Matrix metalloproteinases are involved in C-terminal and interglobular domain processing of cartilage aggrecan in late stage cartilage degradation.

Monoclonal antibody (MAb) technology was used to examine aggrecan metabolites and the role of aggrecanases and matrix metalloproteinases (MMPs) in proteolysis of the interglobular domain (IGD) and C-terminus of aggrecan. An in vitro model of progressive cartilage degradation characterized by early proteoglycan loss and late stage collagen catabolism was evaluated in conjunction with a broad-spectrum inhibitor of MMPs. We have for the first time demonstrated that IGD cleavage by MMPs occurs during this late stage cartilage degeneration, both as a primary event in association with glycosaminoglycan (GAG) release from the tissue and secondarily in trimming of aggrecanase-generated G1 metabolites. Additionally, we have shown that MMPs were responsible for C-terminal catabolism of aggrecan and generation of chondroitin sulfate (CS) deficient aggrecan monomers and that this aggrecan truncation occurred prior to detectable IGD cleavage by MMPs. The onset of this later stage MMP activity was also evident by the generation of MMP-specific link protein catabolites in this model culture system. Recombinant MMP-1, -3 and -13 were all capable of C-terminally truncating aggrecan with at least two cleavage sites N-terminal to the CS attachment domains of aggrecan. Through analysis of aggrecan metabolites in pathological synovial fluids from human, canine and equine sources, we have demonstrated the presence of aggrecan catabolites that appear to have resulted from similar C-terminal processing of aggrecan as that induced in our in vitro culture systems. Finally, by developing a new MAb recognizing a linear epitope in the IGD of aggrecan, we have identified two novel aggrecan metabolites generated by an as yet unidentified proteolytic event. Collectively, these results suggest that C-terminal processing of aggrecan by MMPs may contribute to the depletion of cartilage GAG that leads to loss of tissue function in aging and disease. Furthermore, analysis of aggrecan metabolites resulting from both C-terminal and IGD cleavage by MMPs may prove useful in monitoring different stages in the progression of cartilage degeneration.     

Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells

Amsacrine is an acridine derivative drug applied in haematological malignancies. It targets topoisomerase II enhancing the formation of a cleavable DNA-enzyme complex and leading to DNA fragmentation in dividing cancer cells. Little is known about other modes of the interaction of amsacrine with DNA, by which it could affect also normal cells. Using the alkaline comet assay, we showed that amsacrine at concentrations from the range 0.01 to 10 microM induced DNA damage in normal human lymphocytes, human promyelocytic leukemia HL-60 cells lacking the p53 gene and murine pro-B lymphoid cells BaF3 expressing BCR/ABL oncogene measured as the increase in percentage tail DNA. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Amifostine at 14 mM decreased the level of DNA damage in normal lymphocytes, had no effect on the HL-60 cells and potentiated the DNA-damaging effect of the drug in BCR/ABL-transformed cells. Vitamin C at 10 and 50 microM diminished the extent of DNA damage in normal lymphocytes, but had no effect in cancer cells. Pre-treatment of the cells with the nitrone spin trap, N-tert-butyl-alpha-phenylnitrone or ebselen, which mimics glutathione peroxidase, reduced the extent of DNA damage evoked by amsacrine in all types of cells. The cells exposed to amsacrine and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results obtained suggest that free radicals may be involved in the formation of DNA lesions induced by amsacrine. The drug can also methylate DNA bases. Our results indicate that the induction of secondary malignancies should be taken into account as diverse side effects of amsacrine. Amifostine may potentate DNA-damage effect of amsacrine in cancer cells and decrease this effect in normal cells and Vitamin C can be considered as a protective agent against DNA damage in normal cells.     

