Differential expression of RANK,RANK-L,and osteoprotegerin by synovial fluid neutrophils from patients with rheumatoid arthritis and by healthy human blood neutrophils

Functional links between bone remodeling and the immune system in chronic inflammatory arthritis are mediated, in part, by the ligand of receptor activator of nuclear factor-kappa-B (RANK-L). Because neutrophils play a crucial role in chronic inflammation, the goal of this study was to determine whether proteins of the RANK/RANK-L pathway are expressed by synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA) and to characterize this pathway in normal human blood neutrophils. The expression of RANK-L, osteoprotegerin (OPG), RANK, and tumor necrosis factor receptor-associated factor 6 (TRAF6) was determined by polymerase chain reaction, enzyme-linked immunosorbent assay, Western blotting, and cytofluorometry. RANK signaling was analyzed by the degradation of inhibitor of kappaB-alpha (I-κB-α). SF neutrophils from patients with RA express and release OPG and express the membrane-associated forms of RANK-L and RANK. In contrast, normal blood neutrophils express only the membrane-associated form of RANK-L. They do not express the mRNAs encoding OPG and RANK. SF neutrophils from RA patients and normal blood neutrophils release no soluble RANK-L. They express the mRNA for TRAF6. The expression of OPG and RANK by normal human blood neutrophils, however, can be induced by interleukin-4 + tumor necrosis factor-alpha and by SFs from patients with RA. In contrast, SFs from patients with osteoarthritis do not induce the expression of OPG and RANK. Moreover, the addition of RANK-L to normal blood neutrophils pretreated by SF from patients with RA decreased I-κB-α, indicating that RANK signaling by neutrophils stimulated with SF is associated with nuclear factor-kappa-B activation. In summary, RANK-L is expressed by inflammatory and normal neutrophils, unlike OPG and RANK, which are expressed only by neutrophils exposed to an inflammatory environment. Taken together, these results suggest that neutrophils may contribute to bone remodeling at inflammatory sites where they are present in significantly large numbers.     

Clonal response to cold tolerance in creeping bentgrass and role of proline-associated pentose phosphate pathway

Single seed origin creeping bentgrass (‘Penncross’) clonal lines were screened to find genetic heterogeneity, which reflected diversity of phenolic production linked to cold stress within a cross-pollinated cultivar. In this study, total soluble phenolic and antioxidant activity varied among 20 creeping bentgrass clonal lines, confirming wide heterogeneity in this cross-pollinated species. Correlations between phenolic content and proline-associated pentose phosphate pathway were also found among the clonal lines. The active metabolic role of proline in cellular metabolic adjustment to cold stress and its support for likely energy synthesis via mitochondrial oxidative phosphorylation was inferred in creeping bentgrass clonal lines based on the activity of proline dehydrogenase. Results of photochemical efficiency of these clonal lines after cold temperature treatment (4 °C) also indicated a close association between stress tolerance and proline-associated pentose phosphate pathway regulation for phenolic biosynthesis and antioxidant response. This study provides a sound metabolic based rationale to screen bentgrass clonal lines for enhanced cold stress tolerance.     

The Mycobacteriophage D29 Gene 65 Encodes an Early-Expressed Protein That Functions as a Structure-Specific Nuclease

