Comparison of survival and perioperative outcome of the colonic stent and the transanal decompression tube placement and emergency surgery for left-sided obstructive colorectal cancer: a retrospective multi-center observational study "The CODOMO study".

PURPOSE: Advances in endoscopic technology have led to the reevaluation of self-expandable metallic stent (SEMS) placement as a bridge-to-surgery (BTS) in patients with obstructive colorectal cancer. In Japan, after inclusion of SEMS placement as a BTS in the medical insurance coverage in 2012, this procedure has been increasingly performed. However, a transanal decompression tube (TADT) placement has been used as a BTS. We aimed to retrospectively evaluate the optimal strategy for obstructive left-sided colorectal cancer (OLCRC) by comparing SEMS and TADT placement with emergency surgery. METHODS: We included 301 patients with stage II and III OLCRC from 27 institutions. The study patients were divided into Surgery group (emergency surgery, n = 103), SEMS group (BTS by SEMS, n = 113), and TADT group (BTS by TADT, n = 85). We compared the survival and perioperative outcomes of patients in the Surgery group as a standard treatment with those in the SEMS and TADT groups. RESULTS: The 3-year relapse-free survival rate in patients in the Surgery group was 74.8%, while that in patients in the SEMS group and TADT group were 69.0% (p = 0.39) and 55.3% (p = 0.006), respectively. The technical success rate was not statistically different, but the clinical success rate was significantly higher in the SEMS group than in the TADT group (p = 0.0040). With regard to postoperative complications after curative surgery, the SEMS group had significantly lower of complications (≥ grade 2) than the Surgery group (p = 0.022). CONCLUSION: Patients who underwent SEMS placement for OLCRC had similar oncological outcomes to patients who underwent emergency surgery.

Oxygen ultra-fine bubbles water administration prevents bone loss of glucocorticoid-induced osteoporosis in mice by suppressing osteoclast differentiation.

Oxygen ultra-fine bubbles (OUB) saline injection prevents bone loss of glucocorti\coid-induced osteoporosis in mice, and OUB inhibit osteoclastogenesis via RANK-TRAF6-c-Fos-NFATc1 signaling and RANK-p38 MAPK signaling in vitro. INTRODUCTION: Ultra-fine bubbles (<200 nm in diameter) have several unique properties, and they are tested in various medical fields. The purpose of this study was to investigate the effects of oxygen ultra-fine bubbles (OUB) on glucocorticoid-induced osteoporosis (GIO) model mice. METHODS: Prednisolone (PSL, 5 mg) was subcutaneously inserted in 6-month-old male C57BL/6J mice, and 200 µl of saline, OUB-diluted saline, or nitrogen ultra-fine bubbles (NUB)-diluted saline was intraperitoneally injected three times per week for 8 weeks the day after operations. Mice were divided into four groups; (1) control, sham-operation + saline; (2) GIO, PSL + saline; (3) GIO + OUB, PSL + OUB saline; (4) GIO + NUB, PSL + NUB saline. The effects of OUB on osteoblasts and osteoclasts were examined by serially diluted OUB medium in vitro. RESULTS: Bone mass was significantly decreased in GIO [bone volume/total volume (%): control vs. GIO 12.6 vs. 7.9; p < 0.01] while significantly preserved in GIO + OUB (GIO vs. GIO + OUB 7.9 vs. 12.9; p < 0.05). In addition, tartrate-resistant acid phosphatase (TRAP)-positive cells in the distal femur [mean osteoclasts number/bone surface (mm-1)] was significantly increased in GIO (control vs. GIO 6.8 vs. 11.6; p < 0.01) while suppressed in GIO + OUB (GIO vs. GIO + OUB 11.6 vs. 7.5; p < 0.01). NUB did not affect these parameters. In vitro experiments revealed that OUB significantly inhibited osteoclastogenesis by inhibiting RANK-TRAF6-c-Fos-NFATc1 signaling, RANK-p38 MAPK signaling, and TRAP/Cathepsin K/DC-STAMP mRNA expression in a concentration-dependent manner. OUB did not affect osteoblastogenesis in vitro. CONCLUSIONS: OUB prevent bone loss in GIO mice by inhibiting osteoclastogenesis.