Helicobacter pylori infection and antioxidants can modulate the genotoxic effects of heterocyclic amines in gastric mucosa cells

Helicobacter pylori (H. pylori) infection plays an important role in gastric carcinogenesis. This bacterium may induce cancer transformation and change the susceptibility of gastric mucosa cells to various exogenous dietary irritants. The aim of the study was to evaluate the influence of H. pylori infection on the reaction of the stomach cells to a genotoxic effect of heterocyclic amines (HCAs). These well-known mutagens are formed during cooking of protein-rich foods, primarily meat. Taking into account that persons consuming a mixed-western diet are exposed to these compound nearly an entire lifetime and more than half of human population is infected with H. pylori, it is important to assess the combined effect of H. pylori infection and HCAs in the context of DNA damage in gastric mucosa cells, which is a prerequisite to cancer transformation. We employed 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) because these substances are present in a great amount in cooked and fried meat. Using alkaline comet assay, we showed that the extent of the DNA damage induced by HCAs was significantly higher in H. pylori infected gastric mucosa cells than in non-infected counterparts. We did not observed any difference in the efficiency of repair of DNA lesions induced by HCAs in both type of cells. Vitamin C reduced the genotoxic effects of HCAs in H. pylori infected and non-infected gastric mucosa cells. Melatonin more effectively decreased DNA damage caused by HCAs in H. pylori infected gastric mucosa cells as compared with control. Our results suggest that H. pylori infection may influence the susceptibility of gastric mucosa cells to HCAs and dietary antioxidative substances, including vitamin C and melatonin may inhibit the genotoxic effects of HCAs on gastric mucosa cells and may reduce the risk of carcinogenesis caused by food borne mutagens and H. pylori infection.     

Topical application of capsaicin reduces visceral adipose fat by affecting adipokine levels in high‐fat diet‐induced obese mice

Objective:

Visceral obesity contributes to the development of obesity‐related disorders such as diabetes, hyperlipidemia, and fatty liver disease, as well as cardiovascular diseases. In this study, we determined whether topical application of capsaicin can reduce fat accumulation in visceral adipose tissues.

Methods and Results:

We first observed that topical application of 0.075% capsaicin to male mice fed a high‐fat diet significantly reduced weight gain and visceral fat. Fat cells were markedly smaller in the mesenteric and epididymal adipose tissues of mice treated with capsaicin cream. The capsaicin treatment also lowered serum levels of fasting glucose, total cholesterol, and triglycerides. Immunoblot analysis and RT‐PCR revealed increased expression of adiponectin and other adipokines including peroxisome proliferator‐activated receptor (PPAR) α, PPARγ, visfatin, and adipsin, but reduced expression of tumor necrosis factor‐α and IL‐6.

Conclusions:

These results indicate that topical application of capsaicin to obese mice limits fat accumulation in adipose tissues and may reduce inflammation and increase insulin sensitivity.     

Detection of platelet-associated IgG in chronic immune thrombocytopenic purpura using antibody-coated polyacrylamide beads

Platelet-associated IgG (PAIgG) was detected by means of anti-human IgG coated polyacrylamide beads ("Immunobeads") technique in 32 patients with chronic ITP. Both a direct test (with in vivo sensitized platelets) and an indirect test (with in vitro loaded platelets) were carried out. The percent of rosette forming beads was both in the direct test (41.2%) and in the indirect test (32.6%) significantly higher in the cases of chronic ITP patients than in the controls (2.5% and 3.2%, respectively). These results confirm the diagnostic value of this new, relatively simple and rapid method in routine clinical practice.     

Age-related limitations of interleukin-6 in predicting early mortality in acute ST-elevation myocardial infarction

Interleukin-6 (IL-6) is an inflammatory cytokine whose levels increase significantly during myocardial infarction (MI).It has been hypothesised that the concentrations of IL-6 at admission may be useful in prognosticating long-term outcomes. It is unclear, however, whether IL-6 could improve the prognosis of early mortality in MI.We have compared serum IL-6 levels and analysed the disease course in 158 patients with ST-elevation MI (STEMI) who either survived (n?=?148) or died (n?=?10) within 30 days following the admission. Patients were treated in a single university centre with primary percutaneous coronary intervention (PCI).The non-survivors (6.3%) displayed most of typical risk factors for poor outcome. In addition they had significantly higher concentrations of IL-6 at hospital admission (median values 8.5 vs. 2.0 pg/ml; p?=?0.038). However, they were also significantly older than the survivors (median values 72 vs. 57 years; p?=?0.0001). IL-6 levels are known to increase with age and we could confirm a significant correlation between patients’ calendar age and circulating IL-6 (p?=?0.009). Regression analysis revealed that IL-6 concentrations were significantly affected by patients’ age but they did not independently relate to patients’ outcome.Such results indicate that circulating IL-6 at admission may be of limited value in predicting early mortality in STEMI. It is important to recognize that, because of the small group of patients who died (N?=?10), the results must be interpreted with caution. Therefore, we stress that these results should be viewed as preliminary and further validated in a larger set of patients.     

