Intracellular Protein Synthesis Research Articles

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease-2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm(2)) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm(2)) (P < 0.01). (3)H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.

The influence of hierarchical hybrid micro/nano-textured titanium surface with titania nanotubes on osteoblast functions

Hierarchical hybrid micro/nano-textured titanium surface topographies with titania nanotubes were produced by simple acid etching followed by anodization to mimic the hierarchical structure of bone tissues. Primary rat osteoblasts were used to evaluate the bioactivity. The microtopography formed by acid etching of titanium induced inconsistent osteoblast functions with initial cell adhesion and osteogenesis-related gene expression being dramatically enhanced while other cell behaviors such as proliferation, intracellular total protein synthesis and alkaline phosphatase activity, collagen secretion, and extracellular matrix mineralization being depressed. In comparison, addition of nanotubes to the microtopography led to enhancement of multiple osteoblast functions. Nearly all the cell functions investigated in this study were retained or promoted. Compared to a microtopography, the enhancement of multiple cell functions observed from the hierarchical micro/nano-textured surfaces is expected to lead to faster bone maturation around the titanium implants without compromising the bone mass. In addition, the hierarchical micro/nano-textured surfaces still retain the mechanical interlocking ability of the microtopography thereby boding well for osseointegration. Our study reveals a synergistic role played by the micro and nanotopographies in osteoblast functions and provides insight to the design of better biomedical implant surfaces.

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with K(D) values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution.

The effects of TGF-α, IL-1β and PDGF on fibroblast adhesion to ECM-derived matrix and KGF gene expression

The goal of this study was to elucidate the control mechanisms by which exogenous proteins regulate keratinocyte growth factor (KGF) expression in fibroblasts adhered to differing substrates and thereby provide insights into both fundamental in vitro cell signaling and cell-biomaterial interaction research. A serum-free culture system in which cells maintained their proliferative capacity was established and employed. The addition of transforming growth factor- α (TGF-α), interleukin-1β (IL-1β) and platelet-derived growth factor-BB (PDGF-BB) individually showed no effect on KGF protein release, however, IL-1β addition led to increased KGF mRNA transcription, intracellular KGF protein synthesis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) release. Intracellular KGF protein synthesis and extracellular release were enhanced when fibroblasts were treated with a combination of IL-1β and PDGF-BB which suggests KGF synthesis and release are largely regulated by synergistic mechanisms. Surface-bound fibronectin-derived ligands and individual exogenous proteins promoted fibroblast adhesion to semi-interpenetrating polymer networks (sIPNs) but did not stimulate KGF release despite enhancement of KGF mRNA transcription. Additionally, serum conditioning was found to have a significant impact on KGF synthesis and the subsequent mechanisms controlling KGF release. This study demonstrates that KGF release from fibroblasts is likely regulated by multiple mechanisms involving post-transcriptional and exocytic controls which may be impacted by the presence of serum and how serum is removed from the in vitro cell environment.

Genetic Inactivation of Poliovirus Infectivity by Increasing the Frequencies of CpG and UpA Dinucleotides within and across Synonymous Capsid Region Codons

Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only approximately 9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses, but not MEF-1, was temperature sensitive at 39.5 degrees C. Synthesis and processing of viral intracellular proteins were largely unaltered in most codon replacement constructs. Replacement of natural codons with synonymous codons with increased frequencies of CpG and UpA dinucleotides may offer a general approach to the development of attenuated vaccines with well-defined antigenicities and very high genetic stabilities.

