Development of novel α-chitin/nanobioactive glass ceramic composite scaffolds for tissue engineering applications

Bioactive glass ceramic nanoparticles (nBGC) were prepared by sol–gel technique. The novel chitin/nBGC composite scaffolds were prepared using chitin gel with nBGC by lyophilization technique. The prepared nBGC and composite scaffolds were characterized using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Fourier Transformed Infrared Spectroscopy (FT-IR) and X-ray diffraction (XRD). The composite scaffolds showed adequate porosity where the nBGC nanoparticles were homogenously distributed on the pore walls. The swelling, density, degradation and in vitro biomineralization capability of the composite scaffolds were also evaluated. The developed composite scaffolds showed adequate swelling and degradation properties along with its ability to become bioactive. Cytocompatability of the scaffolds was assessed using MTT assay, direct contact test and cell attachment studies. Results indicated no sign of toxicity and cells found to be attached to the pore walls offered by the scaffolds. These results suggested that the developed composite scaffold possess the prerequisites for tissue engineering scaffolds and it can be used for tissue engineering applications.     

Synthesis, characterization and bioactivity studies of novel beta-chitin scaffolds for tissue-engineering applications

Chitin is a biopolymer and it is non-toxic, biodegradable and biocompatible. Chitin has many potential industrial applications because of its abundance, biodegradability, non-toxicity, chemical inertness. beta-Chitin scaffolds were prepared by using saturated calcium chloride alcoholic solution (CaCl(2).6H(2)O/EtOH) and then followed by dialysis with lyophilization. The prepared beta-chitin scaffolds were characterized by FT-IR, scanning electron microscopy (SEM) and thermogravimetric (TGA). The preliminary bioactivity studies of beta-chitin scaffolds were studied by using simulated body fluid (SBF) solution for 7, 14 and 21 days. We also immersed the beta-chitin scaffolds in saturated aqueous CaCl(2) and Na(2)HPO(4) solution over 12h. After 7, 14 and 21 days, the scaffolds were characterized by SEM and FT-IR studies. The SEM studies showed that there is a calcium phosphate layer in the surface as well as in the cross-section of beta-chitin scaffolds. It seems that the beta-chitin scaffolds are useful in the tissue-engineering field.     

Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications

Chitin is a natural biopolymer have been used for several biomedical applications due to its biodegradability and biocompatibility. By using the calcium solvent system, chitin regenerated hydrogel (RG) was prepared by using -chitin. And also, the swelling hydrogel (SG) was prepared by using β-chitin with water. Then, both RG and SG were mixed with gelatin and N-acetyl-d-(+)-glucosamine (GlcNAc) at 120 °C for 2 h. The chitin/gelatin membranes with GlcNAc were also prepared by using RG and SG with GlcNAc. The prepared chitin/gelatin membranes with or without GlcNAc were characterized by mechanical, swelling, enzymatic degradation, thermal and growth of NIH/3T3 fibroblast cell studies. The stress and elongation of chitin/gelatin membrane with GlcNAc prepared from RG was showed higher than the chitin/gelatin membranes without GlcNAc. But, the chitin/gelatin membranes prepared from SG with GlcNAc was showed higher stress and elongation than the chitin/gelatin membranes without GlcNAc. It is due to the crosslinking effect of GlcNAc. The chitin/gelatin membranes prepared from SG showed higher swelling than the chitin/gelatin membranes prepared from RG. In contrast, the chitin/gelatin membranes prepared from RG showed higher degradation than the chitin/gelatin membranes prepared from SG. And also, these chitin/gelatin membranes are showing good growth of NIH/3T3 fibroblast cell. So these novel chitin/gelatin membranes are useful for tissue engineering applications.     

Differential steric effects in the axial ligation of pyridine and imidazole to α- and β-alkylcobinamides

One subclass of B₁₂-requiring enzymes is now known to bind their B₁₂ coenzymes “base-off,” with a histidine residue from the protein supplying an imidazole ligand to the cobalt center. Recent results from Sirovatka and Finke (J.M. Sirovatka and R.G. Finke, J.Am. Chem. Soc. 119, (1997) 3057) show that imidazole has an extraordinary trans effect on the mode of carbon–cobalt bond cleavage in coenzyme B₁₂ analogs, compared to pyridine or the natural 5,6-dimethylbenzimidazole ligand, and it was suggested that a differential steric effect could, in part, account for the uniqueness of the imidazole ligand. Such a differential steric effect for imidazole and pyridine is now demonstrated by studies of the thermodynamics of ligation of these ligands to the α and β diastereomers of two alkylcobinamides (RCbi⁺s, derivatives of cobalamins which lack the normal axial nucleotide) based on the known differences in steric crowding of the α (“lower”) and β (“upper”) axial ligand positions of cobalt corrinoids. Imidazole binds more tightly than pyridine to both diastereomers of NCCH₂Cbi⁺ and CF₃Cbi⁺, in all cases due to a more favorable entropy change, which is the result of lowered steric interference with corrin side chain thermal motions.     

