Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.

OBJECTIVE: CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes cartilage regeneration in experimental osteoarthritis (OA) models. However, CCN2 production is very low in articular cartilage. The aim of this study was to investigate whether or not CCN2 was promoted by cultured chondrocytes treated with low-intensity pulsed ultrasound (LIPUS) and to clarify its mechanism. METHODS: Human chondrocytic cell line (HCS)-2/8, rat primary epiphyseal and articular cartilage cells, and Ccn2-deficient chondrocytes that impaired chondrocyte differentiation, were treated with LIPUS for 20 min at 3.0 MHz frequency and 60 mW/cm2 power. Expressions of chondrocyte differentiation marker mRNAs were examined by real-time PCR (RT-PCR) analysis from HCS-2/8 cells and Ccn2-deficient chondrocytes at 30 min and 1 h after LIPUS treatment, respectively. CCN2 production was examined by Western blotting after 5 h of LIPUS treatment. Moreover, Ca2+ influx was measured by using a Fluo-4 probe. RESULTS: The gene expression of chondrocyte differentiation markers and CCN2 production were increased in cultured chondrocytes treated with LIPUS. In addition, Ca2+ influx and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 were increased by LIPUS treatment, and the stability of TRPV4 and BKca channel mRNAs was decreased by siRNA against CCN2. Consistent with those findings, the LIPUS-induced the gene expressions of type II collagen (COL2a1) and Aggrecan (ACAN) observed in wild-type cells were not observed in the Ccn2-deficient chondrocytes. CONCLUSION: These data indicate that chondrocyte differentiation represented by CCN2 production was mediated via MAPK pathways activated by LIPUS-stimulated Ca2+ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes.

Evaluation of chlorinated benz[a]anthracene on hepatic toxicity in rats and mutagenic activity in Salmonella typhimurium.

Chlorinated benz[a]anthracenes (Cl-BaA) are halogenated aromatic compounds (typified by dioxins) found in the environment at relatively high concentrations. Fischer 344 rats were intragastrically administered 0, 1, or 10 mg of Cl-BaA or its parent compound benz[a]anthracene (BaA) per kg of body weight for 14 consecutive days. Both chemicals at 10 mg/kg/day inhibited the gain in body weight, and consequent increase in relative liver weight. Hepatic gene expression of cytochrome P450 (CYP) 1A1, 1A2, and 1B1 was significantly stimulated by administration of BaA (10 mg/kg/day) compared with the control. After administration of Cl-BaA, only the CYP1A2 gene was significantly induced, even at the lower dosage; CYP1A1 and 1B1 mRNA levels remained unchanged in Cl-BaA-treated rats compared with controls. To elucidate the role of such Cl-BaA exposure and induced CYPs at toxicity onset, we investigated the mutagenicity of BaA and Cl-BaA using Salmonella typhimurium TA98 and TA100. BaA and Cl-BaA at 10 µg/plate produced positive results in both strains in the presence of rat S-9. Incubation of Cl-BaA with recombinant rat CYP1A2 produced a significantly higher number of revertant colonies in TA98 and TA100 than in controls, but no such change was observed for BaA. In conclusion, BaA changes its own physiological and toxicological actions by its chlorination; (1) daily exposure to Cl-BaA selectively induces hepatic CYP1A2 in rats and (2) Cl-BaA induces frameshift mutations in the presence of CYP1A2, although BaA does not exert mutagenicity. This indicates that CYP1A2 may metabolize Cl-BaA to active forms.

Effects of an immuno-enhanced diet containing antioxidants in esophageal cancer surgery following neoadjuvant therapy.

