Role of conserved active site tryptophan-101 in functional activity and stability of phosphoserine aminotransferase from an enteric human parasite

Site-directed mutagenesis study was performed to elucidate the role of conserved tryptophan-101 present at the active site of phosphoserine aminotransferase from an enteric human parasite Entamoeba histolytica. Fluorescence resonance energy transfer and molecular dynamic simulation show that the indole ring of Trp101 stacks with the cofactor PLP. Loss of enzymatic activity and PLP polarization values suggest that Trp101 plays a major role in maintaining a defined PLP microenvironment essentially required for optimal enzymatic activity. Studies on W101F, W101H and W101A mutants show that only the indole ring of the conserved Trp101 forms most favorable stacking interaction with the pyridine ring of the cofactor PLP. Protein stability was compromised on substitution of Trp101 with Phe/His/Ala amino acids. A difference in conformational free energy of 1.65?kcal?mol(-1) was observed between WT-protein and W101A mutant.     

Antibiofilm activity of an exopolysaccharide from marine bacterium Vibrio sp. QY101

Bacterial exopolysaccharides have always been suggested to play crucial roles in the bacterial initial adhesion and the development of complex architecture in the later stages of bacterial biofilm formation. However, Escherichia coli group II capsular polysaccharide was characterized to exert broad-spectrum biofilm inhibition activity. In this study, we firstly reported that a bacterial exopolysaccharide (A101) not only inhibits biofilm formation of many bacteria but also disrupts established biofilm of some strains. A101 with an average molecular weight of up to 546 KDa, was isolated and purified from the culture supernatant of the marine bacterium Vibrio sp. QY101 by ethanol precipitation, iron-exchange chromatography and gel filtration chromatography. High performance liquid chromatography traces of the hydrolyzed polysaccharides showed that A101 is primarily consisted of galacturonic acid, glucuronic acid, rhamnose and glucosamine. A101 was demonstrated to inhibit biofilm formation by a wide range of Gram-negative and Gram-positive bacteria without antibacterial activity. Furthermore, A101 displayed a significant disruption on the established biofilm produced by Pseudomonas aeruginosa, but not by Staphylococcus aureus. Importantly, A101 increased the aminoglycosides antibiotics' capability of killing P. aeruginosa biofilm. Cell primary attachment to surfaces and intercellular aggregates assays suggested that A101 inhibited cell aggregates of both P. aeruginosa and S. aureus, while the cell-surface interactions inhibition only occurred in S. aureus, and the pre-formed cell aggregates dispersion induced by A101 only occurred in P. aeruginosa. Taken together, these data identify the antibiofilm activity of A101, which may make it potential in the design of new therapeutic strategies for bacterial biofilm-associated infections and limiting biofilm formation on medical indwelling devices. The found of A101 antibiofilm activity may also promote a new recognition about the functions of bacterial exopolysaccharides.     

miR-101 is down-regulated by the hepatitis B virus x protein and induces aberrant DNA methylation by targeting DNA methyltransferase 3A

The hepatitis B virus x (HBx) protein has been implicated in HBV-related hepatocellular carcinoma (HCC) pathogenesis. However, whether HBx regulates miRNA expression that plays important roles in gene regulation during hepatocarcinogenesis remains unknown. The expression of microRNA-101 (miR-101) in HBV-related HCC tissues and HCC cells was evaluated by real-time PCR. The direct target of miR-101, DNA methyltransferase 3A (DNMT3A), was identified in silico and validated using a 3′-UTR reporter assay. miR-101 was functionally characterized in cells with transiently altered miR-101 expression. HBx expression was found to have a significant inverse correlation with miR-101 expression in HBx-expressing HepG2 compared to control HepG2 cells. miR-101 expression was frequently down-regulated in HBV-related HCC tissues compared to adjacent noncancerous hepatic tissues and had a significant inverse correlation with DNMT3A expression in HBV-related HCCs. Further characterization of miR-101 revealed that it negatively regulated DNA methylation partly through targeting DNMT3A. HBx-mediated miR-101 down-regulation and DNMT3A up-regulation supported the enhanced DNA methylation of several tumor-suppressor genes in HBx-expressing cells. Our studies demonstrating the deregulation of miR-101 expression by HBx may provide novel mechanistic insights into HBV-mediated hepatocarcinogenesis and identify a potential miRNA-based targeted approach for treating HBV-related HCC.     

