induced by phorbol-12-myristate-13-acetate pretreatment. Simultaneously, PKC alpha was found to be requisite for the activation of p38MAPK in LPS-stimulated microglia. In addition, the PKC alpha levels in the LPS-stimulated microglia were observed to decrease in response to the p38MAPK inhibitor, indicating that the PKC alpha levels are regulated by the p38MAPK activity. We therefore concluded that PKC alpha and p38MAPK are interactively linked to the signaling cascade inducing TNFalpha in LPS-stimulated microglia, and that in this cascade, PKC alpha is requisite for the activation of p38MAPK, leading to the induction of TNF alpha.     

Gender differences in body-sway factors of center of foot pressure in a static upright posture and under the influence of alcohol intake

This study aimed to examine gender differences in 4 body-sway factors of the center of foot pressure (CFP) during a static upright posture and the influence of alcohol intake on them. Four body-sway factors were interpreted in previous studies using factor analysis (the principal factor method and oblique solution by promax-rotation) on 220 healthy young males and females as follows; unit time sway, front-back sway, left-right sway and high frequency band power. The CFP measurement for 1 min was carried out twice with 1 min rest. The measurements of blood pressure, heart rate, whole body reaction time, standing on one leg with eyes closed, and CFP were carried out before and after the alcohol intake using 11 healthy young males and females. The measurement device used was an Anima's stabilometer G5500. The data sampling frequency was 20 Hz. Reliability of 4 body-sway factors was very high. Significant gender differences were found in the left-right sway and the high frequency band power factors, but the influence on body-sway is, as a whole, can be disregarded. These four sway factors can determine the influence of alcohol intake as efficient as 32 sway parameters.     

Percentage of total body fat as estimated by three automatic bioelectrical impedance analyzers

The present study aimed to compare the accuracy of estimating the percentage of total body fat (%TBF) among three bioelectrical impedance analysis (BIA) devices: a single-frequency BIA with four tactile electrodes (SF-BIA4), a single-frequency BIA with eight tactile electrodes (SF-BIA8) and a multi-frequency BIA with eight tactile electrodes (MF-BIA8). Dual-energy x-ray absorptiometry (DXA) and hydrostatic weighing (HW) were used as references for the measured values. Forty-five healthy college student volunteers (21 males: 172.9 +/- 5.5 cm and 65.8 +/- 9.1 kg and 24 females: 160.7 +/- 6.6 cm, 52.6 +/- 6.2 kg) were the subjects. Correlation coefficients between the BIA measurements and the references were calculated. The standard error of estimation (SEE) was calculated by regression analysis when estimating the reference measures (DXA and HW) from the predictor (SF-BIA4, SF-BIA8 and MF-BIA8). The differences in %TBF between the reference and the predictor, calculated by the reference minus the predictor, were plotted against the %TBF measured by the references. The MF-BIA 8 here showed the highest correspondence to the reference and the least estimation error compared with the other BIA methods. It is considered that there is a limit to directly estimate FFM from a regression equation using impedance, weight, height and age as independent variables, and that %TBF can be more accurately estimated by measuring segmental impedances using eight electrodes and multi-frequency electric currents and then estimating total body water from these impedances.     

Constrained clusters of gene expression profiles with pathological features

MOTIVATION: Gene expression profiles should be useful in distinguishing variations in disease, since they reflect accurately the status of cells. The primary clustering of gene expression reveals the genotypes that are responsible for the proximity of members within each cluster, while further clustering elucidates the pathological features of the individual members of each cluster. However, since the first clustering process and the second classification step, in which the features are associated with clusters, are performed independently, the initial set of clusters may omit genes that are associated with pathologically meaningful features. Therefore, it is important to devise a way of identifying gene expression clusters that are associated with pathological features. RESULTS: We present the novel technique of 'itemset constrained clustering' (IC-Clustering), which computes the optimal cluster that maximizes the interclass variance of gene expression between groups, which are divided according to the restriction that only divisions that can be expressed using common features are allowed. This constraint automatically labels each cluster with a set of pathological features which characterize that cluster. When applied to liver cancer datasets, IC-Clustering revealed informative gene expression clusters, which could be annotated with various pathological features, such as 'tumor' and 'man', or 'except tumor' and 'normal liver function'. In contrast, the k-means method overlooked these clusters.     

Production of transgenic rats by ooplasmic injection of spermatogenic cells exposed to exogenous DNA: a preliminary study

The aim of the present study was to investigate the efficiencies of producing transgenic rats by the ooplasmic injection of sperm heads (intracytoplasmic sperm injection: ICSI) and elongating spermatids (elongating spermatid injection: ELSI) exposed to the EGFP DNA solution. A slightly lower proportion of ICSI oocytes using sperm heads exposed to a concentration of 0.5 microg/ml DNA solution for 1 min developed into offspring (13.3%, 48/361) when compared to that of oocytes injected with nontreated sperm heads (19.4%, 32/165). Eight ICSI offspring were found to be EGFP-carrying transgenic rats (16.7% per offspring; 2.2% per embryo). After a 1-min exposure of the elongating spermatids to 5 microg/ml of DNA solution, 8.8% (45/511) of the ELSI oocytes developed into offspring while 12.7% (22/173) of the ELSI oocytes using nontreated spermatids developed. Six ELSI offspring carried the EGFP DNA (13.3% per offspring; 1.2% per embryo). The conventional pronuclear microinjection of 5 microg/ml of DNA solution resulted in the higher production of offspring (29.7%, 104/350) and the birth of three transgenic rats (2.9% per offspring; 0.9% per embryo). Thus, sperm heads and elongating spermatids were practically useful as the vector of exogenous DNA if the DNA-exposed spermatogenic cells were microinseminated into rat oocytes.     

Enhancement of the endotoxin recognition pathway by ventilation with a large tidal volume in rabbits

Ventilation with a small tidal volume (V(t)) is associated with better clinical outcomes than with a large V(t), particularly in critical settings, including acute lung injury. To determine whether V(t) influences the lipopolysaccaharide (LPS) recognition pathway, we studied CD14 expression in rabbit lungs and the release of TNF-alpha by cultured alveolar macrophages after 240 min of ventilation with a large (20 ml/kg) vs. a small (5 ml/kg) V(t). We also applied small or large V(t) to lungs instilled with 50 microg/kg of LPS. The alveolar macrophages collected after large V(t) ventilation revealed a 20-fold increase in LPS-induced TNF-alpha release compared with those collected after small V(t) ventilation, whereas TNF-alpha was undetectable without LPS stimulation. In animals ventilated with a large V(t), the expression of CD14 mRNA in whole lung homogenates and the expression of CD14 protein on alveolar macrophages, assessed by immunohistochemistry, were both significantly increased in the absence of LPS stimulation. A large V(t) applied to LPS-instilled lungs increased the pulmonary albumin permeability and TNF-alpha release into the plasma. These results suggest that mechanical stress caused by a large V(t) sensitizes the lungs to endotoxin, a phenomenon that may occur partially via the upregulation of CD14.     

Comparative analysis of GFP(UV)-beta1,3-N-acetylglucosaminyltransferase 2 production in two insect-cell-based expression systems

Active beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) was produced in the baculovirus expression system (BES) and in stably transformed insect Tn-5B1-4 cells. beta3GnT2 was expressed as a secreted fusion protein with GFP(UV) with three different types of signal sequence to enhance the secretion of the fusion protein. In the stably transformed cells, the maximal beta3GnT2 activity differed between isolates, but their secretion efficiencies were similar. The difference between the maximal beta3GnT activities of the isolates studied was considered to be due to the presence of a copy number of the fusion gene, as determined on the basis of the results of Southern blot analysis. The beta3GnT activities of the culture supernatant in BES (Tn-5B1-4 cells) without or with the addition of the protease inhibitor, leupeptin, were 0.68 and 2.01 mU/ml, respectively. The stably transformed Tn-5B1-4 cells (Tn-pXme11) exhibited a beta3GnT activity of 6.83 mU/ml, which was 3.4-fold higher than that observed for BES with the leupeptin addition. The purity of fusion protein purified from the culture supernatant of the Tn-pXme11 was higher than 95% on SDS-PAGE, in contrast with that purified from the culture supernatant of the baculovirus-infected cells which contained low-molecular-weight fragments of the fusion protein. The stably transformed cell line is more suitable than BES for the efficient production of the secretory protein, beta3GnT2.