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to DNA synthesis. One such gene is 65, which encodes a protein belonging to the RecA/DnaB helicase superfamily. In this study a recombinant version of the mycobacteriophage D29 gp65 was functionally characterized. The results indicated that it is not a helicase as predicted but an exonuclease that removes 3′ arms from forked structures in an ATP-dependent manner. The gp65 exonuclease acts progressively from the 3′ end, until the fork junction is reached. As it goes past, its progress is stalled over a stretch of seven to eight nucleotides immediately downstream of the junction. It efficiently acts on forked structures with single stranded arms. It also acts upon 5′ and 3′ flaps, though with somewhat relaxed specificity, but not on double-stranded forks. Sequence comparison revealed the presence of a KNRXG motif in the C-terminal half of the protein. This is a conserved element found in the RadA/Sms family of DNA repair proteins. A mutation (R203G) in this motif led to complete loss of nuclease activity. This indicated that KNRXG plays an important role in the nuclease function of not only gp65, but possibly other RadA/Sms family proteins as well. This is the first characterization of a bacteriophage-derived RadA/Sms class protein. Given its mode of action, it is very likely that gp65 is involved in processing branched replication intermediates formed during the replication of phage DNA.Fork structures are intricately associated with DNA replication. Such structures result due to unwinding of the DNA ahead of the replicating machinery. The unwound single strands are then used as templates for the synthesis of the new strands, either continuously (leading strand) or discontinuously (lagging strand). Repair of stalled forks involve complex mechanisms which may vary from one organism to another (5). However, in most cases the process requires nucleases that recognize stalled fork structures and cleave them specifically. Such nucleases are generally referred to as structure-specific nucleases (25). One such nuclease named FEN1 found in eukaryotes has been studied fairly extensively, and it is believed that this nuclease is involved in the removal of 5′ flaps from Okazaki fragments (11, 23). FEN1 belongs to a larger family of structure-specific nucleases, which includes human XPG (17), an endonuclease related to the disease xeroderma pigmentosa. Although the XPG family is associated with the removal of 5′ flaps the XPF type proteins are needed for removing the 3′ flaps (3). Similar proteins have been found in several Archaea (28). In Escherichia coli, the Holliday junction resolving enzyme system RuvABC is believed to be involved in resolving stalled forks by creating double-stranded breaks, which may be repaired through homologous recombination (29). Studies in E. coli have revealed that there are multiple redundant pathways that are capable of repairing stalled forks. One such pathway involves a protein named RadA/Sms, the absence of which results in partial increase in sensitivity to radiation in E. coli (2). Genes encoding RadA/Sms family proteins are present in many bacteria, including mycobacteria. Most of these members carry a conserved element KNRFG. It is believed (2) that RadA/Sms family of proteins may generate double-stranded breaks at fork junctions, although this has not been specifically demonstrated.Mycobacteriophages of the L5 family, which includes D29, BxB1, may be either temperate or potentially temperate (D29) (14, 15, 27). Despite their temperate character these phages share a strong resemblance with lytic phages. An important feature shared by lytic phages in general is their ability to synthesize DNA using phage-encoded DNA polymerases (13). They also possess many genes linked to nucleotide metabolism. It appears that as far as DNA replication is concerned, lytic phages prefer to be self-sufficient. This is apparently an important issue since lytic phages inactivate their host and therefore host-specific functions cannot be used to support phage growth.Following the availability of the genome sequence, many interesting aspects of mycobacteriophages have come to light. The central region of mycobacteriophage L5/D29 genome has been predicted to harbor several genes whose products may contribute directly or indirectly toward synthesis of new DNA strands. In a recent investigation from this laboratory it has been demonstrated (4) that at least some of the genes in this region are involved in the production of deoxyribonucleotide precursors which are probably needed at increased levels during phage replication. Apart from these genes there are several others which probably encode DNA polymerization related functions. One such gene that drew our interest was gene 65, which appears to encode a RecA/DnaB helicase superclass protein (22). The N-terminal region of this protein contains the Walker motifs A and B, which are characteristically present in the members of the RecA/DnaB superfamily. Walker motifs A and B (30) are found in proteins that hydrolyze ATP for executing their respective functions. To investigate the possible function of gp65, its gene was overexpressed in E. coli, and the recombinant protein was purified. Assays performed with the recombinant gp65 revealed that it is a structure-specific nuclease that acts exonucleolytically on fork structures, resulting in truncated forms lacking the 3′ arm. This function was demonstrated to require a particular motif KNRXG that is omnipresent in the RadA/Sms family of proteins (2). This characterization of D29 gp65 could give us better insight into how mycobacteriophages replicate their DNA within their hosts.     

Natural antioxidants synergistically enhance the anticancer potential of AP9-cd, a novel lignan composition from Cedrus deodara in human leukemia HL-60 cells

Antioxidants have been used as adjuvant with anticancer therapy to synergize the potential of the anti-neoplastic therapeutics. Based on the fact, we have studied the effect of three natural antioxidants curcumin, silymarin and acteoside on AP9-cd (standardized lignan composition from Cedrus deodara) induced cytotoxicity in human leukemia HL-60 cells. The antioxidant potential of individual test compounds was first evaluated with ferric reducing antioxidant power (FRAP) test, which revealed that all four molecules behave as antioxidants. The apoptotic potential of AP9-cd was significantly enhanced in HL-60 cells in the presence of curcumin, silymarin and acteoside. It was confirmed by using various models like MTT assay, DNA fragmentation, nuclei condensation, sub-Go DNA population, Annexin-V-FITC binding, ROS depletion and immunoblotting in HL-60 cells. AP9-cd and individual antioxidants alone at low doses (10μg and 10μM, respectively) have meager or no cytotoxicity in HL-60 cells, whereas in mutual combinations, there were 2-3 times enhancement in Annexin-V-FITC and sub-Go DNA population. Moreover, prominent DNA ladders were observed at low doses of AP9-cd in combinations with various antioxidants. The Hoechst staining of the nucleus also revealed the same results for the HL-60 cells treated with AP9-cd and different antioxidants. The molecular diagnostics revealed that the combinations induced a strong antioxidant effect which was correlated with the downregulation of NF-κB expression in the nucleus. Out of the three antioxidants, curcumin was found to be more potent than acteoside and silymarin in terms of enhancing the apoptotic potential of AP9-cd. These results propose an important role of natural antioxidant as adjuvant to enhance the anticancer potential of AP9-cd and more likely other anti-neoplastic therapeutics.     