Synovial mesenchymal stem cells from osteo- or rheumatoid arthritis joints exhibit good potential for cartilage repair using a scaffold-free tissue engineering approach.

OBJECTIVE: To assess whether synovial mesenchymal stem cells (SMSCs) from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) can be used as an alternative cell source for cartilage repair using allogenic tissue engineered construct (TEC). METHODS: Twenty-five patients (17 female, average age 61.8 years) were divided according to their pathology (control trauma group; N = 6, OA group; N = 6) and RA patients were subdivided into two groups to evaluate the impact of biologics in accordance with whether treated with biologics [Bio(+)RA; N = 7] or not [Bio(-)RA; N = 6]. We compared the following characteristics among these groups: (1) The cell proliferation capacity of SMSCs; (2) The influence of passage number on features of SMSCs; (3) The weight and volume of TEC from the same number of SMSCs; (4) Inflammatory cytokine gene expressions levels of TEC; (5) The chondrogenic potential of TEC; and (6) Osteochondral repair using TEC in athymic nude rats. RESULTS: SMSCs from the four groups exhibited equivalent features in the above evaluation items. In in vivo studies, the TEC-treated repair tissues for all groups exhibited significantly better outcomes than those for the untreated group and no significant differences among the four TEC groups. CONCLUSION: SMSCs from OA or RA patients are no less appropriate for repairing cartilage than those from trauma patients and thus, may be an effective source for allogenic cell-based cartilage repair.

Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

Sphingosine kinase 1 expression is downregulated during differentiation of Friend cells due to decreased c-MYB.

Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between -53bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.

Mesp2 initiates somite segmentation through the Notch signalling pathway.

The Notch-signalling pathway is important in establishing metameric pattern during somitogenesis. In mice, the lack of either of two molecules involved in the Notch-signalling pathway, Mesp2 or presenilin-1 (Ps1), results in contrasting phenotypes: caudalized versus rostralized vertebra. Here we adopt a genetic approach to analyse the molecular mechanism underlying the establishment of rostro-caudal polarity in somites. By focusing on the fact that expression of a Notch ligand, Dll1, is important for prefiguring somite identity, we found that Mesp2 initiates establishment of rostro-caudal polarity by controlling two Notch-signalling pathways. Initially, Mesp2 activates a Ps1-independent Notch-signalling cascade to suppress Dll1 expression and specify the rostral half of the somite. Ps1-mediated Notch-signalling is required to induce Dll1 expression in the caudal half of the somite. Therefore, Mesp2- and Ps1-dependent activation of Notch-signalling pathways might differentially regulate Dll1 expression, resulting in the establishment of the rostro-caudal polarity of somites.

Giant Rashba-type spin splitting in bulk BiTeI.

There has been increasing interest in phenomena emerging from relativistic electrons in a solid, which have a potential impact on spintronics and magnetoelectrics. One example is the Rashba effect, which lifts the electron-spin degeneracy as a consequence of spin-orbit interaction under broken inversion symmetry. A high-energy-scale Rashba spin splitting is highly desirable for enhancing the coupling between electron spins and electricity relevant for spintronic functions. Here we describe the finding of a huge spin-orbit interaction effect in a polar semiconductor composed of heavy elements, BiTeI, where the bulk carriers are ruled by large Rashba-like spin splitting. The band splitting and its spin polarization obtained by spin- and angle-resolved photoemission spectroscopy are well in accord with relativistic first-principles calculations, confirming that the spin splitting is indeed derived from bulk atomic configurations. Together with the feasibility of carrier-doping control, the giant-Rashba semiconductor BiTeI possesses excellent potential for application to various spin-dependent electronic functions.

Is lung resection appropriate for late octogenarians? Surgical outcomes of patients aged ≥ 80 years with lung cancer.

PURPOSE: This study aimed to determine the outcomes and prognostic factors associated with octogenarians who underwent pulmonary resection for lung cancer. METHODS/PATIENTS: From 2009 to 2018, 76 octogenarians underwent pulmonary surgery for lung cancer at the Kanazawa Medical University, Japan. They were divided into two groups (early and late octogenarians), and their clinicopathological characteristics and outcomes were investigated. Overall survival rates and recurrence-free survival rates were determined using Kaplan-Meier curves. Univariate and multivariate analyses were performed to identify prognostic factors. RESULTS: Limited surgery was performed more often in the late octogenarian group; however, most perioperative factors were not significantly different between the two groups. The 3-year overall survival and recurrence-free survival rates were 61.2% and 52.8%, respectively. The median observation period was 37.5 (8.9-112.3) months postoperatively. Kaplan-Meier curves showed that age ≥ 85 years (late octogenarian), smoking history, and squamous cell carcinoma on histology were associated with worse survival rates. Multivariate analysis identified age ≥ 85 years (late octogenarian) (p = 0.011) and cigarette smoking (p = 0.025) as unfavorable prognostic factors for overall survival and recurrence-free survival, respectively. CONCLUSIONS: Most octogenarians with an indication for surgery can tolerate pulmonary surgery. However, owing to the limitations of this retrospective, single-center study, future studies involving multiple-institutions are required to confirm our findings.

The critical velocity and 1500-m surface performances in Finswimming.

The purpose of this investigation was to determine whether the concepts of critical velocity (CV) and anaerobic swimming capacity (ASC) could be used by coaches as a reliable index in order to monitor 1500-m Surface (SF) performances in Finswimming. Thirteen Finswimmers (6 males and 7 females, 24+/-6 years), members of the Japanese national team, were instructed to swim three different swimming distances (400-, 800-, and 1500-m) at maximal effort in a 50m long course swimming pool. CV and the ASC were calculated using 400-m and 800-m swim times. Mean height and body mass were 170.2 cm and 69.7 kg in male and 160.5 and 61.0 kg in female. A highly positive correlation was found between the CV and the mean velocity of 1500-m SF (V1500) (r=0.91, P<0.01), but no correlation was found between the ASC and V1500. (r=0.46, P=0.11). However, a high correlation was found between the ASC and the residual error of V1500, calculated from the relationship between V1500 and the CV (r=0.89, P<0.01). These results suggest that the CV is a useful method for evaluating 1500-m SF performance and an aerobic performance expressed as the CV contributes to 1500-m SF performance.

Lipid analysis of isolated plasma membranes of lymphoid cells in benign thymomas of Buffalo/Mna rats.

Plasma membranes were isolated from lymphoid cells of benign thymomas obtained from inbred BUF/Mna rats (21 mo old) and from normal thymocytes obtained from young rats (7 wk old) of the same strain. The isolated plasma membranes were electron microscopically pure, and the specific activities of Na+, K+-ATPase, and 5'-nucleotidase were enhanced. The lipid compositions of the plasma membranes from these two sources were analyzed and compared. The cholesterol and plasmalogen contents of membranes from both sources were similar, but the phospholipid content of the benign thymoma lymphoid cell membranes was slightly lower than that of the normal thymocytes, resulting in a somewhat higher molar ratio of cholesterol to phospholipid. The plasma membranes of the thymoma lymphoid cells also exhibited a slightly higher microviscosity as measured with fluorescence polarization. No significant differences were observed in the phospholipid compositions of the two membrane preparations.

Morphological reversion of sis-transformed NIH3T3 cells by trichostatin A.

Trichostatin A (TSA) induced the normal and flat phenotype of sis-transformed NIH3T3 cells at quite a low concentration of 1 ng/ml. Although morphological changes were found in other oncogene-transformed cells, they were not the same as those seen for the sis-transformed cells. Almost complete reversion into the flat phenotype was seen at 6 h after administration of the compound, suggesting that the morphological change was caused not merely by selection of TSA-resistant cells of the flat phenotype. The effect of TSA was reversible when the cell culture was incubated after its removal. Synthesis of sis-mRNA did not decrease with the treatment of TSA at a concentration sufficient to reverse the transformed morphology. Cycloheximide abolished the activity of TSA, showing that TSA required new protein synthesis to express its activity.