Auditory ossicles from southwest Asian Mousterian sites

The present study describes and analyzes new Neandertal and early modern human auditory ossicles from the sites of Qafzeh and Amud in southwest Asia. Some methodological issues in the measurement of these bones are considered, and a set of standardized measurement protocols is proposed. Evidence of erosive pathological processes, most likely attributed to otitis media, is present on the ossicles of Qafzeh 12 and Amud 7 but none can be detected in the other Qafzeh specimens. Qafzeh 12 and 15 extend the known range of variation in the fossil H. sapiens sample in some metric variables, but morphologically, the new specimens do not differ in any meaningful way from living humans. In most metric dimensions, the Amud 7 incus falls within our modern human range of variation, but the more closed angle between the short and long processes stands out. Morphologically, all the Neandertal incudi described to date show a very straight long process. Several tentative hypotheses can be suggested regarding the evolution of the ear ossicles in the genus Homo. First, the degree of metric and morphological variation seems greater among the fossil H. sapiens sample than in Neandertals. Second, there is a real difference in the size of the malleus between Neandertals and fossil H. sapiens, with Neandertals showing larger values in most dimensions. Third, the wider malleus head implies a larger articular facet in the Neandertals, and this also appears to be reflected in the larger (taller) incus articular facet. Fourth, there is limited evidence for a potential temporal trend toward reduction of the long process within the Neandertal lineage. Fifth, a combination of features in the malleus, incus, and stapes may indicate a slightly different relative positioning of either the tip of the incus long process or stapes footplate within the tympanic cavity in the Neandertal lineage.     

The effects of acute corticosterone administration on anxiety, endogenous corticosterone, and c-Fos expression in the rat brain

The effects of acute pretreatment of rats with corticosterone (5 and 20 mg/kg, s.c.) on emotional behavior, expression of c-Fos protein in brain structures, and serum concentration of corticosterone were studied to model the short-term glucocorticoid-dependent changes in brain functions. Corticosterone was administered 90 min before training of a conditioned fear reaction (a freezing response), and behavioral, hormonal and immunocytochemical effects were examined 1 day later, on the test day. Pretreatment of rats with corticosterone significantly attenuated the freezing reaction in the conditioned fear test. The effect of the corticosterone was accompanied by a selective enhancement of the aversive context-induced c-Fos expression in some brain structures: the parvocellular and magnocellular neurons of the paraventricular hypothalamic nucleus (pPVN and mPVN), the medial amygdala nucleus (MeA), and the cingulate cortex, area 1 (Cg1), as well as an increase in the concentration of aversive context-induced endogenous serum glucocorticoid, 1.5 h and 10 min after the test session, respectively. It is suggested that the behavioral effects of acute pretreatment of rats with corticosterone could be due to changes in the mnemonic processes in the brain, inhibition of brain corticotropin releasing factor (CRF) synthesis, or stimulation of GABA-A receptor modulating neurosteroids synthesis. It is hypothesized that the enhanced activity of Cg1, MeA, pPVN, and mPVN, and the hypothalamic-pituitary-adrenal axis with concomitant increased serum glucocorticoid concentration, might serve to facilitate active coping behavior in a threatening situation.     