Neoadjuvant therapy-induced immunological deterioration may be a key factor in postoperative morbidity in patients with esophageal cancer. This study aimed to determine the effects of perioperative feeding with an immuno-enhanced diet on immune competence in patients treated with neoadjuvant therapy followed by surgery. Because an immuno-enhanced diet that contained several antioxidants was used, perioperative oxidative stress and the effects of the immuno-enhanced diet on this stress were also investigated. Of 39 patients with esophageal cancer who underwent similar surgical procedures, 26 patients who received chemotherapy or chemoradiation therapy before surgery were randomly divided into two groups: group 1 (n= 14) was given an immuno-enhanced diet for 5 days before surgery, and group 2 (n= 12) received no enteral feeding products before surgery. Group 3 (n= 13) consisted of patients that did not receive neoadjuvant therapy and received no enteral feeding products before surgery. Several markers for coagulation and fibrinolysis were determined and immunological assessments were performed for each patient. To measure reactive oxygen metabolites and the total antioxidant capacity, diacron-reactive oxygen metabolites (d-ROMs) and OXY-adsorbent tests were performed using a free radical elective evaluator. Significant depression in lymphocyte numbers was observed in groups 1 and 2 before and early after surgery as compared to group 3. Numbers of B cells, CD4/CD8 ratio, and phytohemagglutinin-induced lymphocyte transformation tests were also significantly decreased in groups 1 and 2 on postoperative day 1. Fibrin and fibrinogen degradation products were significantly elevated in group 2 compared to group 1. d-ROMs and OXY-adsorbent test values were elevated before surgery and were decreased transiently early after surgery. Compared to groups 2 and 3, d-ROMs values were significantly lower in group 1 patients throughout the postoperative period, while OXY-adsorbent test values were significantly higher in group 2 patients. Oxidative index was significantly suppressed in group 1 compared to group 3. No significant intergroup differences were observed with regard to morbidity after surgery. Although the baseline levels of immunological function might have been different because of less-advanced cancer stages in group 3, neoadjuvant therapy significantly affected several immunological parameters. Preoperative administration of an immuno-enhanced diet did not significantly prevent neoadjuvant therapy-induced immunological deterioration prior to esophageal cancer surgery. Patients with esophageal cancer had elevated levels of oxidant and antioxidant activities before surgery, which were transiently decreased early after surgery. Although the underlying mechanisms for these perioperative changes are unclear, this study showed that an immuno-enhanced diet containing several antioxidants may reduce oxidative stress following esophageal cancer surgery. After these mechanisms are studied further, oxidative stress control may become another tool for perioperative management to reduce morbidity after esophageal cancer surgery.

Molecular, anatomical and physiological properties of a genetically modified soybean line transformed with rd29A:AtDREB1A for the improvement of drought tolerance.

We evaluated the molecular, anatomical and physiological properties of a soybean line transformed to improve drought tolerance with an rd29A:AtDREB1A construct. This construct expressed dehydration- responsive element binding protein DREB1A from the stress-inducible rd29A promoter. The greenhouse growth test included four randomized blocks of soybean plants, with each treatment performed in triplicate. Seeds from the non-transformed soybean cultivar BR16 and from the genetically modified soybean P58 line (T(2) generation) were grown at 15% gravimetric humidity for 31 days. To induce water deficit, the humidity was reduced to 5% gravimetric humidity (moderate stress) for 29 days and then to 2.5% gravimetric humidity (severe stress). AtDREB1A gene expression was higher in the genetically modified P58 plants during water deficit, demonstrating transgene stability in T(2) generations and induction of the rd29A promoter. Drought-response genes, including GmPI-PLC, GmSTP, GmGRP, and GmLEA14, were highly expressed in plants submitted to severe stress. Genetically modified plants had higher stomatal conductance and consequently higher photosynthetic and transpiration rates. In addition, they had more chlorophyll. Overexpression of AtDREB1A may contribute to a decrease in leaf thickness; however, a thicker abaxial epidermis was observed. Overexpression of AtDREB1A in soybean appears to enhance drought tolerance.

Association of treatment-achieved HbA1c with incidence of coronary artery disease and severe eye disease in diabetes patients.

AIM: To examine the association between treatment-achieved HbA1c values and incidence of both coronary artery disease (CAD) and severe eye disease with different diabetes treatments. METHODS: Associations of treatment-achieved HbA1c were investigated in various treatment groups [diet only; insulin; sulphonylurea (SU) alone; SU with glinides; and antihyperglycaemic agents other than glinides, SU or insulin] taken from a nationwide claims database of 14,633 Japanese diabetes patients. Cox's regression analysis examined risks over a 5.1-year follow-up. RESULTS: A significant linear trend was associated with HbA1c levels and CAD events in the diet-only group, and CAD risks were significantly higher in insulin and SU groups with HbA1c ≤ 7.0% and > 8.0% than in the diet-only group with HbA1c ≤ 7.0%. In contrast to CAD, a linear association was observed regardless of treatment modality between achieved HbA1c levels and risk of severe diabetic eye disease, but with no significant difference in eye disease risk between groups with HbA1c ≤ 7.0% and 7.1-8.0% in those treated with either SU alone, SU with glinides, or insulin. CONCLUSION: These findings suggest that the relationship between treatment-achieved HbA1c and incidence of both CAD and severe diabetic eye disease differed according to treatment, based on a large-scale real-life database. More research is now needed to confirm these findings and to further investigate the underlying mechanisms.

