The nature of the intercellular material of adult mammalian tissues

the aim of this work was to define the influence of the ageing process on the activity of proteolytic enzymes, such as trypsin, elastase, plasmin and active MMP-9 concentration, as well as the inhibitor alpha 1-antitrypsin. Moreover, we assessed associations between enzyme activity and selected clinical and biochemical parameters. healthy normotensive volunteers (n = 60, 30 women) aged 20-82 years were split into subgroups: young (aged 20-22), middle-aged (49-52) and elderly (77-82). Serum enzyme activity was assessed using fluorometric methods. overall, active MMP-9 concentration and trypsin activity decreased with age, and alpha1-antitrypsin concentration and plasmin activity increased. Activity of elastase increased with age when compared to the young age group. An inverse correlation was identified between MMP-9 concentration and BMI and a direct correlation found between BMI and elastase, plasmin activity and alpha1-antitrypsin concentration. In the middle-aged group, glucose correlated directly with trypsin activity and inversely with MMP-9 concentration. Trypsin activity and MMP-9 concentration correlated inversely with cholesterol concentration and plasmin and elastase activity, and the alpha1-antitrypsin concentration correlated with cholesterol concentration in the overall group. the results confirm the influence of the ageing process on the activity of serum proteolytic enzymes. The activity of individual proteolytic enzymes in the serum changes with age.

Experiments on an ascites hepatoma: I. Enzymatic digestion and alkaline degradation of the cementing substance and separation of cells, in tumor islands

Inhaled ultrafine (nano) particles can translocate into the bloodstream and interact with circulatory cells causing systemic and cardiovascular events. To gain more insight into this potential mechanism, we studied the interaction of diesel exhaust particles (DEP) with human, rat and mouse erythrocytes in vitro. Incubation of erythrocytes with DEP (1, 10 or 100 µg/ml) for 30 min caused the highest hemolytic effect (up to 38%) in rats, compared to small but significant hemolysis in mice (up to 2.5%) and humans (up to 0.7%). Transmission electron microscopy of erythrocytes revealed the presence of variable degrees of ultrafine (nano)-sized aggregates of DEP either internalized and/or adsorbed onto the erythrocytes in the three species. A significant amount of DEP was found in rat and mouse (but not human) erythrocytes. Lipid erythrocyte susceptibility to in vitro peroxidation measured by malondialdehyde showed a significant and dose-dependent increase in erythrocytes of rats, but not humans or mice. Unlike in human erythrocytes, total antioxidant status (TAS) and superoxide dismutase (SOD) activity in rats were significantly and dose- dependently decreased. In mouse erythrocytes, DEP caused a decreased in SOD (at 10 µg/ml) and TAS (at 100 µg/ml) activities. In conclusion, DEP caused species–dependent erythrocyte hemolysis and oxidative stress, and were either taken up and/or adsorbed onto the red blood cells. Rat (and to a lesser degree mouse) erythrocytes were susceptible to DEP. Human erythrocytes showed the highest resistance to the observed effects. These species difference should be noted when using rats and mice blood as models for humans.

The extracellular matrix (ECM) undergoes constant dynamic changes; proteolytic enzymes, particularly the serine proteases plasmin, trypsin and elastase, catalyze critical functions in these processes. Notably, ECM degradation disorders have been reported in various morbid conditions, including cardiac infarction, atheromatosis, and neoplastic diseases, indicating a physiological requirement for proper ECM maintenance. Here we define the role of proteolytic enzymes in the development of aging by assessing changes in proteolytic enzyme activity in serum during aging in rats. The activities of trypsin, elastase and plasmin in rat serum were determined by the fluorometric method using AMC-labeled substrates in 34Wistar rats divided into four age groups: 3 month-olds (n=8), 9 month-olds (n=8), 15 month-olds (n=8) and 24 month-olds (n=10). Analysis of proteolytic enzyme activity in four age-dependent groups revealed that in comparison to their 3, 9, and 24 month-old counterparts, the 15 month-old rats exhibited a statistically significant increase in average elastase activity. In accordance with previous studies, a statistically significant increase in trypsin levels was found in the 3 month-old rats, suggesting that trypsin activity decreases with age. Average plasma plasmin activity in the 24 month-old rats was, moreover, statistically significantly higher than that in the other three age groups. Analysis of combined proteolytic activity indicates that age-dependent patterning of blood serine protease enzyme activity may be related to age-related diseases.

