Chemical constituents of Euphorbia marschalliana Boiss

Acetone extract of aerial parts of Euphorbia marschalliana collected from Iran has been subjected to different chromatography techniques for fractionation and purification. The stereo-structures of the myrsinol esters 15-O-acetyl-3-O-propionyl-5-O-butanoyl-7-O-nicotinoylmyrsinol (1) and 15-O-acetyl-3,5-O-dibutanoyl-7-O-nicotinoylmyrsinol (2) have been probed using ROESY spectroscopy and modified for the stereochemistry at C-6, C-12 and C-13. Beta-sitosterol (3), 29-norcycloart-5-ene (4), 5,8-lanostadiene-3beta-ol (5), 3beta,24(S),25-trihydroxycycloartane (6), 3beta,24(R),25-trihydroxycycloartane (7) and 24-methylenecycloartan-3beta-ol (8) were identified for the first time in this plant.     

Inhibitory Effect of Aspergillus fumigatus Extract on matrix metalloproteinases Expression

BACKGROUND: Matrix metalloproteinases (MMPs) play a role in several physiologic and pathologic events. There are some evidence indicating the involvement of MMPs in the pathophysiology of fungal infections. METHODS: Here we study somatic extract of Aspergillus fumigatus. The influence of Aspergillus vs. two other fungal extracts on MMPs production by Fibrosarcoma cell line was investigated using an in vitro cytotoxicity assay, sodium dodecyl sulfate-polyacrylamide and gelatin zymography. RESULTS: Comparative dose-dependent inhibitory effects on MMPs were seen by A. fumigatus extract and compared with some steroidal and non-steroidal drugs. Cytotoxicity analysis of our extract revealed much lower cell death than other examined agents. CONCLUSIONS: Since inhibition of MMPs activity has been employed in modality therapy in such diseases as cancer, this extract might be promising in the preparation of anti-MMP therapeutic derivatives.     

Relevance of the direct pathway of sensitization in corneal transplantation is dictated by the graft bed microenvironment

Corneal grafts were until recently considered entirely devoid of resident APCs, giving rise to the tenet that alloantigen recognition is mediated exclusively by the indirect (host APC-dependent) pathway. The recent discovery of a resident myeloid corneal dendritic cell population that is normally MHC class II(-) but can readily up-regulate class II expression during inflammation led us to hypothesize that under certain conditions the direct pathway of allosensitization becomes operative. To test this, corneal allotransplants were performed in either inflamed (high-risk (HR)) or uninflamed (low-risk (LR)) host beds in mice, and the frequencies of host T cells activated via the direct pathway were determined. We found that directly primed CD4(+) T cells were detected in the HR but not LR setting, and these cells displayed a clear Th1 phenotype by 2 wk after grafting. Moreover, the use of MHC class II knockout donor tissue led to significantly enhanced survival of HR but not LR allografts. Finally, we show that donor corneal APC demonstrate high expression of CD40, CD80, and CD86 costimulatory molecules when derived from HR but not LR grafts. These data are the first to report that a functional donor APC-dependent direct response is elicited in corneal transplant hosts when the graft bed is inflamed and underscore the relevance of the graft microenvironment in dictating the pathway of allosensitization.     

Conjugation of tetanus toxoid with Salmonella typhimurium PTCC 1735 O-specific polysaccharide and its effects on production of opsonizing antibodies in a mouse model

Serious enteric and extra-intestinal infections with Salmonella typhimurium are very common in many human populations. Phagocytosis is the main defense mechanism against this bacterium; however, the unique structure of S. typhimurium lipopolysaccharide (LPS) makes it resistant to opsonization by complement components. In the present study, the S. typhimurium LPS O-chain was purified and conjugated with tetanus toxoid and the effects of the conjugated vaccine (O-specific polysaccharide tetanus toxoid (O-SP-TT)) on induction of specific antibodies were investigated in a mouse model. In vitro assays measuring phagocytosis in the presence of opsonizing antibodies were performed. Three subcutaneous injections of the O-SP-TT conjugate conferred protection against the intraperitoneal challenge with S. typhimurium and the LD50 was greater for immunized animals than for controls. The mean number of ingested S. typhimirium / mouse peritoneal cell in the presence of sera obtained from immunized mice with purified O-chain, O-SP-TT conjugate, heat-killed bacteria, and negative control were 6.96, 14.24, 15.96, and 6.67, respectively. In summary, we developed an O-SP-TT conjugate that induced opsonizing antibodies that increased phagocytosis, as determined by in vitro assays. In addition, chemiluminescence results, an indicator of oxidative burst, indicated that peritoneal cells respond better to live S. typhimurium cells in the presence of sera obtained from O-SP-TT conjugate immunized mice.     

Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds

Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(L-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions. gene expression; osteogenesis; angiogenesis     

Predation by Allothrombium pulvinum on the spider mites Tetranychus urticae and Amphitetranychus viennensis: Predation Rate, Prey Preference and Functional Response

The deutonymphs of Allothrombium pulvinum Ewing (Acari: Trombidiidae) are among the most important natural enemies of spider mites in North, North East and West Iran. In this study, maximum predation rate and preference experiments were conducted with A. pulvinum deutonymphs on apple leaf discs, to determine their preference for either of two spider mite species: Amphitetranychus viennensis (Zacher) and Tetranychus urticae Koch (Acari: Tetranychidae). Maximum predation rate tests showed that the predatory mite consumed more eggs and females of T. urticae than of A. viennensis. Furthermore, the Manly’s preference index for eggs and females of T. urticae confirmed that T. urticae were the preferred prey. The functional response of A. pulvinum deutonymphs on females of T. urticae was examined over a 24-h period. Predation of A. pulvinum deutonymphs presented with females of T. urticae followed a type III functional response. Estimated handling time for the predatory mites was 4.51 h and attack coefficient b, which describes the changes in attack rate with prey densities in a type III functional response, was 0.021.     

Fluorimetric study of the artificial chaperone-assisted renaturation of carbonic anhydrase: a kinetic analysis

It is now well accepted that ionic detergents along with alpha- or beta-cyclodextrins can enhance protein refolding yields. In this report, we evaluated the effect of detergent's tail length on the kinetics of denatured carbonic anhydrase refolding along with determining the rate-limiting step of the whole refolding process. A sensitive fluorimetric technique was also developed to follow up the second-by-second fate of the denatured protein while undergoing refolding. In this technique, inclusion complexes are formed between the correctly refolded CA and the fluorescent active site probe, 5-dimethylaminonaphtalene-1-sulfonamide. By this specific technique, it became evident that the rate of detergent stripping from the CA-detergent mixed micelles that also appeared to be the rate-limiting step depends on the beta-CD-detergent association constants which are under the influence of detergent's tail length. Based on these findings, appropriate refolding conditions could be designed to kinetically diminish the rate of off-pathway aggregation.     

Concomitant reduction of matrix metalloproteinase-2 secretion and intracellular reactive oxygen species following anti-sense inhibition of telomerase activity in PC-3 prostate carcinoma cells

Background: The level of activity of the telomerase has been shown to correlate with the degree of invasiveness in several tumor types. In addition, cellular redox state is believed to regulate the secretion of matrix metalloproteinase-2 (MMP-2).Aims: To determine the effect of anti-sense telomerase treatment of prostate cancer cells on MMP-2 activity, and the reactive oxygen and nitrogen species (two effectors of cellular redox state).Methods: Anti-sense oligonucleotide against RNA component of human telomerase (hTR) was introduced into the cells using Fugene-6 transfection reagent. The activity of telomerase was assessed using Telomere Repeat Amplification Protocol (TRAP assay). Activity of matrix metalloproteinase-2 (MMP-2) was determined by zymography. Levels of intracellular reactive oxygen species (ROS) and nitric oxide metabolites were measured by dichlorofluorescein diacetate (DCFH-DA) staining and Griess reagent, respectively. The level of apoptosis was determined using TUNEL assay.Results: TRAP assay showed more than 90% inhibition of telomerase activity after 72 h of transfection. Pro-MMP-2 activity was decreased down to 50% of the control levels. Intracellular reactive oxygen species were also significantly decreased. Neither apoptosis rate nor the level of nitric oxide metabolites was significantly different between anti-sense treated and control cells.Conclusions: Concomitant reduction of the pro-MMP-2 secretion and ROS in PC-3 cells following hTR inhibition suggests that over-activity of telomerase in cancer cells might increase the level of matrix metalloproteinase-2 and thus, be directly involved in the invasion process through enhancement of intracellular oxidative stress.