Lack of B-RAF mutations in head and neck squamous cell carcinoma.

B-RAF is one of the most commonly mutated oncogenes in human cancer. However, the mutation status of B-RAF has not been established completely in HNSCC. We have analysed the mutation status of the kinase domain of the B-RAF gene (exons 11 and 15) in 91 Japanese HNSCC patients as well as 12 HNSCC cell lines. DNA was extracted and amplified by PCR. Mutations were then analysed by SSCP mutation detection method. Since V600EB-RAF constitutes 90 % of the mutations identified in B-RAF in human cancers, we also used MASA analysis to specifically detect this mutation in exon 15 of B-RAF. Using both methods, no mutation was found in both exon 11 and 15 in all patients and cell lines. Mu tations are absent or rare in the kinase domain of B-RAF in Japanese HNSCC. However, more studies are still needed to determine its usefulness as a target for molecular therapy in these patients.

Impact of glucose tolerance status on the development of coronary artery disease among working-age men.

AIMS: To examine the impact of glucose tolerance status on the development of coronary artery disease (CAD) in working-age men in Japan. METHODS: This population-based retrospective cohort study included 111,621 men aged 31-60 years [63,558 with normal glucose tolerance (NGT); 37,126 with prediabetes; 10,937 with diabetes]. The Cox proportional-hazards regression model was used to identify variables related to the incidence of CAD. RESULTS: Multivariate analysis showed that, compared with NGT, diabetes increased the risk of CAD by 17.3 times (95% CI: 6.36-47.0) at ages 31-40 years, by 2.74 times (95% CI: 1.85-4.05) at ages 41-50 years and by 2.47 times (95% CI: 1.69-3.59) at ages 51-60 years. The HRs for CAD in men with diabetes aged 31-40 equaled that of men with NGT aged 51-60 [18.2 (7.15-46.4) and 19.4 (8.28-45.4), respectively]. CONCLUSION: The impact of diabetes on CAD was markedly greater in men aged 31-40 years compared with those aged 41-60 years.

Impact of body mass index and metabolic phenotypes on coronary artery disease according to glucose tolerance status.

OBJECTIVE: This study aimed to examine the impact of obesity, as defined by body mass index (BMI), and a metabolically unhealthy phenotype on the development of coronary artery disease (CAD) according to glucose tolerance status. METHODS: This population-based retrospective cohort study included 123,746 Japanese men aged 18-72years (normal glucose tolerance: 72,047; prediabetes: 39,633; diabetes: 12,066). Obesity was defined as a BMI≥25kg/m2. Metabolically unhealthy individuals were defined as those with one or more of the following conditions: hypertension, hypertriglyceridaemia and/or low HDL cholesterol. A Cox proportional hazards regression model identified variables related to CAD incidence. RESULTS: The prevalences of obese subjects with normal glucose tolerance, prediabetes and diabetes were 21%, 34% and 53%, whereas those for metabolically unhealthy people were 43%, 60% and 79%, respectively. Multivariate analysis showed that a metabolically unhealthy phenotype increases hazard ratios (HRs) for CAD compared with a metabolically healthy phenotype, regardless of glucose tolerance status (normal glucose tolerance: 1.98, 95% CI: 1.32-2.95; prediabetes: 2.91, 95% CI: 1.85-4.55; diabetes: 1.90, 95% CI: 1.18-3.06). HRs for CAD among metabolically unhealthy non-obese diabetes patients and obese diabetes patients with a metabolically unhealthy status were 6.14 (95% CI: 3.94-9.56) and 7.86 (95% CI: 5.21-11.9), respectively, compared with non-obese subjects with normal glucose tolerance and without a metabolically unhealthy status. CONCLUSION: A metabolically unhealthy state can associate with CAD independently of obesity across all glucose tolerance stages. Clinicians may need to consider those with at least one or more conditions indicating a metabolically unhealthy state as being at high risk for CAD regardless of glucose tolerance status.

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.

Expression of nestin in rat and human glomerular podocytes.