Carbohydrate Metabolism in Ascites Tumor and HeLa Cells

The effect of the mode of administration of nitrogen mustard and cytosine arabinoside on the production of chromosomal aberrations in mouse bone marrow and ascites tumour cells

Erythrocyte autoantibodies and autoantibody-specific suppressor cells are elicited in mice injected with rat erythrocytes. Here it is shown, first, that modified rat erythrocytes elicit suppressor cells independently of autoantibody production. Haemoglobin-free membranes of rat erythrocytes, but not soluble lysates of rat erythrocytes, elicited autoantibodies and suppressor cells. Solubilization of these rat erythrocyte membranes with solutions of Triton-X-114 produced membrane fractions which induced autoantibody-specific suppression in the absence of detectable autoantibody production. These results are compatible with the view that the activation of suppressor cells in this model involves recognition of structures on the rat erythrocytes. Secondly, trinitrophenyl (TNP) was attached to rat erythrocytes in various doses in attempts to make the immunogenicity of TNP similar to that of the cross-reactive determinants on rat and mouse erythrocytes, to determine whether TNP-specific suppressor cells were produced. The results show that at various doses of TNP, including those at which antibody production to TNP could not be detected, rat erythrocytes coated with TNP induced autoantibody-specific suppressor cells but not suppressor cells with specificity for antibodies to TNP.

Ascitic and solid Ehrlich tumor inhibition by Chenopodium ambrosioides L. treatment

IT has been demonstrated that rabbit anti-mouse erythrocyte serum is capable of agglutinating ascites tumour cells1. Nothing is known, however, about the components of the erythrocyte which are responsible for producing the agglutinin; the present experiment was carried out to clarify this point, and mouse-specific ascites tumour cells and mouse erythrocytes were used.

Natural or deliberate activation of the immune system of pathogen-free mice markedly affected their response to an autoimmune-inducing stimulus. Specifically, mice immunized with rat red blood cells were found to make antibodies reactive with both rat and mouse erythrocytes. Animals housed for an extended period in a conventional environment developed an autoimmune response twice as fast as those kept in isolators. In an attempt to emulate this effect, mice kept in a sterile environment were infected with a potent polyclonal activator of B lymphocytes, lactate dehydrogenase-elevating virus, at the same time as they were inoculated with rat erythrocytes. Whereas uninfected animals developed a progressively increasing autoantibody titer, infected mice quickly attained high anti-erythrocyte autoantibody titers that remained rather constant. Contrary to circulating autoantibodies, bound anti-erythrocyte antibodies decreased with time. Virus infection enhanced all the IgG subclass responses, with the exception of IgG1, to both rat and mouse erythrocytes. None of the modifications of the autoimmune responses resulted in anemia.

Stability and effects of organic solvents on endopeptidases from the gastric fluid of the marine crab Cancer pagurus