Comparative study of the phospholipid composition of plasma membranes isolated from rat primary hepatomas induced by 3'-methyl-4-dimethylaminoazobenzene and from normal growing rat livers.

Plasma membranes (PM's) were isolated from primary hepatomas induced in Wistar rats by 3'-methyl-4-dimethylaminoazobenzene, from nonhepatomal regions of the same rat livers, and also from various normal rat livers, including the resting and regenerating livers of adult rats and developing livers of postnatal rats. Phospholipid analyses of these PM preparations revealed the following differences in the hepatoma PM in comparison with those of the PM of normal adult resting livers: (a) decrease in the content of total phospholipids; (b) large increase in plasmalogen content; (c) relative increase of sphingomyelin and ethanolamine phospholipids, and the decrease of choline phosphoglycerides, i.e., decrease of the ratios of choline phosphoglycerides to choline phosphosphingoside and of choline-containing phospholipids to ethanolamine-containing phospholipids; (d) decrease in phosphatidylserine and phosphatidylinositol. The phospholipid composition of the PM's from normal growing livers showed definite decreases in phosphatidylserine and sphingomyelin and increases in ethanolamine phospholipids; however, no significant alteration was observed in plasmalogen content in comparison to the PM of normal adult resting livers. The PM from the nonhepatomal regions of the hepatoma-bearing livers did not show those differences observed in hepatoma PM.

Functional and morphological characteristics of transplantable rat pituitary tumors established in nude mice.

Two strains of transplantable rat pituitary tumors, MtT SA5 and MtT SA6, have been established in female nude mice from a single original pituitary tumor which had spontaneously occurred in a female Wistar rat at 759 days of age. MtT SA5 tumor produces prolactin (PRL), growth hormone, and adrenocorticotropic hormone, and MtT SA6 tumor secretes PRL and growth hormone. Additionally, both tumors induce severe nephropathy and promote pathogenicity of murine hepatitis virus, resulting in hepatic necrosis. Electron micrographs of MtT SA5 and MtT SA6 tumors revealed three and two types of cells, respectively, in reference to secretory granules. The tumors seem to consist of mixed population, each cell secreting each hormone. Since marked adrenal enlargement and relatively low serum corticosterone levels were found in MtT SA5-bearing rats, it is suggested that MtT SA5 tumor secretes adrenocorticotropic hormone and/or its related peptides which induce adrenal hyperplasia with little or no stimulation of corticoid production. In nude mice bearing MtT SA5 tumor, concentrations of growth hormone in serum and tumor tissue were exceedingly higher than those of PRL, while they were in the same magnitude in MtT SA6-bearing nude mice. We also found that PRL levels in serum and tumor tissue of MtT SA5-bearing nude mice were much higher in males than females, although those of MtT SA5-bearing rats were not significantly different in both sexes.

Characterization of a unique nuclear estrogen-binding component in an estrogen-responsive mouse Leydig cell tumor.

An estrogen-responsive mouse Leydig cell tumor line (Tumor 124958) has been shown to contain only a low-affinity binder for estradiol in the cytosol fraction. This differed from the putative estrogen receptor in terms of its hormone-binding specificity as well as affinity. In addition, the possibility that an estrogen receptor-like molecule exists in the nuclei even without hormonal stimuli was examined using purified nuclei. Scatchard plot analyses showed that these nuclei possessed a large amount of estrogen binder having a high affinity for estradiol and diethylstilbestrol. The content of this nuclear binding component was not diminished by using molybdate, a potent inhibitor for receptor activation, and in vitro incubation of collagenase-dispersed cells with estradiol did not cause significant increase in the number of nuclear binding sites when compared with the values obtained by direct incubation of isolated nuclei with estradiol. These results support the view that this nuclear estrogen binder is not due to artificial migration of the cytosol receptor into nuclei during homogenization. The characterization of this nuclear binding component under cell-free conditions revealed that its affinity for estradiol in Mg2+-containing buffer was temperature dependent (Kd 3 nM at 30 degrees and 12 nM at 0 degrees) without significant alteration in the number of maximum binding sites. Introduction of a chelating agent (ethylenediaminetetraacetate) into the buffer system abolished the temperature effect on the affinity, resulting in high affinity for estradiol at both low and high temperatures. These Mg2+ and temperature effects were reversible. In addition, when compared with putative nuclear estrogen receptors, this nuclear binding was observed to be relatively resistant to high salt or micrococcal nuclease treatments in relation to solubilization from nuclei. However, trypsin digestion was found to result in a marked decrease in the nuclear binding sites, indicating that this unique nuclear binding component contains a protein unit(s). These results suggest the possibility that this tumor line contains a unique unoccupied nuclear estrogen binder which might be able to transmit estrogen signals to tumor cell nuclei with regard to tumor growth.

Genetic pathways in the evolution of morphologically distinct colorectal neoplasms.

Colorectal adenomas can be morphologically classified as exophytic or flat. Polypoid cancers and cancers arising de novo (ie., without any adenomatous component) might be the results of genetic progression from exophytic and flat adenomas, respectively. In this study, we examined 94 morphologically distinct neoplastic specimens for mutations in K-RAS and analyzed 10 microsatellite loci tightly linked to the tumor suppressor genes APC, p53, DCC/SMAD4, hMSH2, and hMLH1. K-RAS mutations were significantly associated with exophytic adenomas [11 of 21 (52%)] compared to flat adenomas [2 of 13(15%), P < 0.03] and polypoid cancers [17 of 25 (68%)] compared to cancers arising de novo [7 of 25 (28%), P < 0.01]. Two polypoid cancer cases demonstrated three and four different K-RAS mutations, respectively, suggesting multiple areas of clonal expansion. Cancers arising de novo were significantly associated with loss of heterozygosity (LOH) at chromosome 3p compared to pol ypoid cancers [6 of 18(33%) versus 1 of 20(5%), P < 0.03], whereas the prevalence of LOH at chromosomes 2p, 5q, 17p, and 18q and microsatellite instability were not different between the groups. For all cancers, LOH at chromosomes 17p and 18q occurred in 47 and 51%, respectively. However, LOH at 17p and 18q occurred in 0 and 16% of benign lesions, respectively, suggesting their role in malignant transformation. There was no difference in LOH at chromosomes 17p and 18q between exophytic and flat lesions. These findings suggest that (a) mutant K-RAS is associated with the exophytic growth of colonic neoplasms, and that (b) some colorectal cancers arising de novo lose chromosome 3p during their evolution, which is not seen in polypoid cancers. Half of all cancers lose chromosomes 17p and 18q at or near the malignant transition of benign lesions as reported previously, irrespective of morphology. There may be more than one genetic avenue for colorectal cancer formation, and this correlates with the morphological characteristics.

Aberrations in the fragile histidine triad (FHIT) gene in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) seems to be closely associated with lung carcinogenesis. To identify the genetic characteristics of precancerous IPF lesions in the peripheral lung, we performed PCR-based microsatellite analysis with DNA extracted from microdissected tissues; fluorescent in situ hybridization (FISH) analysis of the fragile histidine triad (FHIT) gene and immunohistochemical analysis of Fhit protein expression in samples of metaplasias and bronchiolar epithelia obtained from patients with IPF. We used four microsatellite markers of the FHIT gene within or flanking the FHIT gene on chromosome 3p for loss of heterozygosity (LOH) analysis. LOH of the FHIT locus was frequently found among the lesions of metaplasias and bronchiolar epithelia in the patients with IPF [62 (52%) of 119 informative lesions]. Fifty-four (73%) of the 74 lesions of metaplasias and bronchiolar epithelia obtained from the IPF patients with lung carcinoma and 8 (17%) of the 46 samples obtained from the IPF patients without lung carcinoma showed LOH at the FHIT gene (P < 0.0001). We confirmed allelic loss in the metaplasias and bronchiolar epithelia of IPF by FISH analysis of the FHIT gene. Additionally, the level of Fhit protein expression in the metaplastic cells of IPF was frequently reduced. Our findings suggest that allelic loss of the FHIT gene may be involved in carcinogenesis in the peripheral lung of patients with IPF.

Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutant-allele-specific amplification (MASA).

K-ras mutations are found in approximately half of all colorectal tumors examined. To explore the possibility of detecting mutated K-ras rapidly and efficiently in DNAs isolated from fecal material, we applied the mutant allele specific amplification (MASA)-PCR method to DNA from feces of patients with colorectal tumors. Among 55 colorectal adenocarcinomas or adenomas examined, 19 were found to carry K-ras mutations in codons 12 or 13. We were able to PCR-amplify DNAs isolated from feces of 15 of these 19 patients, but in only three of the fecal samples, we were able to detect the K-ras mutations corresponding to tumor DNA by MASA and ethidiumbromide staining of the gel. The carcinomas in these three cases were more than 40 mm x 40 mm in size and located in the sigmoid colon or rectum. However, we identified the K-ras mutations in fecal DNAs of additional seven patients by MASA when the gels were blotted and probed with a radio-labeled oligonucleotide; the tumors in those patients had arisen in the distal half of the colon and the smallest of these tumors was only 7 mm x 5 mm. No K-ras mutations were detectable in feces of the remaining five cases, whose tumors were relatively small and/or located in the proximal region. The results suggested that the MASA-PCR system has potential for development as a simple, rapid and noninvasive method for diagnosing the presence of colorectal tumors that carry mutant K-ras alleles, particularly tumors located in the distal colon.

Effect of different physical states of phospholipid substrates on partially purified platelet phospholipase A2 activity.

Partial purification of alkaline phospholipase A2 (EC 3.1.1.4) from rabbit platelets was carried out and the effect of different physical states of the substrate phosphatidylcholine on the activity was investigated. (1) The enzyme was purified about 1020-fold by means of Sephadex gel chromatography after extraction from a particulate fraction of rabbit platelets, followed by CM-cellulose chromatography, and had a molecular weight of approx. 12 000 as determined by gel chromatography. (2) The activity of the purified enzyme was enhanced by the addition of detergents. Sodium deoxycholate and sodium cholate markedly stimulated the activity, and the effect of these substances was observed well below the critical micelle concentrations. Triton X-100 stimulated the activity moderately, and the activation was observed only above the critical micelle concentration. (3) The addition of negatively charged phospholipids to the substrate egg phosphatidylcholine induced a moderate activation of hydrolysis. (4) The addition of long-chain cation to the substrate induced an inhibition of the activity, whereas the addition of long-chain anion activated the hydrolysis of egg phosphatidylcholine, but did not activate the hydrolysis of phosphatidylcholine in the total lipid extract of rabbit platelets. (5) Hydrolysis of dimyristoyl phosphatidylcholine increased in the temperature region of the phase transition of the substrate. Addition of cholesterol at the concentration of 20 mol% diminished the effect of phase transition. (6) Release of [1-14C]arachidonic acid from an equimolar mixture of egg phosphatidylcholine with dipalmitoyl or distearoyl phosphatidylcholine was activated at the temperature of 0 degrees C or 20 degrees C, respectively. From these results, we suggest that platelet phospholipase A2 can be activated to release fatty acids from the platelet phospholipids at the domains within membranes, where exist the structural irregularities and/or accumulation of negative charge within the bilayers.

Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse.

A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl2 and 5 mM MgCl2, and centrifuged at 52 000 X g for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of (Na+ + K+)-ATPase, Mg2+-ATPase and 5'-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40 degrees C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.