Phosphorylations of αA- and αB-crystallin

In addition to being refractive proteins in the vertebrate lens, the two α-crystallin polypeptides (αA and αB) are also molecular chaperones that can protect proteins from thermal aggregation. The αB-crystallin polypeptide, a functional member of the small heat shock family, is expressed in many tissues in a developmentally regulated fashion, is stress-inducible, and is overexpressed in many degenerative diseases and some tumors indicating that it plays multiple roles. One possible clue to α-crystallin functions is the fact that both polypeptides are phosphorylated on serine residues by cAMP-dependent and cAMP-independent mechanisms. The cAMP-independent pathway is an autophosphorylation that has been demonstrated in vitro, depends on magnesium and requires cleavage of ATP. Disaggregation of αA-, but not αB-crystallin into tetramers results in an appreciable increase in autophosphorylation activity, reminiscent of other heat shock proteins, and suggests the possibility that changes in the aggregation state of αA-crystallin are involved in yet undiscovered signal transduction pathways. The α-crystallin polypeptides differ with respect to their abilities to undergo cAMP-dependent phosphorylation, with preference given to the αB-crystallin chain. These differences and complexities in α-crystallin phosphorylations, coupled with the differences in expression patterns of the two α-crystallin polypeptides, are consistent with the idea that each polypeptide has distinctive structural and metabolic roles.     

Cardiovascular actions of prostaglandins F2α; 15-me-F2α; F1α; F2β and F1β in the cat

Prostaglandin F_2α (5μg/kg, i.v.) causes an increase in pulmonary arterial pressure, decrease in systemic arterial pressure, and reflex bradycardia in the anesthetized cat. The same dose of the 15-methyl analogue of PGF_2α produces the same triad of effects but of greater magnitude and duration. Although prostaglandins F_1α, F_2β and F_1β also cause the same cardiovascular effects as F_2α, there is a decrease in potency for all parameters measured, with PGF_2α>PGF_1α>PGF_2β>PGF_1β. When compared to the actions of PGF_2α in producing an increase in pulmonary arterial pressure, PGs F_1α, F_2β and F_1β were less potent by approximately 10, 100, and 1000 fold respectively.     

Separation and concentration of Δ-6-keto-PGF1α using monoclonal antibody to ω3-olefin structure of trienoic prostanoids

A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate Δ¹⁷-6-keto-prostaglandin F_1α (PGF_1α) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal afntibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for ω3-olefin structure. The 4G9-12B antibody became more specific for Δ¹⁷-6-keto-PGF_1α than 6-keto-PGF_1α by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, Δ¹⁷-6-keto-PGF_1α was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, ω3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating Δ¹⁷-6-keto-PGF_1α in the human blood or urine.     

α3β1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins

Laminin-5 and α3β1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/α3β1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 α3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes, which is initiated by α3β1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3ζ and σ, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3ζ with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes is initiated by laminin-5 stimulation via the α3β1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.     

Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.     

The contribution of β1 integrins to neuronal migration and differentiation depends on extracellular matrix molecules

The interaction of β1 integrin receptors and different extracellular matrix molecules during neuronal development was investigated by comparing both migration and morphological differentiation of D3 wild-type embryonic stem (ES) cell line-derived neural precursor cells with those of the β1 integrin knockout ES cell line G201. Analysing neurosphere explants on laminin and fibronectin as major β1 integrin ligands, the maximal spreading of outward migrating neuronal cells was determined. Compared with gelatine as a standard substrate, migration was found to be significantly increased for D3-derived neurospheres on fibronectin and laminin-1. These matrix effects were found to be even enhanced for G201 preparations. In addition, also the differentiation of wild-type and β1 integrin −/− neurones – as determined by MAP-2- and HNK-1-immunoreactive processes – was found to be increased on fibronectin and laminin when compared to gelatine standards. In the respective knockout preparations on these matrices, again perturbation effects were less pronounced than on gelatine. Our observations indicate that laminin and fibronectin are involved both in β1 integrin-dependent and -independent signalling mechanisms during neurogenesis. Upregulation of compensatory mechanisms such as β1 integrin-independent receptors for laminin and fibronectin might be responsible for the much less pronounced perturbations of G201 neural precursor migration and differentiation on these two substrates than on gelatine.     