Epithelial ovarian cancer (EOC) is still one of the deadliest malignancies in women frequently involving peritoneal tumor spread and ascites. Despite the heterogeneity of EOC, patients are mostly treated with standard therapy (cytoreductive surgery and platinum based chemotherapy). Understanding molecular mechanisms of EOC and its peritoneal metastasis is essential to develop new targeted therapies. In this study, we extended the transcriptome analysis of large RNA (Auer et al. Oncotarget 2015, 6:17261) to small RNA including micro RNAs (miRNAs) and piwi protein interacting RNAs (piRNAs). The transcriptome was analyzed concerning peritoneal tumor spread as introduced in our previous study: miliary – i.e. numerous small millet-like tumor seedings - versus non-miliary – i.e. big, bulky implants. Small RNA-sequencing (sRNA-seq) was performed with a total of 43 tumor cell enriched samples isolated from ascites and solid tumors from 23 chemo-naïve high grade serous EOC (HGSOC) patients and analyzed together with corresponding RNA-seq data from our previous study. qPCR was applied for validation of sRNA-seq data (n=48). Expression differences were determined (I) between miliary and non-miliary tumor spread in (Ia) ascitic and (Ib) solid tumor cells and (II) between ascitic and solid tumor cells in (IIa) miliary and (IIb) non-miliary on three levels: single miRNAs, gene-miR sets, and general types of RNAs (especially those of the competing endogenous RNA network, ceRNA). sRNA-seq yielded evidence for expression of 649 known and 2,306 newly predicted miRNAs and 22,987 known and 783 newly predicted piRNAs. qPCR validation rate was 90% for 48 selected sRNAs. (Ia) 401 miRNAs, but no gene-miR set and (Ib) one miRNA, but >19,000 gene-miR sets were differentially expressed between miliary and non-miliary in ascitic and solid tumor cells, respectively. 72% of these deregulated gene-miR sets were characterized by down-regulated coding genes and up-regulated miRNAs. (IIa) 109 miRNAs and 49 gene-miR sets and (IIb) 55 miRNAs and no gene-miR set were differentially expressed between ascitic and solid tumor cells in miliary and non-miliary, respectively. These numbers indicate varying strategies for differential expression between miliary and non-miliary, one the one hand, and between e. g. ascitic and solid tumor cells, on the other hand: thousands of small changes in miRNA and gene expression versus few, but greater changes in individual miRNA and gene expression. Global analyses of coding, long non-coding (lncRNAs), circular (circRNAs) RNAs, and miRNAs revealed a down-regulated ceRNA network (i.e. less circRNAs and lncRNAs compared to coding RNAs), but an unchanged total amount of miRNAs. Given the sponge function of the ceRNA network, this indicates more free miRNAs available for regulation in miliary compared to non-miliary. In addition, a 13 sRNA signature was developed to predict the peritoneal tumor spread type from FFPE tissues. It was used for validation of the impact on overall survival (OS) in an independent cohort of 32 HGSOC patients. In accordance with our previous study, miliary tumor spread had a negative impact on OS independent of age and FIGO stage. We are the first to perform an integrative analysis of large and small RNA-seq data of ascitic and solid tumor cells from 23 HGSOC patients. We found evidence for hundreds of known mi- and piRNAs and thousands of newly predicted mi- and piRNAs. We could show a negative prognostic impact of the miliary tumor spread in a validation cohort. Solid tumors of the miliary type seem to be more influenced by miRNA regulation than solid tumors of the non-miliary type which implies different spread strategies. This fundamentally different mechanism of gene regulation and the different prognosis of these two spread types underline the need for alternative treatment regiments. Citation Format: Anna Bachmayr-Heyda, Katharina Auer, Nyamdelger Sukhbaatar, Stefanie Aust, Simon Deycmar, Agnes T. Reiner, Stephan Polterauer, Sabine Dekan, Dietmar Pils. Peritoneal tumor spread in high grade serous ovarian cancer: An effect of the competing endogenous RNA network? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B56.