The impact of diamond nanocrystallinity on osteoblast functions

Nanocrystalline diamond has been proposed as an anti-abrasive film on orthopedic implants. In this study, osteoblast (bone forming cells) functions including adhesion (up to 4 h), proliferation (up to 5 days) and differentiation (up to 21 days) on different diamond film topographies were systematically investigated. In order to exclude interferences from changes in surface chemistry and wettability (energy), diamond films with nanometer and micron scale topographies were fabricated through microwave plasma enhanced chemical-vapor-deposition and hydrogen plasma treatment. Scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy and water contact angle measurements verified the similar surface chemistry and wettability but varied topographies for all of the diamond films prepared on silicon in this study. Cytocompatibility assays demonstrated enhanced osteoblast functions (including adhesion, proliferation, intracellular protein synthesis, alkaline phosphatase activity and extracellular calcium deposition) on nanocrystalline diamond compared to submicron diamond grain size films for all time periods tested up to 21 days. An SEM study of osteoblast attachment helped to explain the topographical impact diamond had on osteoblast functions by showing altered filopodia extensions on the different diamond topographies. In summary, these results provided insights into understanding the role diamond nanotopography had on osteoblast interactions and more importantly, the application of diamond films to improve orthopedic implant lifetimes.

Preliminary quantitative profile of differential protein expression between rat L6 myoblasts and myotubes by stable isotope labeling with amino acids in cell culture

Defining the mechanisms governing myogenesis has advanced in recent years. Skeletal-muscle differentiation is a multi-step process controlled spatially and temporally by various factors at the transcription level. To explore those factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high-accuracy mass spectrometry (LTQ-Orbitrap), was applied successfully. Rat L6 cell line is an excellent model system for studying muscle myogenesis in vitro. When mononucleate L6 myoblast cells reach confluence in culture plate, they could transform into multinucleate myotubes by serum starvation. By comparing protein expression of L6 myoblasts and terminally differentiated multinucleated myotubes, 1170 proteins were quantified and 379 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressed proteins are mainly involved in inter-or intracellular signaling, protein synthesis and degradation, protein folding, cell adhesion and extracellular matrix, cell structure and motility, metabolism, substance transportation, etc. These findings were supported by many previous studies on myogenic differentiation, of which many up-regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time in our study. In summary, our results provide new clues for understanding the mechanism of myogenesis.

Sendai Virus C Protein Plays a Role in Restricting PKR Activation by Limiting the Generation of Intracellular Double-Stranded RNA

Sendai virus (SeV) C protein is a multifunctional protein that plays important roles in regulating viral genome replication and transcription, antagonizing the host interferon system, suppressing virus-induced apoptosis, and facilitating virus assembly and budding. We here report a novel role of SeV C protein, the limitation of double-stranded RNA (dsRNA) generation for maintaining the rate of protein synthesis in infected cells. It was found that the intracellular protein synthesis rate was maintained even after wild-type (wt) SeV infection, but markedly suppressed following C-knockout SeV infection. This indicates the requirement of C protein for maintaining protein synthesis after infection. In contrast to wt SeV infection, C-knockout SeV infection caused phosphorylation of both the translation initiation factor eIF2alpha and dsRNA-dependent protein kinase (PKR). Phosphorylation of eIF2alpha occurred mainly due to the action of PKR, since knockdown of PKR by small interfering RNA limited eIF2alpha phosphorylation. C protein, however, could inhibit neither poly(I):poly(C)-activated nor Newcastle disease virus-induced phosphorylation of PKR and eIF2alpha, suggesting that C protein does not target common pathways leading to PKR activation. Immunofluorescent staining experiments with a monoclonal antibody specifically recognizing dsRNA revealed generation of a large amount of dsRNA in cells infected with C-knockout SeV but not wt SeV. The dsRNA generation as well as phosphorylation of PKR and eIF2alpha induced by C-knockout SeV was markedly suppressed in cells constitutively expressing C protein. Taken together, these results demonstrate that the SeV C protein limits generation of dsRNA, thereby keeping PKR inactive to maintain intracellular protein synthesis.

