Characterization of subpopulated articular chondrocytes separated by Percoll density gradient

The aim of this study was to develop a method for fractionation of articular chondrocytes from the entire thickness of the tissue. Isolated chondrocytes from rabbit articular cartilage fractionated by centrifugation in a discontinuous Percoll gradient resulted in four cell fractions with two differing properties. The lowest-density fraction consisted mainly of large cells with small nuclei proliferated actively, maintained the chondrocytic phenotype, and secreted larger amounts of proteoglycan. In contrast, the highest-density fraction consisted of small cells with large nuclei proliferated slowly, did not express the chondrocytic phenotype, and produced larger amounts of interleukin 1-induced nitric oxide. Comparing our results with other previous reports, we find that fraction 1 cells are likely originated from the deep layer of the articular cartilage, whereas fraction 4 cells are tentatively categorized as chondrocytes from the superficial layer of cartilage. Centrifugal fractionation of articular chondrocytes via Percoll density gradient permits clear separation of these heterogeneous cells into different phenotypic populations and allows distinguishing of cells from the different layers of articular cartilage. This simple novel method will provide ready separation of articular chondrocytes for the investigation of the pathogenesis of articular cartilage.     

Response of recombinant Chinese hamster ovary cells to hyperosmotic pressure: effect of Bcl-2 overexpression

In an attempt to use the hyperosmotic pressure for improved foreign protein production in recombinant Chinese hamster ovary (rCHO) cells, the response of rCHO cells producing a humanized antibody (SH2-0.32-(Delta)bcl-2 cells) to hyperosmotic pressure was determined in regard to cell growth and death, and antibody production. Further, the feasibility of Bcl-2 overexpression in improving rCHO cell viability under hyperosmotic pressure was also determined by comparing control cells (SH2-0.32-(Delta)bcl-2) with Bcl-2 overexpressing cells (14C6-bcl-2). After 3 days of cultivation in the standard medium (294 mOsm x kg(-1)), the spent medium was exchanged with the fresh media with various osmolalities (294-640 mOsm x kg(-1)). The results obtained show that hyperosmotic pressure inhibited cell growth in a dose-dependent manner, though 14C6-bcl-2 cells were less susceptible to hyperosmotic pressure than SH2-0.32-(Delta)bcl-2 cells. At 522 mOsm x kg(-1), SH2-0.32-(Delta)bcl-2 cells underwent a gradual cell death mainly through apoptosis due to the cytotoxic effect of hyperosmotic pressure. In contrast, Bcl-2 overexpression in 14C6-bcl-2 cells could delay the apoptosis induced by 522 mOsm x kg(-1) by inhibiting caspase-3 activation. Bcl-2 overexpression could also improve the cellular membrane integrity of 14C6-bcl-2 cells. When subjected to hyperosmotic pressure, the specific antibody productivity of SH2-0.32-(Delta)bcl-2 cells and 14C6-bcl-2 cells was increased in a similar extent. As a result, the final antibody concentration achieved in 14C6-bcl-2 cells at 522 mOsm x kg(-1) was 2.5-fold higher than that at 294 mOsm x kg(-1). At 580 mOsm x kg(-1), acute hyperosmotic pressure induced the rapid loss of viability in both SH2-0.32-(Delta)bcl-2 and 14C6-bcl-2 cells through necrosis rather than through apoptosis. Taken together, Bcl-2 overexpression and optimized hyperosmotic pressure could improve the antibody production of rCHO cells.     

Iron activates NF-kappaB in Kupffer cells

Iron exacerbates various types of liver injury in which nuclear factor (NF)-kappaB-driven genes are implicated. This study tested a hypothesis that iron directly elicits the signaling required for activation of NF-kappaB and stimulation of tumor necrosis factor (TNF)-alpha gene expression in Kupffer cells. Addition of Fe2+ but not Fe3+ (approximately 5-50 microM) to cultured rat Kupffer cells increased TNF-alpha release and TNF-alpha promoter activity in a NF-kappaB-dependent manner. Cu+ but not Cu2+ stimulated TNF-alpha protein release and promoter activity but with less potency. Fe2+ caused a disappearance of the cytosolic inhibitor kappaBalpha, a concomitant increase in nuclear p65 protein, and increased DNA binding of p50/p50 and p65/p50 without affecting activator protein-1 binding. Addition of Fe2+ to the cells resulted in an increase in electron paramagnetic resonance-detectable.OH peaking at 15 min, preceding activation of NF-kappaB but coinciding with activation of inhibitor kappaB kinase (IKK) but not c-Jun NH2-terminal kinase. In conclusion, Fe2+ serves as a direct agonist to activate IKK, NF-kappaB, and TNF-alpha promoter activity and to induce the release of TNF-alpha protein by cultured Kupffer cells in a redox status-dependent manner. We propose that this finding offers a molecular basis for iron-mediated accentuation of TNF-alpha-dependent liver injury.     