Chemical, structural and biological studies of cis-[Pt(3-Acpy)2Cl2]

Recent developments in the field of platinum anticancer drugs have revealed that compounds containing derivates of pyridine may exhibit highly cytotoxic activity against a variety of tumor cells, with AMD473 (cis-PtCl₂(NH₃)(2-methylpyridine)) as one of the most relevant examples. Following these advances, this paper describes the synthesis, characterization and X-ray structure of the square-planar compound cis-[Pt(3-Acpy)₂Cl₂] (1, Acpy stands for acetylpyridine), where the coordination of 3-acetylpyridine takes places through the pyridine nitrogen of the ligand. The structural arrangement of this compound is highly peculiar and it is the first example with two of these 3-acetylpyridine molecules in a cis disposition. In addition, the anticancer and antibacterial activities of this compound together with studies of DNA binding are also described in detail, with selective activity of compound 1 against A2780R cells. cis-[Pt(3-Acpy)₂Cl₂] apparently coordinates to the DNA double helix upon exchange of at least one of the Cl⁻ ions with the media and shows very interesting bacteriolytic and bacteriostatic activity against Escherichia coli and Streptomyces, respectively.     

Genomic organization of four β-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications

The genomic organization of genes encoding β-1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated. HG-eng1 and GR-eng1 both contained eight introns and structural domains of 2151 and 2492 bp, respectively. HG-eng2 and GR-eng2 both contained seven introns and structural domains of 2324 and 2388 bp, respectively. No significant similarity in intron sequence or size was observed between HG-eng1 and HG-eng2, whereas the opposite was true between GR-eng1 and GR-eng2. Intron positions among all four cyst nematode cellulase genes were conserved identically in relation to the predicted amino acid sequence. HG-eng1, GR-eng1, and GR-eng2 had several introns demarcated by 5′-GC…AG-3′ in the splice sites, and all four nematode cellulase genes had the polyadenylation and cleavage signal sequence 5′-GAUAAA-3′—both rare occurences in eukaryotic genes. The 5′- flanking regions of each nematode cellulase gene, however, had signature sequences typical of eukaryotic promoter regions, including a TATA box, bHLH-type binding sites, and putative silencer, repressor, and enhancer elements. Database searches and subsequent phylogenetic comparison of the catalytic domain of the nematode cellulases placed the nematode genes in one group, with Family 5, subfamily 2, glycosyl hydrolases from Scotobacteria and Bacilliaceae as the most homologous groups. The overall amino acid sequence identity among the four nematode cellulases was from 71 to 83%, and the amino acid sequence identity to bacterial Family 5 cellulases ranged from 33 to 44%. The eukaryotic organization of the four cyst nematode cellulases suggests that they share a common ancestor, and their strong homology to prokaryotic glycosyl hydrolases may be indicative of an ancient horizontal gene transfer.     

α-Latrotoxin stimulates glutamate release from cortical astrocytes in cell culture

The mechanism responsible for the ability of bradykinin to cause calcium-dependent release of glutamate from astrocytes in vitro was investigated. The glutamate transport inhibitor, dihydrokainate, did not block bradykinin-induced glutamate release, and bradykinin did not cause cell swelling. These data exclude the involvement of glutamate transporters or swelling mechanisms as mediating glutamate release in response to bradykinin. α-Latrotoxin (3 nM), a component of black widow spider venom, stimulated calcium-independent glutamate release from astrocytes. Since α-latrotoxin induces vesicle fusion and calcium-independent neuronal neurotransmitter release, our data suggest that astrocytes may release neurotransmitter using a mechanism similar to the neuronal secretory process.     

Increase of isoprostane 8-epi-PGF2 αafter restarting smoking

Isoprostanes are known as reliable markers of in vivo oxidation injury. Cigarette smoking has been shown to be associated with a significant increase in 8-epi-PGF(2alpha), a major member of this family of compounds. Quitting smoking reduces 8-epi-PGF(2alpha) values to normal within a couple of weeks only. In this follow-up we checked the 8-epi-PGF(2alpha), values in plasma, serum and urine in 28 people who restarted smoking after a quitting attempt of various duration. 8-epi-PGF(2alpha)shows a certain increase after restarting smoking reaching a maximum after already 1 week. Continuation of smoking does not significantly further increase 8-epi-PGF(2alpha). These data indicate a fast response of restarting as on quitting smoking on in vivo oxidation injury. The oxidation injury reflected by 8-epi-PGF(2alpha)may be a key pathogenetic mechanism in smoking-induced vascular injury.     