P450 enzymes from the bacterium Novosphingobium aromaticivorans

Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported.     

Crystallographic location and mutational analysis of Zn and Cd inhibitory sites and role of lipidic carboxylates in rescuing proton path mutants in cytochrome c oxidase

Cytochrome c oxidase (CcO) transfers protons from the inner surface of the enzyme to the buried O2 reduction site through two different pathways, termed K and D, and from the outer surface via an undefined route. These proton paths can be inhibited by metals such as zinc or cadmium, but the sites of inhibition have not been established. Anomalous difference Fourier analyses of Rhodobacter sphaeroides CcO crystals, with cadmium added, reveal metal binding sites that include the proposed initial proton donor/acceptor of the K pathway, Glu-101 of subunit II. Mutant forms of CcO that lack Glu-101II (E101A and E101A/H96A) exhibit low activity and eliminate metal binding at this site. Significant activity is restored to E101A and E101A/H96A by adding the lipophilic carboxylic compounds, arachidonic acid and cholic acid, but not by their non-carboxylic analogues. These amphipathic acids likely provide their carboxylic groups as substitute proton donors/acceptors in the absence of Glu-101II, as previously observed for arachidonic acid in mutants that alter Asp-132I of the D pathway. The activity of E101A/H96A is still inhibited by zinc, but this remaining inhibition is nearly eliminated by removal of subunit III, which is known to alter the D pathway. The results identify the Glu-101/His-96 site of subunit II as the site of metal binding that inhibits the uptake of protons into the K pathway and indicate that subunit III contributes to zinc binding and/or inhibition of the D pathway. By removing subunit III from E101A/H96A, thereby eliminating zinc inhibition of the uptake of protons from the inner surface of CcO, we confirm that an external zinc binding site is involved in inhibiting the backflow of protons to the active site.     

Alanine 101 and alanine 110 of the alpha subunit of Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase influence the regiospecific oxidation of aromatics

Saturation mutagenesis was used to generate 10 mutants of toluene-o-xylene monooxygenase (ToMO) at alpha subunit (TouA) positions A101 and A110: A101G, A101I, A101M, A101VE, A101V, A110G, A110C, A110S, A110P, and A110T; by testing the substrates toluene, o-cresol, m-cresol, p-cresol, phenol, naphthalene, o-methoxyphenol, m-methoxyphenol, p-methoxyphenol, o-xylene, and nitrobenzene, these positions were found to influence the regiospecific oxidation of aromatics. For example, compared to wild-type ToMO, TouA variant A101V produced threefold more 3-methoxycatechol from m-methoxyphenol as well as produced methylhydroquinone from o-cresol whereas wild-type ToMO did not. Similarly, variant A110C synthesized 1.8-fold more o-cresol from toluene and 1.8-fold more 3-methoxycatechol from m-methoxyphenol, and variant A110G synthesized more m-nitrophenol and twofold less p-nitrophenol from nitrobenzene. The A101V and A110C mutations did not affect the rate of reaction with the natural substrate toluene, so the variants had high activity. This is the first report that these or analogous residues influence the catalysis with this class of enzymes. Wild-type ToMO was found to oxidize o-methoxyphenol to methoxyhydroquinone (60%) and 4-methoxyresorcinol (40%), m-methoxyphenol to 4-methoxycatechol (96%) and 3-methoxycatechol (4%), and p-methoxyphenol to 4-methoxycatechol (100%).     