Large-scale study of phosphoproteins involved in long-term potentiation in the rat dentate gyrusin vivo

The mechanisms underlying the induction of synaptic plasticity and the formation of long-term memory involve activation of cell-signalling cascades and protein modifications such as phosphorylation and dephosphorylation. Based on a protein candidate strategy, studies have identified several protein kinases and their substrates, which show an altered phosphorylation state during the early phases of long-term potentiation (LTP), yet only a limited number of synaptic phosphoproteins are known to be implicated in LTP. To identify new phosphoproteins associated with LTP, we have undertaken a proteomic study of phosphoproteins at different time points following the induction of LTP in the dentate gyrus in vivo (0, 15 and 90 min). For each time point, proteins from the dentate gyrus were separated by two-dimensional gel electrophoresis and stained with Pro-Q Diamond, a fluorescent stain specific for phosphoproteins. Fourteen proteins whose phosphorylation state varied significantly following LTP were identified using matrix-assisted laser desorption ionization/time of flight mass spectrometry and electrospray ionization-Orbitrap tandem mass spectrometry (MS/MS). They are involved in various cellular functions implicated in synaptic plasticity, such as intracellular signalling, axonal growth, exocytosis, protein synthesis and metabolism. Our results highlight new proteins whose phosphorylation or dephosphorylation is associated with LTP induction or maintenance. Further studies focusing on the regulation of specific phosphorylation sites will lead to greater understanding of the individual implications of these proteins in LTP as well as of their molecular interactions.

Translocon closure to calcium leak in vascular smooth muscle cells

The translocon is an endoplasmic reticulum (ER) membrane protein complex that mediates translocation of nascent polypeptide into the ER. Recent studies have suggested it also acts as a calcium leak channel of the ER (Flourakis et al 2006 FASEB J, 20, 1215–7). Our objective was to investigate the relevance of this hypothesis to proliferating vascular smooth muscle cells. Intracellular calcium and protein synthesis were measured using fura‐2 and 35S‐methionine respectively. Chemical inhibitors of protein synthesis (puromycin, emetine and anisomycin) all act via the translocon and were confirmed as strong inhibitors of protein synthesis in smooth muscle cells after 1 hr of incubation. Puromycin (which holds the translocon open) evoked a transient increase in calcium that was inhibited by pre‐incubation with thapsigargin. Emetine and anisomycin (which close the translocon) inhibited calcium‐release evoked by puromycin, but not the calcium‐release evoked by extracellular ATP or ionomycin. Thapsigargin‐evoked calcium release was slightly reduced by emetine but unaffected by anisomycin. The data suggest the translocon is not a physiological calcium leak channel in vascular smooth muscle cells but that it can enable calcium‐release when acted on by puromycin.The research is supported by the Wellcome Trust and The Egyptian Ministry of Higher Education.

Farnesyltransferase inhibitors inhibit T-cell cytokine production at the posttranscriptional level

AbstractSeveral cytoplasmic proteins, such as GTPases of the Ras family, containing a C-terminal CAAX motif are prenylated by farnesyltransferase to facilitate localization to cellular membranes where activation occurs. Farnesyltransferase inhibitors (FTIs) interfere with this farnesylation process, thereby preventing proper membrane localization and rendering the proteins unavailable for activation. Currently, FTIs are being explored as antineoplastic agents for the treatment of several malignancies. However, since farnesylated proteins like Ras are also involved in intracellular signaling in lymphocytes, FTIs might interfere with T-cell activation. Based on this hypothesis we examined the effect of several FTIs on cytokine production in response to anti-CD3 + anti-CD28 monoclonal antibodies or PMA + ionomycin. Murine Th1 and Th2 clones, stimulated in the presence of FTIs, showed a dose-dependent reduction of lineage-specific cytokine secretion (IFN-γ, IL-2, IL-4, IL-5). However, no inhibition of ERK or JNK MAP kinases was observed, nor was induction of cytokine mRNA affected. Rather, intracellular cytokine protein synthesis was blocked. Inhibition of human T-cell INF-γ production also was observed, correlating with reduced phosphorylation of p70S6K. These results indicate that FTIs inhibit T-cell activation at the posttranscriptional level and also suggest that they may have potential as novel immunosuppressive agents.

