Induction of superficial zone protein (SZP)/lubricin/PRG 4 in muscle-derived mesenchymal stem/progenitor cells by transforming growth factor-β1 and bone morphogenetic protein-7

Introduction

Articular cartilage (AC) is an avascular tissue with precise polarity and organization. The three distinct zones are: surface, middle and deep. The production and accumulation of the superficial zone protein (SZP), also known as lubricin, by the surface zone is a characteristic feature of AC. To date, there is a wealth of evidence showing differentiation of AC from mesenchymal stem cells. Most studies that described chondrogenic differentiation did not focus on AC with characteristic surface marker SZP/lubricin. The present investigation was initiated to determine the induction of SZP/lubricin in skeletal muscle-derived mesenchymal stem/progenitor cells (MDMSCs) by transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7).

Methods

MDMSCs were cultured as a monolayer at a density of 1 × 10⁵ cells/well in 12-well tissue culture plates. Cell cultures were treated for 3, 7 and 10 days with TGF-β1 and BMP-7. The medium was analyzed for SZP. The cells were used to isolate RNA for RT-PCR assays for SZP expression.

Results

The SZP/lubricin increased in a time-dependent manner on Days 3, 7 and 10 in the medium. As early as Day 3, there was a three-fold increase in response to 3 ng/ml of TGF-β1 and 300 ng/ml of BMP-7. This was confirmed by immunochemical localization of SZP as early as Day 3 after treatment with TGF-β1. The expression of SZP mRNA was enhanced by TGF-β1.

Conclusions

The present investigation demonstrated the efficient and reproducible induction of SZP/lubricin accumulation by TGF-β1 and BMP-7 in skeletal MDMSCs. Optimization of the experimental conditions may permit the utility of MDMSCs in generating surface zone-like cells with phenotypic markers of AC and, therefore, constitute a promising cell source for tissue engineering approaches of superficial zone cartilage.     

Influence of aflatoxin B1 on seed germination, seedling growth, chlorophyll and carotenoid contents of mustard (brassica juncea L. var. pusa bold) seeds

Five different concentrations (100, 250, 500, 1000 and 2000 μg/L of aflatoxin B₁ were found to be inhibitory to seed germination and seedling growth (root and shoot lengths) of mustard seeds (variety Pusa bold). These also lowered the levels of chlorophyll and carotenoids in the emerging leaves during seedling growth. The inhibitory effect was correlated with the concentration of applied toxin.     

Stimulation of the side population fraction of ATDC5 chondroprogenitors by hypoxia

The influence of oxygen tension on the side population (SP) fraction sorted from ATDC5 chondroprogenitor cells was investigated. ATDC5 cells cultured in normoxia (20%) or hypoxia (1% oxygen) were compared. The SP fraction was significantly higher in the cells cultured in hypoxia. The gene expression of 3 ABC transporters, abcb1a/b (mdr1a/b) and abcg2 (bcrp1) was quantified by RT-PCR. SP cells were characterized by the higher expression of abcb1a. The expression levels of abcb1b and abcg2 were higher than abcb1a. However, there was no significant difference between SP and non-SP fractions in the expression of abcb1b and abcg2. The telomeric repeat amplification protocol assay showed that SP cells tended to show lower telomerase activity than non-SP cells. Chondrogenic properties of ATDC5 cells derived from SP or non-SP were assessed by micromass culture. There were not significant differences between SP and non-SP derived cells in Alcian blue staining and sox9, Aggrecan, Col2a1 and SZP mRNA expression. The results demonstrate that the SP fraction was stimulated by hypoxia and chondrogenic properties of SP and non-SP fraction of ATDC5 cells were similar.     

Meso-substituted tetra-cationic porphyrins photosensitize the death of human fibrosarcoma cells via lysosomal targeting

In this paper we present a study on the intracellular localisation and the efficiency of cell photoinactivation of a series of derivatives of 5,10,15,20-tetrakis-(4-N-methylpyridyl)-porphine (C1), whose degree of lipophilicity was varied through replacement of one methyl group with an alkyl chain of various length. Human HT1080 fibrosarcoma cells exposed to the various C1 derivatives (0.25 microM) for 24 h and irradiated with increasing doses of red-light (0.45-27 J/cm2) were inactivated with different efficiencies. The efficiency of cell photoinactivation increased with the increasing length of the hydrocarbon tail and lipophilicity and correlated with the efficiency of the porphyrin accumulation into the cells. Despite the presence of positive charges, these porphyrins did localise rather selectively in lysosomes while mitochondrial localisation was not evident, as demonstrated by fluorescence microscopy studies. Studies on isolated mitochondria provided evidence that the porphyrin uptake and distribution in these organelles were not modulated by the transmembrane potential but were exclusively controlled by partitioning phenomena which might have prevented mitochondria localization in whole cells. Our findings demonstrated that these porphyrins entered the cells through the endocytotic pathway and were transported to lysosomes whose pH increased rapidly upon irradiation. Lysosomal damage did not cause any intracellular redistribution of the porphyrin and represented the primary event causing cell death, very likely via necrosis.     

Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N

Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.     

Bioavailability of aspirin and salicylamide following oral co-administration in human volunteers.

BC powder (I) is a commercially available analgesic containing the active ingredients aspirin and salicylamide. The kinetics of I, BC powder minus aspirin (II), and BC powder minus salicylamide (III) were evaluated in 13 volunteers. Ten minutes after administration of I, aspirin reached a maximum concentration of 12.9 micrograms/mL, while salicylamide concentration reached a peak value of 3.4 micrograms/mL. However, when III was administered, aspirin was not detected at 10 min and only reached a concentration of 0.4 microgram/mL at 2 and 6 h. Furthermore, the area under the plasma concentration versus time curve for aspirin when III was administered was sixfold less compared with treatment with I. The area under the curve for aspirin metabolites was significantly different in I versus III. After treatment with II, a delay in salicylamide peak concentration was observed. Gentisamide was not detected throughout the study. This study demonstrates that salicylamide significantly enhances plasma levels of aspirin with potential therapeutic implications.     

Vitamin and micronutrient concentrations in cyclosporine-induced renal tumor from diabetic rats

Concentrations of vitamins, biopterin, free inositol and acid-soluble carnitine were determined in cyclosporine A induced renal adenocarcinoma and uninvaded renal tissue from streptozotocin diabetic rats. Vitamin B6, thiamin, riboflavin, nicotinate, free inositol and acid-soluble carnitine were significantly decreased in tumor than nontumor tissue. Concentrations of folic acid, B12, biotin, pantothenate and biopterin were similar in both tissues. These studies suggest that renal adenocarcinoma affects concentrations of only certain vitamins and micronutrients.