Role of the cyclic lipopeptide massetolide A in biological control of Phytophthora infestans and in colonization of tomato plants by Pseudomonas fluorescens

Pseudomonas strains have shown promising results in biological control of late blight caused by Phytophthora infestans. However, the mechanism(s) and metabolites involved are in many cases poorly understood. Here, the role of the cyclic lipopeptide massetolide A of Pseudomonas fluorescens SS101 in biocontrol of tomato late blight was examined. Pseudomonas fluorescens SS101 was effective in preventing infection of tomato (Lycopersicon esculentum) leaves by P. infestans and significantly reduced the expansion of existing late blight lesions. Massetolide A was an important component of the activity of P. fluorescens SS101, since the massA-mutant was significantly less effective in biocontrol, and purified massetolide A provided significant control of P. infestans, both locally and systemically via induced resistance. Assays with nahG transgenic plants indicated that the systemic resistance response induced by SS101 or massetolide A was independent of salicylic acid signalling. Strain SS101 colonized the roots of tomato seedlings significantly better than its massA-mutant, indicating that massetolide A was an important trait in plant colonization. This study shows that the cyclic lipopeptide surfactant massetolide A is a metabolite with versatile functions in the ecology of P. fluorescens SS101 and in interactions with tomato plants and the late blight pathogen P. infestans.     

Antigenic modulation by anti-CD5 immunotoxins

We evaluated the modulation of T101 immunotoxins (IT) and free T101 antibody from the surface of normal and leukemic cells to determine whether the presence of toxin on antibody affected antigenic modulation. Reagents were made by conjugating T101, which binds to the T cell antigen CD5, to either intact ricin or purified ricin A chain. We found that T101-A chain modulated CD5 more efficiently than T101-ricin, which modulated CD5 more efficiently than T101 alone. Kinetic studies showed that maximal modulation of IT was reached within 3 hr. When toxicity of the reagents was tested in protein synthesis inhibition assays, T101-ricin in the presence of lactose inhibited 99% of the protein synthesis of CEM cells. T101-A chain was less toxic, inhibiting protein synthesis only 23 to 43%. The addition of the potentiating agent monensin nearly doubled the toxicity of T101-A chain, but did not affect T101-A chain modulation. To determine the fate of bound IT, T101 and T101-ricin were labeled with 125I. Cells were incubated under modulating conditions in the presence of radiolabeled reagents. T101 and T101-ricin were internalized into CEM cells. In contrast, T101, but not T101-ricin, appeared to be shed from peripheral blood mononuclear cells. Our findings show clearly that: 1) the presence of toxin on antibody does not inhibit--and may actually enhance--modulation; 2) T101-IT are internalized, not shed from the cell surface; 3) the lack of toxicity of T101-A chain is not attributed to inability to modulate; 4) there is no correlation between enhancement of T101-A chain toxicity by monensin and antigenic modulation by A chain reagents; and 5) modulation, which is undesirable in monoclonal antibody therapy, may be advantageous in the therapeutic use of IT.     

Attenuating mutations of the matrix gene of influenza A/WSN/33 virus

The matrix protein (M1) of influenza virus plays an essential role in viral replication. Our previous studies have shown that basic amino acids 101RKLKR105 of M1 are involved in RNP binding and nuclear localization. For the present work, the functions of 101RKLKR105 were studied by introducing mutations into the M gene of influenza virus A/WSN/33 by reverse genetic methods. Individual substitution, R101S or R105S, had a minimal effect on viral replication. In contrast, the double mutation R101S-R105S was synergistic and resulted in temperature sensitivity reflected by reduced viral replication at a restrictive temperature. To investigate the in vivo effect on infection, BALB/c mice were infected with either A/WSN/33 wild-type (Wt) or mutant viruses and assessed for signs of illness, viral replication in the lungs, and survival rates. The results from mouse studies indicated that the R101S-R105S double mutant virus was strongly attenuated, while single mutant viruses R101S and R105S were minimally attenuated compared to A/WSN33 Wt under the same conditions. In challenge studies, mice immunized by infection with R101S-R105S were fully protected from lethal challenge with A/WSN/33. The replication and attenuating properties of R101S-R105S suggest its potential in development of live influenza virus vaccines.     

The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101

The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interfere with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co-expression with mutated forms of Vps4, Nedd4 and Tsg101. A dominant-negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV-1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 did not prevent interaction; however, three-dimensional spatial imaging suggested that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 was not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101-Nedd4 interaction.     