Enhanced osteoblast functions on anodized titanium with nanotube‐like structures

Previous studies have demonstrated increased osteoblast (bone-forming cells) adhesion on titanium and Ti-6Al-4V anodized to possess nanometer features compared with their unanodized counterparts. In this study, osteoblast long-term functions (specifically, synthesis of intracellular proteins, synthesis of intracellular collagen, alkaline phosphatase activity, and deposition of calcium-containing minerals) were determined on titanium anodized to possess either heterogeneous nanoparticles or ordered nanotubes. Titanium was anodized in dilute hydrofluoric acid at 20 V for 20 min to possess nanotubes, while titanium was anodized at 10 V for 20 min to possess nanoparticles. Most importantly, results showed that calcium deposition significantly increased on anodized titanium with nanotube-like structures compared with unanodized titanium and anodized titanium with nanoparticulate structures after 21 days of osteoblast culture. In this manner, the results of the present in vitro study indicated that anodization might be a promising quick and inexpensive method to modify the surface of titanium-based implants to induce better bone cell functions important for orthopedic applications.

Role of connective tissue growth factor in extracellular matrix degradation in renal tubular epithelial cells

In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming growth factor beta1 (TGF-beta1) and to explore the role of CTGF in the degradation of renal extracellular matrix (ECM), a human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-beta1 (5 microg/L), the mRNA level of PAI-1 was detected by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-beta1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-beta1 was significantly inhibited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may be a novel way in preventing renal fibrosis.

Function and Structure of the Molybdenum Cofactor Carrier Protein from Chlamydomonas reinhardtii

The molybdenum cofactor (Moco) forms the catalytic site in all eukaryotic molybdenum enzymes and is synthesized by a multistep biosynthetic pathway. The mechanism of transfer, storage, and insertion of Moco into the appropriate apo-enzyme is poorly understood. In Chlamydomonas reinhardtii, a Moco carrier protein (MCP) has been identified and characterized recently. Here we show biochemical evidence that MCP binds Moco as well as the tungstate-substituted form of the cofactor (Wco) with high affinity, whereas molybdopterin, the ultimate cofactor precursor, is not bound. This binding selectivity points to a specific metal-mediated interaction with MCP, which protects Moco and Wco from oxidation with t((1/2)) of 24 and 96 h, respectively. UV-visible spectroscopy showed defined absorption bands at 393, 470, and 570 nm pointing to ene-diothiolate and protein side-chain charge transfer bonds with molybdenum. We have determined the crystal structure of MCP at 1.6 Angstrom resolution using seleno-methionated and native protein. The monomer constitutes a Rossmann fold with two homodimers forming a symmetrical tetramer in solution. Based on conserved surface residues, charge distribution, shape, in silico docking studies, structural comparisons, and identification of an anionbinding site, a prominent surface depression was proposed as a Moco-binding site, which was confirmed by structure-guided mutagenesis coupled to substrate binding studies.

Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro

Connective tissue growth factor (CTGF) has been reported to play an important role in mediating the profibrotic effects of transforming growth factor-beta (TGF-beta) in various renal diseases. To elucidate the role of CTGF in renal tubular epithelial-myofibroblast transdifferentiation, we examined the expression of alpha-smooth muscle actin (alpha-SMA), vimentin, tenascin-C, and collagen IV expression upon the stimulation of CTGF in cultured human proximal tubular epithelial cell line (HKC), and further investigated the effects of endogenous CTGF blockade on the transdifferentiation process induced by TGF-beta. It is revealed that upon the stimulation of recombinant human CTGF (rhCTGF, 2.5 or 5.0 microg/L), the expression of alpha-SMA and tenascin-C mRNA increased significantly (p<0.01), while collagen IV gene expression decreased significantly (p<0.01), all in a dose-dependent manner. The percentage of alpha-SMA-positive cells was significantly larger in the rhCTGF-stimulated groups than that in negative control (38.9%, 65.5% vs. 2.4%, respectively, p<0.01) as confirmed by flow cytometry. Both cytoplasmic and secretory tenascin-C expression was upregulated by the stimulation of rhCTGF (p<0.01). Under this condition, collagen IV secreted into the culture media was lowered markedly (p<0.01). On RT-PCR analysis, TGF-beta1 upregulated CTGF gene expression, preceding that of alpha-SMA. The alpha-SMA mRNA expression induced by TGF-beta1 was significantly inhibited by CTGF antisense oligodeoxynucleotide (ODN) transfection (p<0.01). With prolonged incubation time, CTGF antisense ODN also inhibited intracellular alpha-SMA protein synthesis, as demonstrated by indirect immuno-fluorescence. So it is concluded that CTGF could promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblasts in vitro, both directly and as a downstream mediator of TGF-beta, and CTGF blockade would be a possible therapeutic target against tubulointerstitial fibrosis.

Structural and functional changes in gastric epithelium in Helicobacter pylori-associated chronic gastroduodenal pathologies

Complex structural analysis of the gastric mucosa was carried out in patients with Helicobacter pylori-associated chronic gastroduodenal ulcers, chronic gastritis, and vibration gastropathy. Microscopic examination showed stereotypical changes in the epithelium in all diseases: degeneration, focal intestinal metaplasia, dysplasia, and glandular atrophy. The severity of these changes depended on the disease entity. The most typical ultrastructural modifications of epitheliocytes were damage to the apical plasmalemma, heterogeneity of the secretory compartment of the cytoplasm, dilatation of the cytoplasmic reticulum, vacuolation, and signs of cytolysis. Plastic reactions of the gastric epithelium reflected disproportional changes in all cell metabolites caused by increased proliferative activity of the epithelium under conditions of uneven inhibition of intracellular protein synthesis.

Role of connective tissue growth factor in renal tubular epithelial-myofibroblast transdifferentiation and extracellular matrix accumulation in vitro

Connective tissue growth factor (CTGF) has been reported to play an important role in mediating the profibrotic effects of transforming growth factor-β (TGF-β) in the pathogenesis of renal fibrosis. To further elucidate the role of CTGF in renal tubular transdifferentiation and extracellular matrix (ECM) metabolism, we examined the time-course of CTGF, α-smooth muscle actin (α-SMA), fibronectin and plasminogen activator inhibitor-1(PAI-1) gene expression upon the stimulation of TGF-β1 (5 μg/L) in cultured human proximal tubular epithelial cell line (HKC), and further investigated the effects of endogenous CTGF blockade. On reverse transcriptional-polymerase chain reaction (RT-PCR) analysis, TGF-β1 upregulated CTGF gene expression, preceding that of α-SMA, fibronectin and PAI-1. The α-SMA, fibronectin and PAI-1 mRNA expression induced by TGF-β1 were significantly inhibited by CTGF antisense oligodeoxynucleotide (ODN) transfection. With prolonged incubation time, CTGF antisense ODN also inhibited intracellular α-SMA and PAI-1 protein synthesis, lowered the level of fibronectin and PAI-1 protein secreted into the media, as confirmed by indirect immuno-fluorescence, flow cytometry, enzyme-linked immunosorbent assay (ELISA) and Western blot methods respectively. These results suggested that CTGF may play a crucial role in the renal tubular epithelial-transdifferentiation and the following deposition/degradation process of ECM during tubulointerstitial fibrosis.