Isolation of soluble yeast beta-glucans that inhibit human monocyte phagocytosis mediated by beta-glucan receptors

The trypsin-sensitive receptor that mediates phagocytosis of unopsonized zymosan particles by human monocytes has been designated as a beta-glucan receptor because of its functional inhibition by specific algal and plant beta-glucans. Soluble ligands that are chemically and structurally identical to beta-glucan constituents of zymosan were isolated from a carbohydrate-enriched fraction of yeast extract by sequential chromatography on DE-cellulose, SP-Sephadex, and Con A-Sepharose. Preincubation of adherent human monocytes with 278, 210, and 2.5 micrograms/ml hexose equivalents in pooled chromatographic fractions from DE-cellulose, SP-Sephadex, and Con A-Sepharose, respectively, effected 50% reductions in subsequent phagocytosis of zymosan particles without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (ESIgG). The purified yeast extract-derived beta-glucans, which contained 92% glucose and 8% mannose by gas chromatographic analysis and eluted from a Sephacryl S-200 column as a broad peak with a Kav of 0.39 and estimated molecular sizes of from 20,000 to 70,000 m.w., required only 3.5 +/- 0.9 micrograms/ml (mean +/- SD, n = 6), as compared with 31.5 micrograms/ml of the algal beta-glucan laminarin to achieve 50% decreases in zymosan ingestion. Alternatively, soluble yeast beta-glucans with estimated molecular sizes of from 2 X 10(5) to 2 X 10(6) were prepared from yeast glucan particles, which contained 98% glucose and 0% mannose, by sonication and sequential centrifugation at 15,000 and 100,000 X G for 30 and 60 min, respectively. Monocyte ingestion of zymosan was reduced by 50% by pretreatment with 60 ng/ml of the soluble beta-glucans in 15,000 X G supernatants, whereas ingestion of ESIgG was unaffected by as much as 50 micrograms/ml of this material. Partial acid hydrolysis of soluble glucan-derived beta-glucans in 15,000 X G supernatants followed by gel filtration on Bio-Gel P-4 revealed two well-defined peaks within the inclusion volume of the column with phagocytosis-inhibiting activity. Oligoglucosides that eluted at a Kav of 0.46 had an estimated molecular size of 2,000 m.w. and effected a 48% reduction in zymosan ingestion at inputs of 2 to 5 micrograms/ml, and smaller oligoglucosides with a Kav of 0.82 and an estimated molecular size of 1,000 m.w. effected a 50% reduction at inputs of 25 micrograms/ml. Preincubation of monocytes for 2 min with 25 micrograms/ml of the oligoglucosides with estimated molecular size of 1,000 m.w. and with 50 ng/ml of soluble glucan-derived beta-glucans in 100,000 X G supernatants reduced zymosan ingestion by 41% +/- 4 and 44% +/- 3 (mean +/- SD, n = 3), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activated Ras prevents downregulation of Bcl-X(L) triggered by detachment from the extracellular matrix. A mechanism of Ras-induced resistance to anoikis in intestinal epithelial cells

Detachment of epithelial cells from the extracellular matrix (ECM) results in a form of apoptosis often referred to as anoikis. Transformation of intestinal epithelial cells by oncogenic ras leads to resistance to anoikis, and this resistance is required for the full manifestation of the malignant phenotype. Previously, we demonstrated that ras-induced inhibition of anoikis in intestinal epithelial cells results, in part, from the ras-induced constitutive downregulation of Bak, a pro-apoptotic member of the Bcl-2 family. Since exogenous Bak could only partially restore susceptibility to anoikis in the ras-transformed cells, the existence of at least another component of the apoptotic machinery mediating the effect of activated ras on anoikis was suggested. Indeed, here we show that, in nonmalignant rat and human intestinal epithelial cells, detachment from the ECM or disruption of the cytoskeleton results in a significant downregulation of the antiapoptotic effector Bcl-X(L), and that activated H- or K-ras oncogenes completely abrogate this downregulation. In addition, we found that enforced downregulation of Bcl-X(L) in the ras-transformed cells promotes anoikis and significantly inhibits tumorigenicity, indicating that disruption of the adhesion-dependent regulation of Bcl-X(L) is an essential part of the molecular changes associated with transformation by ras. While the ras-induced downregulation of Bak could be reversed by pharmacological inhibition of phosphatidylinositol 3 kinase (PI 3-kinase), the effect of ras on Bcl-X(L) was PI 3-kinase- and mitogen-activated protein kinase (MAP kinase)-independent. We conclude that ras-induced resistance to anoikis in intestinal epithelial cells is mediated by at least two distinct mechanisms: one that triggers downregulation of Bak and another that stabilizes Bcl-X(L) expression in the absence of the ECM.