Cloning of new members of heat shock protein HSP101 gene family in wheat (Triticum aestivum (L.) Moench) inducible by heat, dehydration, and ABA(1)

We have cloned two cDNAs, TaHSP101B and TaHSP101C, encoding two heat stress-inducible members of HSP101/ClpB family in bread wheat (Triticum aestivum (L.) Moench.). Proteins encoded by these cDNAs are highly similar at the primary sequence level and diverged from the previously reported TaHSP101 (designated TaHSP101A) both in the consensus ATP/GTP-binding region II and in the carboxy terminal region. The HSP101 gene was determined to be a single copy gene or a member of a small gene family in hexaploid wheat. Messages encoding HSP101 proteins were inducible by heat stress treatments in both wheat leaves and roots. Accumulation of the TaHSP101C mRNA was less abundant than that of TaHSP101B mRNA. We are showing for the first time that in addition to heat stress, expression of HSP101 mRNAs in wheat leaves was induced by a 2-h dehydration and a treatment with 5x10(-5)M ABA, but not affected by chilling or wounding, indicating that HSP101 proteins may be involved in both heat and drought responses in wheat.     

Molecular cloning of two linked loci that increase the transformability of transformation-deficient mutants of Haemophilus influenzae.

A plasmid containing a 13.3-kb insert (pER194) was isolated from an EcoRI genomic library of Haemophilus influenzae on the basis of its ability to increase the transformability of the transformation-deficient mutants Com-78 and Com-101. The plasmid failed to increase the transformability of the Rec-1 and Rec-2 mutants, indicating that the mutations producing the Com-78 and Com-101 phenotypes are distinct from those giving rise to the Rec-1 and Rec-2 phenotypes. The physical mapping of the cloned fragment on the H. influenzae chromosome was found to be consistent with the genetic mapping of the Com-101 trait. A 2.8-kb EcoRI-BglII subfragment, representing one end of the 13.3-kb clone, was found to increase the transformation frequency of the Com-78 and Com-101 mutants when supplied in trans, indicating that the subfragment carries one or more loci required for chromosomal transformation. The corresponding region of the Com-101 chromosome was determined by hybridization analysis to contain a 0.3-kb insertion, suggesting that the Com-101 strain may contain an insertion mutation at this locus. A 3.0-kb EcoRI-MluI subfragment, representing the other end of the 13.3-kb EcoRI fragment, was found to increase the transformation frequency of the Com-101 mutant but not of the Com-78 mutant, suggesting that the Com-101 phenotype results from a complex genotype involving mutations at two or more transformation-related loci. This conclusion is consistent with data indicating that the Com-101 trait can be genetically separated into at least two components.     

Identification and mapping of regions of the plasmid pKM101 which influence the growth rate and resistance to phleomycin E of Escherichia coli WP2.

The effects of deletion of various regions of the pKM101 genome on several phenotypes conferred by pKM101 in Escherichia coli WP2 cells were investigated. Differences in the response of cells carrying pKM101 or various pKM101 deletion derivatives to the mutagenic effects of phleomycin E can be attributed to differences in sensitivity to the lethal effects of phleomycin E. Resistance to phleomycin E is conferred by the pKM101 mucAB genes (or an adjacent gene) but observed only with pKM101 derivatives which have lost a 2.2-kilobase (BalI-KpnI-2) segment which completely includes the pKM101 endonuclease gene nuc. A pKM101 slow-growth determinant, distinct from the slo gene, has also been identified and localized in the 2.4-kilobase (BalI-KpnI-3) segment which is adjacent to the nuc gene. Loss of this region does not appear to substantially influence the toxic or mutagenic effects of phleomycin E.     

Preservation of Xenopus laevis rDNA-containing plasmid, pXlr101A, injected into the fertilized egg of Xenopus laevis

A circular rDNA-containing recombinant plasmid, pXlr101A, and its vector pBR322 were microinjected into the cytoplasm of fertilized Xenopus laevis eggs. The DNAs extracted from injected embryos at various stages of development were analyzed by hybridization with 32P-labeled pBR322 as the probe. Neither pXlr101A nor pBR322 were replicated, but they were maintained until the tailbud stage, disappearing during the muscular response stage. When pXlr101A-injected embryos were raised until the 2-week-old tadpole stage, sequences homologous to pBR322 were detectable in two Eco RI fragments of the pXlr101A-injected tadpole DNA. The sizes of the Eco RI fragments suggested that the plasmid sequences were preserved most probably in the chromosome-integrated form.     