Structural perturbation of α-crystallin and its chaperone-like activity

α-Crystallin is a multimeric lenticular protein that has recently been shown to be expressed in several non-lenticular tissues as well. It is shown to prevent aggregation of non-native proteins as a molecular chaperone. By using a non-thermal aggregation model, we could show that this process is temperature-dependent. We investigated the chaperone-like activity of α-crystallin towards photo-induced aggregation of γ-crystallin, aggregation of insulin and on the refolding induced aggregation of β- and γ-crystallins. We observed that α-crystallin could prevent photo-aggregation of γ-crystallin and this chaperone-like activity of α-crystallin is enhanced several fold at temperatures above 30°C. This enhancement parallels the exposure of its hydrophobic surfaces as a function of temperature, probed using hydrophobic fluorescent probes such as pyrene and 8-anilinonaphthalene-1-sulfonate. We, therefore, concluded that α-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30°C involving enhanced or re-organized hydrophobic surfaces of α-crystallin is important for its chaperone-like activity. We also addressed the issue of conformational aspects of target proteins and found that their aggregation prone molten globule states bind to α-crystallin. We trace these developments and discuss some new lines that suggest the role of tertiary structural aspects in the chaperone process.     

β-secretase (BACE1)-inhibiting stilbenoids from Smilax Rhizoma

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of Smilax Rhizoma (the dried rhizomes of Smilax china L.) showed potent inhibitory activity. The active compounds were identified as a trans/cis-resveratrol mixture, oxyresveratrol, veraphenol, and cis-scirpusin A. They were shown to non-competitively inhibit BACE1 with the Ki values of 5.4 x 10(-6), 5.4 x 10(-6), 3.4 x 10(-6), and 5.4 x 10(-6)M and IC(50) values of 1.5 x 10(-5), 7.6 x 10(-6), 4.2 x 10(-6), and 1.0 x 10(-5)M, respectively. The active compounds were less inhibitory to alpha-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, suggesting that they were relatively specific inhibitors of BACE1.     

Integrins and the activation of latent transforming growth factor β1 – An intimate relationship

Integrins are crucial for the ability of cells to sense mechanical perturbations and to transmit intracellular stress to their environment. We here review the more recently discovered role of integrins in activating the pleiotrophic cytokine transforming growth factor beta 1 (TGF-β1). TGF-β1 controls tissue homeostasis in embryonic and normal adult tissues and contributes to the development of fibrosis, cancer, autoimmune and vascular diseases when being mis-regulated. In most of these conditions, active TGF-β1 is generated by dissociation from a large latent protein complex that sequesters latent TGF-β1 in the extracellular matrix (ECM). Two main models are proposed how integrins contribute to latent TGF-β1 activation: (1) In a protease-dependent mechanism, integrins αvβ8 and αvβ3 are suggested to simultaneously bind the latent TGF-β1 complex and proteinases. This close vicinity at the cell surface improves enzymatic cleavage of the latent complex to release active TGF-β1. (2) Integrins αvβ3, αvβ5, αvβ6, and αvβ8 appear to change the conformation of the latent TGF-β1 complex by transmitting cell traction forces. This action requires association of the latent complex with a mechanically resistant ECM and is independent from proteolysis. Understanding that different integrins use different mechanisms to activate latent TGF-β1 opens new possibilities to develop cell-specific therapeutic strategies for TGF-β1-induced pathologies.     

Enhanced oxidizability of ubiquinol and α-tocopherol during lovastatin treatment

A double-blinded, placebo-controlled cross-over trial was carried out with 27 hypercholesterolemic men with coronary heart disease. During the 6-week treatment period lovastatin (60 mg/day) decreased fasting serum LDL cholesterol by 45%, LDL phosphorus by 38% and apoB by 33%. Ubiquinol content diminished by 13% as measured per LDL phosphorus. When LDL was oxidized ex vivo with AMVN both LDL ubiquinol and α-tocopherol were exhausted faster after lovastatin treatment compared to placebo, by 24% (P<0.005) and 36% (P<0.0001), respectively. Lag time in copper-induced oxidation of LDL decreased by 7% (P<0.01). This suggests diminished antioxidant-dependent resistance of LDL to the early phase of oxidative stress.     

Transformation of 3-hydroxy-steroids by Fusarium moniliforme 7α-hydroxylase

Transformation of physiologically important 3-hydroxy-steroids by the DHEA-induced 7α-hydroxylase of F. moniliforme was investigated. Whereas DHEA was almost totally 7α-hydroxylated, PREG, EPIA and ESTR were only partially converted into their 7α-hydroxylated derivatives because hydroxylation at other undetermined positions as well as reduction of ketone at C17 or C20 into hydroxyl also occurred. Cholesterol was not transformed by the enzyme. Kinetic parameters of the 7α-hydroxylation for these substrates were determined and confirmed that DHEA was the best substrate of the 7α-hydroxylase. Inhibition studies of DHEA 7α-hydroxylation by the other 3-hydroxy-steroids were also carried out and proved that DHEA, PREG, EPIA and ESTR shared the same active site of the enzyme. Induction effects of these steroids were compared, and DHEA appeared to be the best inducer of the 7α-hydroxylase of F. moniliforme.