Crocetin from Saffron: An Active Component of an Ancient Spice

The known properties of saffron (Crocus sativus, L.) and its components have been examined. Recently, hormone like effects in green algae and the anti-cancerogenic and anti-toxic effects, have been observed. In particular, the effects of crocetin, a carotenoids (8,8′-diapo-8,8′-carotenoic acid) present in saffron and characterized by a diterpenic and symmetrical structure with seven double bonds and four methyl groups, have been taken into consideration. It has been found that this compound enhances the oxygen diffusivity through liquids, such as plasma. As a consequence of this property, it has been observed that crocetin increases alveolar oxygen transport and enhances pulmonary oxygenation. It improves cerebral oxygenation in hemorrhaged rats and positively acts in the atherosclerosis and arthritis treatment. It inhibits skin tumor promotion in mice (i.e., with benzo(a)pyrene); it has an inhibitory effect on intracellular nucleic acid and protein synthesis in malignant cells, as well as on protein-kinase-C and prorooncogene in INNIH/3T3 cells. This is most likely due to its anti-oxidant activity. Furthermore, crocetin protects against oxidative damage in rat primary hepatocytes. It also suppresses aflatoxin B1-induced hepatotoxic lesions and has a modulatory effect on aflatoxin, B1 cytotoxicity, and DNA adduct formation on C3H10/T1/2 fibroblast cells. It also has a protective effect on the bladder toxicity, induced by cyclophosphamide. The experiments reported in the scientific literature and the interesting results obtained have been carried out in vitro or on laboratory animals, but not yet on man.

Interleukin-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors: role of Mcl-1 and decreased activity of caspase-3 and caspase-8.

Interleukin-15 (IL-15) induces the de novo protein synthesis of intracellular polypeptides and delays neutrophil apoptosis by a mechanism that is still unclear. Herein, we investigated the potential antiapoptotic role of newly synthesized proteins released into the external milieu in IL-15-induced neutrophils. We found that IL-15 induces the de novo synthesis of an approximately 23-kDa protein, representing the predominant protein detected in the milieu, and identified it as IL-1 receptor antagonist (IL-1Ra) by Western blot and immunoprecipitation. We quantified IL-1Ra, IL-1alpha, and IL-1beta concentrations by enzyme-linked immunosorbent assay in intracellular and extracellular fractions from IL-15-induced neutrophils and found that IL-15 does not increase IL-1alpha or IL-1beta production but induces IL-1Ra release. Also, we demonstrated that IL-1Ra does not modulate apoptosis, even at a concentration 250 times greater than that measured in the external milieu. In contrast to granulocyte macrophage-colony stimulating factor, the supernatant harvested from IL-15-induced neutrophils was devoid of antiapoptotic activity. Addition of cycloheximide demonstrates that IL-15 delays apoptosis via de novo synthesis of intracellular proteins and that it increases myeloid cell differentiation factor-1 stability. We demonstrated also that IL-15 decreases the activity of caspase-3 and caspase-8, resulting in an inhibition of vimentin cleavage. Our results indicate that IL-15 can activate an anti-inflammatory loop, based on its ability to induce the synthesis of IL-1Ra by neutrophils. We conclude that IL-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors.

Altered responses of chondrocytes to nanophase PLGA/nanophase titania composites

Chondrocyte (cartilage-synthesizing cells) cell density and synthesis of select intracellular proteins by chondrocytes were investigated on novel nanophase poly-lactic/glycolic acid (PLGA) and titania composites in the present in vitro study. Nanophase PLGA films were created by chemically treating conventional (or micron-structured) PLGA films with 10 n NaOH for 1 h. Titania particle dimensions in ceramic compacts were controlled by utilizing either conventional (i.e., micron) or nanometer grain size titania. Composites of either conventional or nanophase PLGA with either conventional or nanophase titania at 70/30 wt% were also created. Compared to surfaces with a conventional or micron topography, results provided the first evidence of stagnant confluent cell densities on nanostructured surfaces at time points between 1 and 7 days. Moreover, compared to surfaces with a conventional topography, increased chondrocyte intracellular synthesis of alkaline phosphatase and chondrocyte expressed protein-68 (proteins that have been correlated with the functions of chondrocytes) were observed on nanophase PLGA/nanophase titania composites. The present study, thus, provided the first evidence of different chondrocyte responses to nanostructured PLGA/nanophase titania composites; in light of other reports demonstrating increased functions of bone cells on the same materials, such data indicates that further investigation of these materials at the bone-cartilage interface should be conducted.