Nestin is a neuroepithelial precursor cell marker expressed in a variety of human cell types during development. However, no information exists on the expression of nestin in mature glomeruli as well as during the glomerular development. Here, we examined nestin expression in rat and human glomerular tissues in quiescent states using RT-PCR and immunohistochemical methods. Nestin mRNA was detected in the rat glomeruli in parallel with its expression in developing rat brains. In the normal mature rat glomeruli, WT-1 positive cells expressed nestin. Co-expression of nestin and vimentin was observed in mature rat podocytes. Immunoelectron microscopy revealed nestin localization in the cell bodies and primary processes of podocytes. A similar expression pattern was observed for vimentin. In matured glomeruli, nestin was not expressed by mesangial and endothelial cells. In the newborn rat, early developing glomeruli (metanephric cap, metanephric vesicle, comma-shaped vesicle and S-shaped body phases) expressed nestin. In the capillary loop stage, Bowman's capsules also expressed nestin. Immunoelectron microscopy demonstrated that developing podocytes and endothelial cells in S-shaped phase glomeruli expressed nestin. Additionally, in immature glomeruli, the mesangial cells in capillary stage of glomerulus also expressed nexin. As in the rat, WT-1 positive cells in human glomeruli also expressed nestin and immunoelectron microscopy confirmed nestin expression in human glomerular podocytes. These results reveal that in normal condition nestin is expressed in several glomerular cell types at early stage of development and becomes confined to podocytes in mature glomeruli, thus implicating nestin in podocyte functions.

Enhancement of DNA chain breakage by bleomycin A2 in the presence of microsomes and reduced nicotinamide adenine dinucleotide phosphate.

Chain breakage in DNA induced by bleomycin A2 (BLM) was enhanced more than 150-fold by the reduced nicotinamide adenine dinucleotide phosphate-dependent electron transport system of rat liver microsomes. However, the enhancement effect on DNA was partially reduced by the preincubation of BLM with the microsomal systems. BLM-Cu2+ was found to stimulate considerably microsomal reduced nicotinamide adenine dinucleotide phosphate-dependent oxygen consumption and malondialdehyde formation, whereas BLM inhibited both of the effects. These findings suggest that the pharmacological action of BLM is strongly affected by a membrane system, such as microsomes, that produces free radicals.

Increase of antitumor effect on bleomycin by reduced nicotinamide adenine dinucleotide phosphate and microsomes in vitro and in vivo.

The DNA chain breakage effect of bleomycin A2 on isolated nuclei was enhanced over 150-fold by the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent electron transport system of isolated microsomes. The enhancement of bleomycin A2-induced DNA chain breakage by NADPH was also shown at the level of the intact AH66 cells. Furthermore, it was found that the simultaneous administration of bleomycin and NADPH apparently inhibited the growth of Erhlich tumors in mice compared with the administration of bleomycin alone. These results indicate that the action of bleomycin on DNA is stimulated by the microsomal NADPH-dependent electron transport system not only in vitro but also in vivo.

Expression and secretion of the beta subunit of human chorionic gonadotropin by bladder carcinoma in vivo and in vitro.

Expression and secretion of the beta subunit of human chorionic gonadotropin (hCG) by bladder carcinoma cell lines were investigated in vitro and in vivo. As an in vitro study, immunoreactive hCG beta (IR-hCG beta) secreted into the culture media of two bladder transitional cell lines (KoTCC-1 and HT-1197) was analyzed using three kinds of enzyme immunoassays which were specific for intact hCG, free hCG beta, and beta core fragment (beta-CF). Both of the cell lines were determined to secrete IR-hCG beta into the media, which consisted principally of free hCG beta, but detectable levels of intact hCG and beta-CF were not present in the media. Northern blot analysis revealed that the hCG beta gene was expressed in both KoTCC-1 and HT-1197 cells where the sizes of mRNA from these cells were smaller than those from placental and NJG choriocarcinoma cells. As an in vivo study, distribution of IR-hCG beta was analyzed in the tumor tissues, sera, and urine of the mice and the rats transplanted with KoTCC-1 cells. By the immunohistochemical study, the IR-hCG beta was clearly observed in transitional cell carcinoma cells of the transplanted tumor. High levels of IR-hCG beta were detected in both the serum and urine from the animals, but there were quantitative and qualitative differences between serum and urinary IR-hCG beta. Quantitatively, the concentrations of IR-hCG beta in the urine were consistently much higher than those in the serum. Qualitatively, free hCG beta was exclusively detected in the serum whereas high levels of beta-CF in addition to free hCG beta were found in the urine. Intact hCG could not be detected in the serum and urine. These distributions of IR-hCG beta in the animals transplanted with KoTCC-1 cells were completely analogous to those in a patient with hCG beta-producing bladder carcinoma. The present study shows that the same metabolic pathway of IR-hCG beta is operating in mice and rats as in humans, indicating that IR-hCG beta found in patients with bladder carcinoma originates from the tumor and it may be recognized as a tumor marker when beta-CF is measured in the patient's urine.

Reducing needlestick injuries through safety-engineered devices: results of a Japanese multi-centre study.

BACKGROUND: Quantitative information on the effectiveness of safety-engineered devices (SEDs) is needed to support decisions regarding their implementation. AIM: To elucidate the effects of SED use in winged steel needles, intravenous (IV) catheter stylets and suture needles on needlestick injury (NSI) incidence rates in Japan. METHODS: Japan EPINet survey data and device utilization data for conventional devices and SEDs were collected from 26 participating hospitals between 1 April 2009 and 31 March 2014. The NSI incidence rate for every 100,000 devices was calculated according to hospital, year and SED use for winged steel needles, IV catheter stylets and suture needles. Weighted means and 95% confidence intervals (CI) were used to calculate overall NSI incidence rates. FINDINGS: In total, there were 236 NSIs for winged steel needles, 152 NSIs for IV catheter stylets and 180 NSIs for suture needles. The weighted NSI incidence rates per 100,000 devices for SEDs and non-SEDs were as follows: winged steel needles, 2.10 (95% CI 1.66-2.54) and 14.95 (95% CI 2.46-27.43), respectively; IV catheter stylets, 0.95 (95% CI 0.60-1.29) and 6.39 (95% CI 3.56-9.23), respectively; and suture needles, 1.47 (95% CI -1.14-4.09) and 16.50 (95% CI 4.15-28.86), respectively. All devices showed a significant reduction in the NSI incidence rate with SED use (P < 0.001 for winged steel needles, P = 0.035 for IV catheter stylets and P = 0.044 for suture needles). CONCLUSION: SED use substantially reduces the incidence of NSIs, and is therefore recommended as a means to prevent occupational infections in healthcare workers and improve healthcare safety.

Protease digestion of membranes. Ultrastructural and biochemical effects.

(1) Nagarse, a bacterial protease, was permitted to react with sarcoplasmic reticulum, submitochondrial and plasma membranes. Gel electrophoresis indicated that all polypeptides were labile to the enzyme, and therefore must be at least partially exposed at membrane surfaces. However, hydrolysis did not proceed to completion, and in each membrane 30-50% of the original protein mass remained after extensive digestion. Gel patterns showed that remaining polypeptide fragments were in the range of 10000 molecular weight. (2) Amino acid analysis of the original protein and membrane-bound digestion product was performed. Only minor changes were observed following digestion, suggesting that the peptide fragments remaining with the membrane did not have specialized amino acid compositions. (3) freeze-fracture analysis of Nagarse-treated sarcoplasmic and plasma membranes showed that particulate structures were present, although particle density and asymmetry of fistribution between fracture faces were decreased. In submitochondrial membranes, digested membranes were indistinguishable from the original membranes in particle density and distribution. We conclude that high molecular weight polypeptides are not required for the production particulate structures in freeze-fracture images of membranes.

Labelling of sarcoplasmic reticulum membranes with 1-dimethylaminonaphthalene-5-sulfonyl chloride.

1. Sarcoplasmic reticulum membranes were labelled with 1-dimethylaminonaphtalene-5-sulfonyl chloride (DnsCl). Analyses of the dansylated membranes demonstrated that the most of the dye was associated with ATPase (ATP phosphohydrolase, EC 3.6.1.3) and phosphatidylethanolamine in the membranes. 2. Dansylation of the membranes could be performed without significant decrease in the ATPase activity. 3. Partial differentiation of fluorescence of Dns-phosphatidylethanolamine from that of Dns-ATPase could be achieved by changing excitation wavelength; Dns-ATPase emmitted in the shorter wavelength region, while Dns-phosphatidylethanolamine emmitted in the longer wavelength region. 4. Fluorescence polarization of the dye bound to the membranes indicated that both the ATPase and phosphatidylethanolamine were strongly immobilized in the membranes, while the ratio of freely rotating dye to the "frozen" dye bound to the ATPase was larger than that bound to the phosphatide.

Prorenin processing and restricted endoproteolysis by mouse tissue kallikrein family enzymes (mK1, mK9, mK13, and mK22).

Four members of the tissue kallikrein family, mK1, mK9, mK13, and mK22, all of which exhibit extensive homology in amino acid sequence among themselves, were obtained from the submandibular gland of ICR mice and examined for their ability to cleave prorenin. Tissue kallikrein mK13 was confirmed to be a prorenin-converting enzyme; and mK9, which was earlier shown to be an EGF-binding protein, was found to cleave mouse Ren 2 prorenin specifically and convert it to mature renin with an activity of approximately 1/10 of that of mK13. With the same substrate, mK22 (beta-NGF endopeptidase) gave two products, renin and arginyl-renin; whereas mK1 (true tissue kallikrein) did not process it at all. The endoproteolytic activity of tissue kallikreins was examined with various peptide-MCA substrates. The substrates contained three key structures; X(Y)-Arg-Arg, X(Y)-Lys-Arg and X-Lys-Lys motifs (where X and Y are hydrophilic and hydrophobic amino acids, respectively). We found that mK1, mK9 and mK13 preferentially cleaved the former two types of substrate, except Y-Arg-Arg-MCA. The substrate X-Lys-Lys-MCA was hardly cleaved by these three tissue kallikreins but was preferentially cleaved by mK22. The four tissue kallikreins seem to have the ability to process precursor proteins containing a pair of basic amino acid residues; the specificities of three of the enzymes (mK1, mK9 and mK13) were similar to each other but were different from that of mK22.

Lipids of plasma membranes from rat thymic lymphoid cells: deficiency of sphingomyelin.

Plasma membranes were isolated from normal thymocytes of Wistar-King-A rats and from Moloney virus-induced rat thymic leukemias (RML11 and RML30 cells) using a simplified method developed by us. All the isolated plasma membranes were electron-microscopically pure and enriched in the specific activities of (Na+ + K+)-ATPase, Mg2+-ATPase and 5'-nucleotidase in comparison with those of the corresponding whole cell homogenates. These plasma membranes as well as the original cells were analyzed for phopholipid composition and contents of phospholipid, cholesterol and plasmalogen. There was no difference in the phospholipid composition among the three plasma membranes. However, all the plasma membranes were deficient in sphingomyelin, namely, 1.8% for the normal thymocytes, 2.2% for the RML11 cells and 1.9% for the RML30 cells as percentage of the total phospholipid phosphorus. The contents of phospholipid (mumol per mg protein), cholesterol (mumol per mg protein) and plasmalogen (mol% to phospholipid) of the plasma membranes from both lines of malignant cells were lower than those of the normal thymocyte membranes. The molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes, because in the former membranes the degree of decrease in the cholesterol content was higher than that in phospholipid content.

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.

Reversal of mesangial enlargement in rats with long-standing diabetes by whole pancreas transplantation.

An important unanswered question about clinical use of pancreas transplantation is: can pancreas transplants reverse or, at least, stabilize well-established lesions of insulin-dependent diabetes mellitus (IDDM)? To answer this question, we performed whole pancreas transplantations in 190 highly inbred rats 6, 9, 12, 15, 18, and 21 mo after induction of diabetes mellitus (DM) with alloxan. We then studied the effect on renal mesangial enlargement (ME) for 24 mo after onset of DM by a quantitative morphologic technique in which camera lucida tracings of the mesangium were made at X 1250 and were analyzed using an electronic planimeter connected to a calculator/computer. A pretransplant kidney biopsy was obtained so that the rats served as their own controls. In addition, studies were performed for 28 mo in 57 untreated diabetic controls and in 55 nondiabetic controls. Monthly metabolic studies showed that whole pancreas transplantation maintained very tight, lifelong metabolic control of diabetes. Kidney sections obtained for 2 yr from diabetic controls and for 21 mo from diabetic rats before transplantation showed highly significant increases in total mesangial area, nuclear-free mesangial area, and percentage of glomerular area occupied by nuclear-free mesangial area. Pancreas transplantation consistently produced a highly significant reversal of well-established ME, regardless of when it was performed.(ABSTRACT TRUNCATED AT 250 WORDS)