Effects of 3,4-benzopyrene on the course of cell cycle events in the chlorococcal alga Scenedesmus quadricauda

Synchronous cultures of the chlorococcal alga Scenedesmus quadricauda were grown under optimal growth conditions. The mean length of their cell cycle was approximately 20 h. The cultures were treated at the start, at the 4th, and 8th hour of the cell cycle with 3,4-benzo(a)pyrene (BP) in the range of 0.1–0.5 g ml^-1 of final concentration. A period about 4 h was found within which no inhibitory effects could be detected even at the highest BP concentrations used. In presence of BP the rates of RNA and protein syntheses gradually decreased until complete inhibition of net syntheses occurred. In a similar way chlorophyll synthesis was inhibited, and this was followed by gradual degradation of the chlorophyll. The higher the concentration of BP the more rapid the decrease of the rates of syntheses and the earlier their complete inhibition. At low BP concentrations while DNA replications were initiated, the number of replications was lowered. At higher concentrations the initiations of DNA replications were delayed or completely suppressed. Syntheses of saccharides were the least inhibited processes in presence of BP. Starch synthesis was slowed down at the end of the cell cycle and fructose synthesis (free and sucrose bound) was even stimulated later in the cell cycle. The release of daughter coenobia, and protoplast fissions were most susceptible to BP treatment, being affected at concentrations which produced no measureble disturbances of macromolecular syntheses. At BP concentrations at which the inhibition of macromolecular syntheses occurred, the delay or suppression of mitoses was observed.Abbreviations BP 3,4-benzo(a)pyrene - PhAR photosynthetically active radiation     

Oxy intermediates of homoprotocatechuate 2,3-dioxygenase: facile electron transfer between substrates

Substrates homoprotocatechuate (HPCA) and O(2) bind to the Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O(2) via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD?HPCA and H200N?HPCA, respectively) with O(2). A blue intermediate forms within 20 ms of mixing of O(2) with H200N?HPCA (H200N(Int1)(HPCA)). Parallel mode electron paramagnetic resonance and Mo?ssbauer spectroscopies show that this intermediate contains high-spin Fe(III) (S = 5/2) antiferromagnetically coupled to a radical (S(R) = 1/2) to yield an S = 2 state. Together, optical and Mo?ssbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200N(Int1)(HPCA) decays over the next 2 s, possibly through an Fe(II) intermediate (H200N(Int2)(HPCA)), to yield the product and the resting Fe(II) enzyme. Reaction of FeHPCD?HPCA with O(2) results in rapid formation of a colorless Fe(II) intermediate (FeHPCD(Int1)(HPCA)). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence of a spin-coupled species in FeHPCD(Int1)(HPCA) suggests it is an intermediate occurring after O(2) activation and attack. The similar Mo?ssbauer parameters for FeHPCD(Int1)(HPCA) and H200N(Int2)(HPCA) suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O(2) via the iron does occur, leading to aromatic ring cleavage.     

Differential effects of light and fungal elicitor on chlorogenic acid and caffeoylshikimic acid metabolism

Upon irradiation an increase in the extractable activity of hydroxycinnamoyl-CoA:d-quinate hydroxycinnamoyl transferase (CQT) and a decrease in the activity of hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (CST) was observed in cell suspension cultures and seedling of carrot (Daucus carota L). Conversely, CST was induced and CQT repressed in the cell cultures upon treatment with fungal elicitor (i.e. cell wall preparations from Phytophthora megasperma). In the cell cultures irradiation led to a continuous accumulation of 5-O-caffeoyl-d-quinic (chlorogenic) acid, while a transient accumulation of 5-O-caffeoylshikimic acid took place in response to elicitor treatment. Cell wall bound 4-hydroxybenzoic, 4-coumaric and also ferulic acid were increased after treatment with Pmg elicitor. These wall bound phenolics may be involved in protection against microbil attack.     