Relating repair susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic stability

A key issue in the nucleotide excision repair (NER) of bulky carcinogen–DNA adducts is the ability of the NER machinery to recognize and repair certain adducts while failing to repair others. Unrepaired adducts can survive to cause mutations that initiate the carcinogenic process. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region polycyclic aromatic hydrocarbon, can be metabolically activated to the enantiomeric benzo[c]phenanthrene diol epoxides (B[c]PhDEs), (+)-(1S,2R,3R,4S)-3,4- dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phe nanthrene and the corresponding (–)-(1R,2S,3S,4R) isomer. These react predominantly with adenine residues in DNA to produce the stereoisomeric 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts. Duplexes containing the 1R (+) or 1S (–) B[c]Ph-dA adduct in codon 61 of the human N-ras mutational hotspot sequence CA*A, with B[c]Ph modification at A*, are not repaired by the human NER system. However, the analogous stereoisomeric DNA adducts of the bay region benzo[a]pyrene diol epoxide (B[a]PDE), 10S (+)- and 10R (–)-trans-anti-B[a]P-N⁶-dA, are repaired in the same base sequence. In order to elucidate structural and thermodynamic origins of this phenomenon, we have carried out a 2 ns molecular dynamics simulation for the 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts in an 11mer duplex containing the human N-ras codon 61 sequence, and compared these results with our previous study of the B[a]P-dA adducts in the same sequence. The molecular mechanics Poisson– Boltzmann surface area (MM-PBSA) method was applied to calculate the free energies of the pair of stereoisomeric B[c]Ph-dA adducts, and a detailed structural analysis was carried out. The different repair susceptibilities of the B[a]P-dA adducts and the B[c]Ph-dA adducts can be attributed to different degrees of distortion, stemming from combined effects of differences in the quality of Watson–Crick hydrogen bonding, unwinding, stretching and helix backbone perturbations. These differences are due to the different intrinsic topologies of the rigid, planar bay region adducts versus the twisted, sterically hindered fjord region adducts.     

Preliminary crystallographic studies of two C-terminally truncated copper-containing nitrite reductases from Achromobacter cycloclastes: changed crystallizing behaviors caused by residue deletion

The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR. C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion. In this study, the two mutants were crystallized using the hanging drop vapor diffusion method. Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2). NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees. The crystallizing behaviors of the two mutants differed from that of the native enzyme. Such change in combination with residue deletion is also discussed here.     

Activity enhancement of Cel5Z from Pectobacterium chrysanthemi PY35 by removing C-terminal region

The phytopathogenic bacterium Pectobacteium chrysanthemi PY35 secretes Cel5Z endoglucanase belonging to the glycoside hydrolase family 5 of EC 3.2.1.4. The mutation of cel5Z::Omega gene was constructed by cloning the 2.0-kb SmaI fragment containing the streptomycin/spectinomycin-resistance gene of pHP45(Omega) into the BalI site of pPY100. The insertion of Omega fragment generated a new stop codon, removing the Ser/Thr-rich linker region and the cellulose binding domain (CBD) in the C-terminal region of cel5Z gene. By subsequent subcloning from this 4.9-kb fragment (pPY1001), a 1.0-kb (pPY1002) fragment was obtained and designated as cel5Z::Omega. The cel5Z::Omega gene had an open reading frame (ORF) of 1011 bp, encoding 336 amino acids, starting with an ATG codon and ending with a new TGA stop codon. The molecular mass of the Cel5Z::Omega protein in E. coli transformant appeared to be 32 kDa by SDS-PAGE analysis in the presence of carboxymethyl-cellulose (CMC). The Cel5Z::Omega protein hydrolyzed CMC with 1.7-fold higher activity than the intact Cel5Z cellulase.     

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.     

Structural analysis of an insect lysozyme exhibiting catalytic efficiency at low temperatures

Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.     

Regulation of WNK1 by an autoinhibitory domain and autophosphorylation

WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity.