Type IIβ Regulatory Subunit of cAMP-Dependent Protein Kinase: Purification Strategies to Optimize Crystallization

To elucidate the structural basis for important differences between types I and II regulatory subunit isoforms (RI and RII) of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase, the full-length RIIβ isoform and five RIIβ deletion mutants were constructed, expressed, purified, and screened for crystallization. Only one of these six proteins yielded diffraction quality crystals. Crystals were grown of the RIIβ deletion mutant (Δ1–111) monomer potentially in complex with two cAMP molecules. X-ray diffraction quality data were obtained only after significant modification to existing purification procedures. Modifications required a Sepharose, not agarose, support for cAMP affinity chromatography followed by rapid, quantitative removal of free cAMP by size-exclusion chromatography under reducing conditions. Data to 2.4 Å resolution were collected at 29°C using synchrotron radiation on a single crystal measuring 0.2 × 0.3 × 1.2 mm³. Data were 99% complete. The hexagonal crystal belonged to space group P6₍₁₎ or P6₍₅₎ with unit cell dimensions a = b = 161.62 Å and c = 39.66 Å.     

The role of hepatitis B virus × gene in development of primary hepatocellular carcinoma

Primary hepatocellular carcinoma (HCC) is one of the most common cancers occurring in human, and there is strong epidemiological evidence suggesting that persistent hepatitis B virus (HBV) infection is the most important risk factor for its development.HBx gene was found to be a transactivator recently. Its continuous expression in hepatocytes may transactivate cellular genes which can play a certain role in development of HCC. TheHBx gene fragment was used to construct a recombinant eukaryotic expression vector pCEP4 and introduced into HepG2 cells. The effect ofHBx gene on HCC cells growth and its molecular mechanism in HCC cells regulation were investigated.     

PAF-antagonistic bicyclo[3.2.1]octanoid neolignans from leaves of Ocotea macrophylla Kunth. (Lauraceae)

Di-nor-benzofuran neolignan aldehydes, Δ⁷-3,4-methylenedioxy-3′-methoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal A) 1, Δ⁷-3,4,5,3′-tetramethoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal B) 2, and macrophyllin-type bicyclo[3.2.1]octanoid neolignans (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-5′-methoxy-3,4-methylenedioxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol A) 3, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5′-trimethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol B) 4, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5,5′-tetramethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol C) 5, as well as 2′-epi-guianin 6 and (+)-licarin B 7, were isolated and characterized from leaves of Ocotea macrophylla (Lauraceae). The structures and configuration of these compounds were determined by extensive spectroscopic analyses. Inhibition of platelet activating factor (PAF)-induced aggregation of rabbit platelets were tested with neolignans 1–7. Although compound 6 was the most potent PAF-antagonist, compounds 3–5 showed some activity.     

Expression of the First N-Glycosylation Gene in the Dolichol Pathway, ALG7, Is Regulated at Two Major Control Points in the G1 Phase of the Saccharomyces cerevisiae Cell Cycle

The Saccharomyces cerevisiae ALG7 gene, which functions by initiating the dolichol pathway of protein N-glycosylation, displays properties of an early growth-response gene. To initiate studies of the involvement of ALG7 in cellular proliferation, we have now more precisely analyzed ALG7 expression in the G1 phase of cell cycle. We show that the rapid rate of ALG7 mRNA accumulation following growth stimulation was attenuated soon thereafter and that ALG7 growth induction occurred irrespective of α-factor. ALG7 growth induction was observed in mutants conditionally defective for reentry into the cell cycle from the stationary phase, indicating that the induction occurred prior to the performance of START. In addition, the steady-state levels of ALG7 mRNAs declined four-fold in response to START-I cell division arrest brought about by α-factor treatment later in G1. Importantly, deregulated expression of ALG7 resulted in an aberrant α-factor response. Our data not only indicate that ALG7 expression is regulated at two critical control points in G1 that determine the proliferative potential of cells, but also provide a link between ALG7 and START.     

Antiproliferative Effect of DNA Polymerase α Antisense Oligodeoxynucleotides on Breast Cancer Cells

Antisense oligonucleotides appear to offer considerable promise as sequence-specific inhibitors of gene expression. Different cellular targets for oligodeoxynucleotides with oncologic interest have been identified such as oncogenes, growth factors, and cell cycle-related genes. DNA polymerase α (polα) plays a relevant role in DNA synthesis and cell proliferation. Polα gene expression is constitutive throughout the cell cycle and its mRNA content and activity are related to the growth rate and neoplastic phenotype. The effects of a 18-mer polα antisense oligomer on the proliferation of the MDA-MB 231 breast cancer cell line have been investigated. After 48 h in culture with oligomers (10 μM), about 50% growth inhibition was observed in antisense-treated cells, as evaluated by 3-(4, 5-dimethythiazol-2yl)-2, 5-diphenyltetrazolium bromide assay and cell count. [³H]Thymidine incorporation exhibited a 90% inhibition of DNA synthesis associated to 64% accumulation of cells at the G1-S border of the cycle as by flow cytometry, at 24 h. Northern hybridization and SDS-PAGE of immunoprecipitated MDA-MB 231 cell lysates revealed a decreased expression of polα mRNA and a reduction of the 180-kDa polypeptide, respectively. Collectively, the data further confirm the relevance of polα in the replicative cycle, as well as strengthen the potentiality of the antisense strategy for the control of gene expression and cell growth.     

Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNA interaction

This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and tRNA^Gln. Temperature-sensitive mutants located in glnS, the gene for GlnRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNA^Gln: GlnRS complex. In order to characterize the specificity of the aminoacylation reaction, mutant tRNA^Gln species were synthesized with either a 2′-deoxy AMP or 3′-deoxy AMP as their 3′-terminal nucleotide. Subsequent assays for aminoacylation and ATP/PP_i exchange activity established the esterification of glutamine to the 2′-hydroxyl of the terminal adenosine: there is no glutaminylation of the 3′-OH group. This correlates with the classification of GlnRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNA^Gln are discussed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed.     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of λ DNA have been compared. A common ^5′GAGACC_3′CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5 ′ G G T C T C N ↓ 3 ′ C C A G A G N N N N N ↑     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

Improving Adaptive and Memory Immune Responses of an HIV/AIDS Vaccine Candidate MVA-B by Deletion of Vaccinia Virus Genes (C6L and K7R) Blocking Interferon Signaling Pathways

Poxvirus vector Modified Vaccinia Virus Ankara (MVA) expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (termed MVA-B) is a promising HIV/AIDS vaccine candidate, as confirmed from results obtained in a prophylactic phase I clinical trial in humans. To improve the immunogenicity elicited by MVA-B, we have generated and characterized the innate immune sensing and the in vivo immunogenicity profile of a vector with a double deletion in two vaccinia virus (VACV) genes (C6L and K7R) coding for inhibitors of interferon (IFN) signaling pathways. The innate immune signals elicited by MVA-B deletion mutants (MVA-B ΔC6L and MVA-B ΔC6L/K7R) in human macrophages and monocyte-derived dendritic cells (moDCs) showed an up-regulation of the expression of IFN-β, IFN-α/β-inducible genes, TNF-α, and other cytokines and chemokines. A DNA prime/MVA boost immunization protocol in mice revealed that these MVA-B deletion mutants were able to improve the magnitude and quality of HIV-1-specific CD4⁺ and CD8⁺ T cell adaptive and memory immune responses, which were mostly mediated by CD8⁺ T cells of an effector phenotype, with MVA-B ΔC6L/K7R being the most immunogenic virus recombinant. CD4⁺ T cell responses were mainly directed against Env, while GPN-specific CD8⁺ T cell responses were induced preferentially by the MVA-B deletion mutants. Furthermore, antibody levels to Env in the memory phase were slightly enhanced by the MVA-B deletion mutants compared to the parental MVA-B. These findings revealed that double deletion of VACV genes that act blocking intracellularly the IFN signaling pathway confers an immunological benefit, inducing innate immune responses and increases in the magnitude, quality and durability of the HIV-1-specific T cell immune responses. Our observations highlighted the immunomodulatory role of the VACV genes C6L and K7R, and that targeting common pathways, like IRF3/IFN-β signaling, could be a general strategy to improve the immunogenicity of poxvirus-based vaccine candidates.     

The A3 adenosine receptor as a new target for cancer therapy and chemoprotection

Adenosine, a purine nucleoside, acts as a regulatory molecule, by binding to specific G-protein-coupled A₁, A_2A, A_2B, and A₃ cell surface receptors. We have recently demonstrated that adenosine induces a differential effect on tumor and normal cells. While inhibiting in vitro tumor cell growth, it stimulates bone marrow cell proliferation. This dual activity was mediated through the A3 adenosine receptor. This study showed that a synthetic agonist to the A3 adenosine receptor, 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyl-uronamide (Cl-IB-MECA), at nanomolar concentrations, inhibited tumor cell growth through a cytostatic pathway, i.e., induced an increase number of cells in the G0/G1 phase of the cell cycle and decreased the telomeric signal. Interestingly, Cl-IB-MECA stimulates murine bone marrow cell proliferation through the induction of granulocyte-colony-stimulating factor. Oral administration of Cl-IB-MECA to melanoma-bearing mice suppressed the development of melanoma lung metastases (60.8 ± 6.5% inhibition). In combination with cyclophosphamide, a synergistic anti-tumor effect was achieved (78.5 ± 9.1% inhibition). Furthermore, Cl-IB-MECA prevented the cyclophosphamide-induced myelotoxic effects by increasing the number of white blood cells and the percentage of neutrophils, demonstrating its efficacy as a chemoprotective agent. We conclude that A3 adenosine receptor agonist, Cl-IB-MECA, exhibits systemic anticancer and chemoprotective effects.     

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae

Resistance to penicillin in non-β-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42–581) into an ATG expression vector. When the recombinant PBP 2 molecules were over-expressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2′ (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2′ for [³H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other β-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.     

Gentisic acid conjugates of Medicago truncatula roots

Three phenolic glycosides 5-O-{[5′′-O-E-(4′′′-O-threo-guaiacylglycerol)-feruloyl]-β-apiofuranosyl-(1→2)-β-xylopyranosyl} gentisic acid, 5-O-[(5′′-O-vanilloyl)-β-apiofuranosyl-(1→2)-β-xylopyranosyl] gentisic acid and 1-O-[E-(4′′′-O-threo-guaiacylglycerol)-feruloyl]-3-O-β-galacturonopyranosyl glycerol were isolated and identified from the roots of Medicago truncatula together with four known 5-O-β-xylopyranosyl gentisic acid, vicenin-2, hovetrichoside C and pterosupin identified for the first time in this species. Structural elucidation was carried out on the basis of UV, mass, ¹H and ¹³C NMR spectral data.     

Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation

Leishmania mexicana has a large family of cyclin‐dependent kinases (CDKs) that reflect the complex interplay between cell cycle and life cycle progression. Evidence from previous studies indicated that Cdc2‐related kinase 3 (CRK3) in complex with the cyclin CYC6 is a functional homologue of the major cell cycle regulator CDK1, yet definitive genetic evidence for an essential role in parasite proliferation is lacking. To address this, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) to target CRK3 and elucidate its role in the cell cycle of L. mexicana. Induction of diCre activity in promastigotes with rapamycin resulted in efficient deletion of floxed CRK3, resulting in G2/M growth arrest. Co‐expression of a CRK3 transgene during rapamycin‐induced deletion of CRK3 resulted in complementation of growth, whereas expression of an active site CRK3^T178E mutant did not, showing that protein kinase activity is crucial for CRK3 function. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thereby confirming CRK3 as a useful therapeutic target and diCre as a valuable new tool for analyzing essential genes in Leishmania.     