Molecular Biology and Cell-free Synthesis of Poliovirus

Abstract. Poliovirus is a small icosahedral particle consisting of only five species of macromolecules: 60 copies each of the capsid protein VP1-4; and one copy of single-stranded RNA, approximately 7500 nt long. The genome, linked at the 5′ end to a small protein VPg and 3′ polyadenylylated, is of plus strand polarity. After receptor-mediated uptake of the virus and release of the RNA into the cytoplasm, the genome serves as mRNA, encoding only a single polypeptide, the polyprotein. The polyprotein is cleaved co-translationally into numerous polypeptides by its own, internal proteinases 2A^pro, 3C^pro and 3CD^pro. Initiation of translation is mediated by a novel genetic element, called internal ribosomal entry site (IRES). IRES elements, which are 400 nt long RNA segments located within the 5′ non-translated region of the viral genome, are common to all picornaviruses. Their function renders translation of picornavirus mRNAs cap- and 5′-independent, an observation that has upset the dogma of cap-dependent translation in eukaryotic cells. IRES elements have also been used to genetically dissect the viral genome and to construct novel expression vectors. Genome replication is not fully understood, the major conundrum being the initiation of RNA synthesis by the primer-dependent viral RNA polymerase 3D^pol, a process leading to VPg-linked RNA products. Nearly all non-structural proteins appear to be involved in initiation, the proteinases 2A^pro and 3CD^pro included. A HeLa cell-free system has been developed that, on programming with plasmid-transcribed viral RNA, will perform viral translation, protein processing, RNA replication, and assembly of capsid protein and newly made genomic RNA. The final yield is infectious poliovirus. This result has nullified the dictum that no virus can replicate in a cell-free medium.     

EXAFS and Mössbauer characterization of the Diiron(III) site in stearoyl-acyl carrier protein Δ9– desaturase

Stearoyl-acyl carrier protein (ACP) Δ⁹-desaturase (Δ9D) from the castor plant is the best characterized soluble acyl-ACP desaturase. This enzyme utilizes a diiron center to catalyze the O₂- and NADPH-dependent introduction of a cis double bond between carbons 9 and 10 of stearoyl-ACP, yielding oleoyl-ACP. In the present study, we have used X-ray absorption spectroscopy to provide the first metrical information for the diferric oxidation state. These studies reveal distinct diiron clusters that have Fe-Fe distances of either 3.12 or 3.41?Å. The species having the 3.12?Å Fe-Fe distance also exhibits a 1.8?Å Fe-O bond and is thus proposed to represent Δ9D molecules containing a (μ-oxo)bis(μ-carboxylato)diiron(III) cluster. The species having the 3.41?Å Fe-Fe distance exhibits no short Fe-O bond, and thus likely represents Δ9D molecules containing a (μ-hydroxo)diiron(III) cluster. Mössbauer studies of the extended X-ray absorption fine structure (EXAFS) samples revealed three quadrupole doublets (ΔE _Q(1)=1.53?mm/s, 72%;ΔE _Q(2)=0.72?mm/s, 21%;ΔE _Q(3)=2.20?mm/s, 7%) that originate from three distinct dinuclear clusters. From analysis of spectral intensities and by comparison with previous studies of (μ-oxo)- and (μ-hydroxo)diiron(III) clusters in both model complexes and proteins, doublet 1, the Mössbauer majority species, is likely associated with the EXAFS majority species having a 3.12?Å Fe-Fe separation and a 1.8?Å Fe-μ-oxo bond, while doublet 2 likely results from one iron site (or both) of a cluster associated with the EXAFS species having a 3.41?Å Fe-Fe separation. The presence of multiple diiron center conformations in diferric Δ9D may reflect the necessity for the active site to allow access of the substrate stearoyl-ACP (～9?kDa) during desaturation catalysis.     

cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome

The cis-replicating RNA elements in the 5' and 3' nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3' NTR RNA, and only poorly by 5' NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.     

A nematode immunomodulator suppresses grass pollen-specific allergic responses by controlling excessive Th2 inflammation

Helminth parasites modulate the immune system by complex mechanisms to ensure persistence in the host. Released immunomodulatory parasite components lead to a beneficial environment for the parasite by targeting different host cells and in parallel to a modulation of unrelated inflammatory responses in the host, such as allergy. The aim of this study was to investigate the effect of the potent helminth immunomodulator, filarial cystatin, in a murine model of airway inflammation and hyperreactivity induced by a clinically relevant aeroallergen (timothy grass (Phleum pratense) pollen) and on the function of peripheral blood mononuclear cells (PBMCs) from timothy grass pollen allergic patients. BALB/c mice were systemically sensitised with a recombinant major allergen of timothy grass pollen (rPhl p 5b) and then challenged with timothy grass pollen extract (GPE) via the airways. Filarial cystatin was applied i.p. during the sensitisation phase. Airway hyperresponsiveness to methacholine challenges, inflammation of airways, inflammatory cell recruitment, cytokine production and lung histopathology were investigated. In a translational approach, PBMCs from allergic subjects and healthy controls were treated in vitro with cystatin prior to stimulation with GPE. Administration of filarial cystatin suppressed rPhl p 5b-induced allergen-specific Th2-responses and airway inflammation, inhibited local recruitment of eosinophils, reduced levels of allergen-specific IgE and down-regulated IL-5 and IL-13 in the bronchoalveolar lavage (BAL). Ex vivo restimulation with cystatin of spleen cells from cystatin-treated mice induced the production of IL-10, while cystatin inhibited allergen-specific IL-5 and IL-13 levels. Human PBMCs from timothy grass pollen allergic patients displayed a shift towards a Th1 response after treatment with cystatin. These results show that filarial cystatin ameliorates allergic inflammation and disease in a clinically relevant model of allergy. This data indicate that filarial cystatin has a modulatory effect on grass pollen-specific responses warranting further investigation of potential preventive and therapeutic options in the treatment of allergies.     

Effects of cellular iron deficiency on the formation of vascular endothelial growth factor and angiogenesis.

Background  

Young women diagnosed with breast cancer are known to have a higher mortality rate from the disease than older patients. Specific risk factors leading to this poorer outcome have not been identified. In the present study, we hypothesized that iron deficiency, a common ailment in young women, contributes to the poor outcome by promoting the hypoxia inducible factor-1α (HIF-1α and vascular endothelial growth factor (VEGF) formation. This hypothesis was tested in an in vitro cell culture model system.     

HIV- 1 Protease Inhibits Cap- and Poly(A)-Dependent Translation upon eIF4GI and PABP Cleavage

A number of viral proteases are able to cleave translation initiation factors leading to the inhibition of cellular translation. This is the case of human immunodeficiency virus type 1 protease (HIV-1 PR), which hydrolyzes eIF4GI and poly(A)-binding protein (PABP). Here, the effect of HIV-1 PR on cellular and viral protein synthesis has been examined using cell-free systems. HIV-1 PR strongly hampers translation of pre-existing capped luc mRNAs, particularly when these mRNAs contain a poly(A) tail. In fact, HIV-1 PR efficiently blocks cap- and poly(A)-dependent translation initiation in HeLa extracts. Addition of exogenous PABP to HIV-1 PR treated extracts partially restores the translation of polyadenylated luc mRNAs, suggesting that PABP cleavage is directly involved in the inhibition of poly(A)-dependent translation. In contrast to these data, PABP cleavage induced by HIV-1 PR has little impact on the translation of polyadenylated encephalomyocarditis virus internal ribosome entry site (IRES)-containing mRNAs. In this case, the loss of poly(A)-dependent translation is compensated by the IRES transactivation provided by eIF4G cleavage. Finally, translation of capped and polyadenylated HIV-1 genomic mRNA takes place in HeLa extracts when eIF4GI and PABP have been cleaved by HIV-1 PR. Together these results suggest that proteolytic cleavage of eIF4GI and PABP by HIV-1 PR blocks cap- and poly(A)-dependent initiation of translation, leading to the inhibition of cellular protein synthesis. However, HIV-1 genomic mRNA can be translated under these conditions, giving rise to the production of Gag polyprotein.