PKC-δ controls the fMLF-induced overproduction of superoxide by neutrophils

Antimicrobial defense by neutrophils implicates the production of reactive oxygen species. Neutrophil responses can be modulated by agonists such as bacterial peptides and proinflammatory factors that modulate neutrophil activity and survival. We evaluated the production of superoxide anions (O₂^?) in response to fMLF by normal human neutrophils after 3 days of preincubation with GM-CSF + IL-4 + TNF-α (survival medium). After 3 days of incubation in survival medium, long-lived neutrophils produced up to six times more O₂^? relative to control neutrophils in response to fMLF and WKYMVM. This augmented response to fMLF was preferentially linked to formyl peptide receptor (FPR), whereas the response to WKYMVM was dependent on formyl peptide receptor-like 1 (FPRL-1). Real-time RT-PCR revealed a diminution of FPR and FPRL-1 expression in neutrophils incubated in survival medium. fMLF-induced overproduction of O₂^? by long-lived neutrophils was independent of intracellular calcium mobilization. The protein tyrosine phosphorylation profile of long-lived neutrophils was modified with respect to control cells. Pharmacological inhibitors of intracellular signals indicated that mechanisms of the excessive fMLF-induced production of O₂^? by long-lived neutrophils implicated the protein kinase C (PKC) pathway, preferentially the PKC-δ isoform, whose protein was augmented in these cells. Thus, long-term cytokine exposure modifies molecular pathways and functional characteristics of the neutrophil.     

β-Galactosidase of Pediococcus species: induction,purification and partial characterization

Summary Six strains of Pediococcus pentosaceus and two of P. acidilactici had intracellular -galactosidase (-gal) activity when grown in the presence of lactose; all but two strains of P. pentosaceus and one of P. acidilactici had such activity when grown in the presence of glucose. Synthesis of -gal by P. pentosaceus ATCC 25745 was inducible with lactose, galactose, melibiose, lactobionic acid and possibly cellobiose but not with glucose, sucrose, maltose, glycerol, fructose or mannose. Lactose, galactose and possibly maltose, melibiose and lactobionic acid but not glucose, sucrose, glycerol, cellobiose, fructose or mannose induced -gal synthesis by P. acidilactici ATCC 25740. Synthesis of -gal was partially inhibited in P. pentosaceus ATCC 25745 and P. acidilactici ATCC 25740 by glucose added to the medium during growth in the presence of galactose or lactose. Isopropyl -d-thiogalactopyranoside failed to induce synthesis of -gal by either strain during growth on glucose. -Gal from P. pentosaceus ATCC 25745 had a molecular weight of 66,000 and activity optima of pH 6.5 and 45° C. Activity of the enzyme was stimulated by reducing agents, Mg²⁺, Mn²⁺, Zn²⁺ and Co²⁺ but not by Ca²⁺, and was markedly inhibited by ethylenediaminetetraacetate (EDTA), HgCl₂, 1,10-phenanthroline, and an oxidizing agent. The K _mvalues of the enzyme for o-nitrophenol--d-galactopyranoside and lactose were 3.07 and 7.0 mM, respectively, suggesting its low affinity for lactose. Offprint requests to: E. H. Marth     

Rational mutagenesis of Thermobifida fusca cutinase to modulate the enzymatic degradation of polyethylene terephthalate

Thermobifida fusca cutinase (TfCut2) is a carboxylesterase (CE) which degrades polyethylene terephthalate (PET) as well as its degradation intermediates [such as oligoethylene terephthalate (OET), or bis-/mono-hydroxyethyl terephthalate (BHET/MHET)] into terephthalic acid (TPA). Comparisons of the surfaces of certain CEs (including TfCut2) were combined with docking and molecular dynamics simulations involving 2HE-(MHET)_3, a three-terephthalate OET, to support the rational design of 22 variants with potential for improved generation of TPA from PET, comprising 15 single mutants (D12L, E47F, G62A, L90A, L90F, H129W, W155F, ΔV164, A173C, H184A, H184S, F209S, F209I, F249A, and F249R), 6 double mutants [H129W/T136S, A173C/A206C, A173C/A210C, G62A/L90F, G62A/F209I, and G62A/F249R], and 1 triple mutant [G62A/F209I/F249R]. Of these, nine displayed no activity, three displayed decreased activity, three displayed comparable activity, and seven displayed increased (~1.3- to ~7.2-fold) activity against solid PET, while all variants displayed activity against BHET. Of the variants that displayed increased activity against PET, four displayed more activity than G62A, the most-active mutant of TfCut2 known till date. Of these four, three displayed even more activity than LCC (G62A/F209I, G62A/F249R, and G62A/F209I/F249R), a CE known to be ~5-fold more active than wild-type TfCut2. These improvements derived from changes in PET binding and not changes in catalytic efficiency.