Cytoplasmic domain of influenza B virus BM2 protein plays critical roles in production of infectious virus

Influenza B virus BM2 is a type III integral membrane protein that displays H⁺ ion channel activity. Analysis of BM2 knockout mutants has suggested that this protein is a necessary component for the capture of M1-viral ribonucleoprotein (vRNP) complex at the plasma membrane and for incorporation of vRNP complex into the virion during the assembly process. BM2 comprises 109 amino acid residues and possesses a longer cytoplasmic domain than the other 3 integral membrane proteins (hemagglutinin, neuraminidase, and NB). To explore whether the cytoplasmic domain of BM2 is important for infectious virus production, a series of BM2 deletion mutants lacking three to nine amino acid residues at the carboxyl terminus, BM2Δ107-109, BM2Δ104-109, and BM2Δ101-109, was generated by reverse genetics. Intracellular transport and incorporation into virions were indistinguishable between truncated BM2 proteins and wild-type BM2. The BM2Δ107-109 mutant produced levels of infectious virus similar to those of wild-type virus and displayed a spherical shape. However, the BM2Δ104-109 and BM2Δ101-109 mutants produced viruses containing dramatically reduced vRNP complex, as with BM2 knockout mutants, and formed enlarged, irregularly shaped virions. Moreover, gradient separation of membranes indicated that membrane association of M1 from mutants was greatly affected by carboxyl-terminal truncations of BM2. Studies of alanine substitution mutants further suggested that amino acid sequences in the 98-109 region are variable while those in the 86-97 region are a prerequisite for innate BM2 function. These results indicate that the cytoplasmic domain of the BM2 protein is required for firm association of the M1 protein with lipid membranes, vRNP complex incorporation into virions, and virion morphology.     

Characterization of the heat shock protein 90 gene in the plant parasitic nematode Meloidogyne artiellia and its expression as related to different developmental stages and temperature

The full-length cDNA and the corresponding gene of the heat shock protein 90, Mt-Hsp90, were isolated and characterized in the plant parasitic nematode Meloidogyne artiellia. The full-length Mt-Hsp90 cDNA contained a 5′ untranslated region (UTR) of 45 bp with the 22 bp trans-spliced leader SL1, an ORF of 2172 bp encoding a polypeptide of 723 amino acids and a 3′ UTR of 191 bp. The deduced amino acid sequence of Mt-hsp90 showed high similarity with other known Hsp90s. Five conserved amino acid signatures indicated that Mt-hsp90 is a cytosolic member of the Hsp90 family. The gene consists of 10 exons and 9 introns, a more expanded gene structure compared to the corresponding Caenorhabditis elegans gene, daf-21. Mt-hsp90 gene was constitutively expressed at high levels in all developmental stages of M. artiellia. Egg masses and second stage juveniles (J2s) were exposed at 5° and 30 °C for different periods of times in order to explore the impact of adverse temperature on Mt-hsp90 gene expression. Expression levels of Mt-hsp90 were examined by fluorescent real-time PCR. At 30 °C a burst of expression for Mt-hsp90 was observed in J2s after 2 h of heat shock treatment, then expression dropped with longer exposing times, although remaining still relatively high after 24 h. This temperature did not affect Mt-hsp90 gene expression in the egg masses. However, egg masses exposed at 5 °C showed a little but gradual increase in the mRNA level with time. By contrast, no significant changes in the Mt-hsp90 level were observed in J2s exposed to cold. These data show that egg masses and J2s exposed to cold and heat stresses have different expression profiles suggesting that Mt-Hsp90 may provide a link between environmental conditions and the life cycle of the nematode.