Specific human CYP 450 isoform metabolism of a pentachlorobiphenyl (PCB-IUPAC# 101)

Polychlorinated biphenyl IUPAC# 101-PCB 101 (chlorination pattern-2,2',4',5,5') is a common, persistent non-coplanar PCB congener found in the ambient environment but information related to its metabolism in humans is lacking. Previous studies indicate PCB 101 is rapidly metabolized in mammals through CYP 2B and 3A family enzymes. Recently, PCB metabolism through a 2A family isoform in hamsters was also reported. To specifically identify the human CYP 450 isoforms responsible for PCB 101 metabolism, we compared human microsome metabolism to metabolism using several specific recombinant human CYP isoforms. These data characterized selective and extensive metabolism by human CYP 2A6. The product formed was the 4-hydroxy-PCB 101 metabolite (4-hydroxy-2,2',4',5,5') and was the only major metabolite observed in the recombinant and human microsome investigation. This is important information for predicting human specific toxicokinetics of PCBs.     

A model for effector activity in a highly specific biological electron transfer complex: the cytochrome P450(cam)-putidaredoxin couple

The camphor hydroxylase cytochrome P450(cam) (CYP101) catalyzes the 5-exo hydroxylation of camphor in the first step of camphor catabolism by Pseudomonas putida. CYP101 forms a specific electron transfer complex with its physiological reductant, the Cys(4)Fe(2)S(2) ferredoxin putidaredoxin (Pdx). Pdx, along with other proteins and small molecules, has also been shown to be an effector for turnover by CYP101. Multidimensional nuclear magnetic resonance (NMR) techniques have been used to make extensive sequential (1)H, (15)N, and (13)C resonance assignments in CYP101 that permit a more complete characterization of the complex formed by CYP101 and Pdx. NMR-detected perturbations in CYP101 upon Pdx binding encompass regions of the CYP101 remote from the putative Pdx binding site, including in particular a region of the CYP101 molecule that has been implicated in substrate access to the active site via dynamical processes. A model for effector activity is proposed in which the primary role of the effector is to prevent uncoupling (formation of reduced oxo species without formation of hydroxycamphor) by enforcing conformations of CYP101 that prevent loss of substrate and/or intermediates prior to turnover. A secondary role could also be to enforce conformations that permit efficient proton transfer into the active site for coupled proton/electron transfer.     

Effect of systemic lupus erythematosus (SLE) treatment drugs on GI-101A breast tumor cell growth

The effect of systemic lupus erythematosus (SLE) treatment drugs on PKC (protein kinase C) activity and cell growth was studied using GI-101A breast tumor cells. Both hydroxychloroquine (HCQ) and prednisone treatments significantly increased the PKC activity in GI-101A cells after 60 min. Treatment of cells with a combination of HCQ/prednisone also produced the highest increase in PKC activity following 60 min incubation. When the GI-101A cells were treated with the same drugs, HCQ (10 ng/ml) prednisone (10 ng/ml) and HCQ/prednisone combination (10 ng/ml of each), for 72 hr the total PKC activity in the cells was significantly elevated and consequently the GI-101A cell growth was stimulated. As a result of drug induced cell growth stimulation the total number of cells in the treatment groups increased significantly compared to the non-treated controls. Interestingly HCQ and prednisone treatment induced cell growths were completely blocked by PKC specific inhibitor chelerythrine (50 microM). Our results suggest that HCQ and prednisone treatment can induce GI-101A cell growth via activating PKC.     

A gene encoding an acyl hydrolase is involved in leaf senescence in Arabidopsis

SAG101, a leaf senescence-associated gene, was cloned from an Arabidopsis leaf senescence enhancer trap line and functionally characterized. Reporter gene and RNA gel blot analyses revealed that SAG101 was not expressed until the onset of senescence in leaves. A recombinant SAG101 fusion protein overexpressed in Escherichia coli displayed acyl hydrolase activity. Antisense RNA interference in transgenic plants delayed the onset of leaf senescence for approximately 4 days. Chemically induced overexpression of SAG101 caused precocious senescence in both attached and detached leaves of transgenic Arabidopsis plants. These data suggest that SAG101 plays a significant role in leaf senescence.