Three different variants of the Ehrlich ascites tumour (EAT) cell were derived and the lectin surface reactivities, as well as the malignant characteristics of each variant, were studied. Wild-type cells (EAT-wt) were selected for growth on basement membranes and tissue culture plastic to give EAT-c cells. The EAT-c were passaged in mice by i.p. injection, giving rise to a third variant (EAT-c/m). Each of these three cell variants was characterized for: (i) specific lectin agglutinability patterns; (ii) the ability to produce ascites tumours in mice; (iii) the ability to produce solid tumours; and (iv) the attachment to and growth on basement membranes and purified extracellular matrix molecules. Analysis of the total protein and carbohydrate content of each cell line showed that there was an increase in the glycosylation of the EAT-c cells compared to EAT-wt cells. After repeated passage of the EAT-c/m cells in mice, the glycosylation level of the EAT-c/m cells returned to that of the EAT-wt cell line. In addition, the EAT-c cells displayed an increase in the number of terminal non-reducing sugars which could indicate either an increase degree of branching or the presence of additional N- and/or O-linked oligosaccharide chains of the cellular glycoproteins. This phenotype was retained by the EAT-c/m cells which had been passaged repeatedly in mice. The most significant increase was in the content of sialic acid-containing glycoproteins found in the EAT-c cells. The sialic acid-binding lectin Maackia amurensis leukoagglutinin (MAL) agglutinated all three EAT cell variants, while the sialic acid-binding Sambucus nigra (elderberry bark) lectin (SNA) agglutinated only the EAT-c and early-passage EAT-c/m cells. These findings indicate the presence of alpha 2,3-linked sialic acid on all three variants, but only the cultured cells and early-passage EAT-c/m cells possess the Neu5Ac alpha 2,6 linkage. The EAT-c cells attached avidly to wells coated with either laminin or fibronectin, as well as extracellular matrix produced by cultured bovine endothelial cells, but the EAT-wt and EAT-c/m cells did not. Paradoxically, the EAT-c cells were incapable of producing solid tumours when injected into a basement membrane-rich skeletal muscle bed, whereas the EAT-wt and EAT-c/m cells produced rapidly growing tumours when injected into the same environment. Lectin agglutination patterns established that ascitic tumour cells within the peritoneal cavity were derived from injected EAT-c cells.

Pyridoxamine (pyridoxine) phosphate oxidase activity in mammalian tissues

Summary Centrifugated, or filtered, extracts prepared from mammary carcinomas that developed spontaneously in old female mice were found to possess a hemolytic potency directed specifically against mouse erythrocytes. When 0.5 ml of a tumor extract of 20 per cent concentration was mixed with 1 ml of a 1 per cent suspension of washed mouse erythrocytes, and placed in an incubator at 37 C for 2½ to 3 hours, hemolysis resulted in practically all instances. The red blood cells of rabbits, guinea pigs, chickens and man appear to be resistant to the hemolytic action of the mouse tumor extracts. The hemolytic potency of the tumor extracts prepared from mouse mammary carcinomas was destroyed by heating of the extracts, prior to the tests, to 68 C for 30 minutes. At room temperature (23 C) the centrifugated tumor extracts lose practically all their hemolytic potency after 5 hours. Extracts prepared from mouse mammary carcinomas that had been transplanted for 8 to 60 successive generations, were found to possess in most instances only a weak hemolytic potency; they were occasionally found, however, to clump slightly mouse erythrocytes in vitro. This clumping potency was to a lesser degree, and in some instances only, directed also against erythrocytes of other species of animals. Centrifugated extracts prepared from transplanted rat tumors (a sarcoma and a carcinoma) were found to exert only a weak hemolytic action on rat erythrocytes in vitro; they had, however, a strong hemolytic action on the red blood cells of the mouse. A slight clumping potency of these extracts on rat erythrocytes was also noticed. Extracts prepared from spontaneous mouse leukemia had a hemolytic potency on mouse erythrocytes. In a series of experiments, centrifugated extracts were prepared from various normal organs, such as liver, spleen, lungs, kidneys, muscle, etc., removed from healthy animals of either a high-tumor, a leukemic, or a low-tumor inbred line of mice. Twenty two females, which were either virgins, or did not have any litters for more than 3 months prior to the tests, were used for these experiments. No hemolysis resulted in 59 of the 61 tests. Similar negative results were obtained with extracts prepared from organs removed from 7 healthy males. Hemolysis resulted, however, when extracts prepared from normal mammary glands of nursing, healthy females of either a high-tumor, a leukemic, or a low-tumor line, were mixed with mouse erythrocytes. To a lesser degree this was also observed when mammary tissue extracts were prepared from pregnant female mice. Mouse placenta extracts were also found to possess a moderate hemolytic potency on mouse erythrocytes. Centrifugated extracts prepared from normal liver removed from healthy mice were found to inhibit the hemolytic potency of the tumor extracts, when mixed with the extracts in equal volumes, and incubated at room temperature for 30 minutes prior to the tests. Heating of the liver extracts to 56 C for 30 minutes was found to destroy their inhibiting potency. Extracts prepared from normal mouse kidneys were found to possess a similar, though less constant, inhibiting ability.