Process/technology modifications in water pollution control

This article discusses the importance of modification in process/technology in various industries in order to control the pollution produced by those industries. Case studies on the modification of rinsing procedures for metal aprts and for the product line between two kinds of yogurts, changes in the mode of transportation in poultry industry and the introduction of biological degreasing of metals show huge benefit due to those modifications. Changing the products as well as input materials, too, bring waste minimization along with sustainable development.

An in vitro comparative assessment with a series of new triphenyltin(IV) 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates endowed with anticancer activities: Structural modifications, analysis of efficacy and cytotoxicity involving human tumor cell lines

Four new triphenyltin(IV) complexes of composition Ph₃SnLH (where LH = 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoate) (1–4) were synthesized and characterized by spectroscopic (¹H, ¹³C and ¹¹⁹Sn NMR, IR, ¹¹⁹Sn Mössbauer) techniques in combination with elemental analysis. The ¹¹⁹Sn NMR spectroscopic data indicate a tetrahedral coordination geometry in non-coordinating solvents. The crystal structures of three complexes, Ph₃SnL¹H (1), Ph₃SnL³H (3), Ph₃SnL⁴H (4), were determined. All display an essentially tetrahedral geometry with angles ranging from 93.50(8) to 124.5(2)°; ¹¹⁹Sn Mössbauer spectral data support this assignment. The cytotoxicity studies were performed with complexes 1–4, along with a previously reported complex (5) in vitro across a panel of human tumor cell lines viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. The screening results were compared with the results from other related triphenyltin(IV) complexes (6–7) and tributyltin(IV) complexes (8–11) having 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates framework. In general, the complexes exhibit stronger cytotoxic activity. The results obtained for 1–3 are also comparable to those of its o-analogs i.e. 4–7, except 5, but the advantage is the former set of complexes demonstrated two folds more cytotoxic activity for the cell line MCF-7 with ID₅₀ values in the range 41–53 ng/ml. Undoubtedly, the cytotoxic results of complexes 1–3 are far superior to CDDP, 5-FU and ETO, and related tributyltin(IV) complexes 8–11. The quantitative structure-activity relationship (QSAR) studies for the cytotoxicity of triphenyltin(IV) complexes 1–7 and tributyltin(IV) complexes 8–11 is also discussed against a panel of human tumor cell lines.

Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Because it plays a key role in the in vitro dimerization of short HIV-1 RNA transcripts (M. Laughrea and L. Jette, Biochemistry 35:1589-1598, 1996, and references therein; M. Laughrea and L. Jette, Biochemistry 35:9366-9374, 1996, and references therein) and because dimeric RNAs may be preferably encapsidated into the HIV-1 virus, alterations of the kissing-loop hairpin might affect the in vivo dimerization and encapsidation processes. Accordingly, substitution and deletion mutations were introduced into the kissing-loop hairpin of an infectious HIV-1 molecular clone in order to produce viruses by transfection methods. The infectivity of the resulting viruses was decreased by at least 99%, the amount of genomic RNA packaged per virus was decreased by 50 to 75%, and the proportion of dimeric genomic RNA was reduced from >80 to 40 to 50%, but the dissociation temperature of the genomic RNA was unchanged. There is evidence suggesting that the deletion mutations moderately inhibited CAp24 production but had no significant effect on RNA splicing. These results are consistent with the kissing-loop model of HIV-1 RNA dimerization. In fact, because intracellular viral RNAs are probably more concentrated in transfected cells than in cells infected by one virus and because the dimerization and encapsidation processes are concentration dependent, it is likely that much larger dimerization and encapsidation defects would have been manifested within cells infected by no more than one virus.

Maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity

Production of the human immunodeficiency virus type 1 (HIV_-1) Gag-Pol precursor protein results from a −1 ribosomal frameshifting event. In infected cells, this generates Gag and Gag-Pol in a ratio that is estimated to be 20:1, a ratio that is conserved among retroviruses. To examine the impact of this ratio on HIV_-1 replication and viral assembly, we altered the Gag/Gag-Pol ratio in virus-producing cells by cotransfecting HIV_-1 proviral DNA with an HIV_-1 Gag-Pol expression vector. Two versions of the Gag-Pol expression vector were used; one contains an active protease [PR(+)], and the other contains an inactive protease [PR(−)]. In an attempt to produce viral particles with Gag/Gag-Pol ratios ranging from 20:21 to 20:1 (wild type), 293T cells were cotransfected with various ratios of wild-type proviral DNA and proviral DNA from either Gag-Pol expression vector. Viral particles derived from cells with altered Gag/Gag-Pol ratios via overexpression of PR(−) Gag-Pol showed a ratio-dependent defect in their virion protein profiles. However, the defects in virion infectivity were independent of the nature of the Gag-Pol expression vector, i.e., PR(+) or PR(−). Based on equivalent input of reverse transcriptase activity, we estimated that HIV_-1 infectivity was reduced 250- to 1,000-fold when the Gag/Gag-Pol ratio in the virion-producing cells was altered from 20:1 to 20:21. Although virion RNA packaging was not affected by altering Gag/Gag-Pol ratios, changing the ratio from 20:1 to 20:21 progressively reduced virion RNA dimer stability. The impact of the Gag/Gag-Pol ratio on virion RNA dimerization was amplified when the Gag-Pol PR(−) expression vector was expressed in virion-producing cells. Virions produced from cells expressing Gag and Gag-Pol PR(−) in a 20:21 ratio contained mainly monomeric RNA. Our observations provide the first direct evidence that, in addition to proteolytic processing, the ratio of Gag/Gag-Pol proteins is also important for RNA dimerization and that stable RNA dimers are not required for encapsidation of genomic RNA in HIV_-1.

Proteolytic processing of the P2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

The conformation of the mature dimeric human immunodeficiency virus type 1 RNA genome requires packaging of pol protein

The packaging of a mature dimeric RNA genome is an essential step in human immunodeficiency virus type 1 (HIV-1) replication. We have previously shown that overexpression of a protease (PR)-inactive HIV-1 Gag-Pro-Pol precursor protein generates noninfectious virions that contain mainly monomeric RNA (M. Shehu-Xhilaga, S. M. Crowe, and J. Mak, J. Virol. 75:1834-1841, 2001). To further define the contribution of HIV-1 Gag and Gag-Pro-Pol to RNA maturation, we analyzed virion RNA dimers derived from Gag particles in the absence of Gag-Pro-Pol. Compared to wild-type (WT) dimeric RNAs, these RNA dimers have altered mobility and low stability under electrophoresis conditions, suggesting that the HIV-1 Gag precursor protein alone is not sufficient to stabilize the dimeric virion RNA structure. The inclusion of an active viral PR, without reverse transcriptase (RT) and integrase (IN), rescued the stability of the virion RNA dimers in the Gag particles but did not restore the mobility of the RNAs, suggesting that RT and IN are also required for virion RNA dimer maturation. Thin-section electron microscopy showed that viral particles deficient in RT and IN contain empty cone-shaped cores. The abnormal core structure indicates a requirement for Gag-Pro-Pol packaging during core maturation. Supplementing viral particles with either RT or IN via Vpr-RT or Vpr-IN alone did not correct the conformation of the dimer RNAs, whereas expression of both RT and IN in trans as a Vpr-RT-IN fusion restored RNA dimer conformation to that of the WT virus and also restored the electron-dense, cone-shaped virion core characteristic of WT virus. Our data suggest a role for RT-IN in RNA dimer conformation and the formation of the electron-dense viral core.

Proline residues within spacer peptide p1 are important for human immunodeficiency virus type 1 infectivity, protein processing, and genomic RNA dimer stability

The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.

Analysis of the contribution of reverse transcriptase and integrase proteins to retroviral RNA dimer conformation

All retroviruses contain two copies of genomic RNA that are linked noncovalently. The dimeric RNA of human immunodeficiency virus type 1 (HIV-1) undergoes rearrangement during virion maturation, whereby the dimeric RNA genome assumes a more stable conformation. Previously, we have shown that the packaging of the HIV-1 polymerase (Pol) proteins reverse transcriptase (RT) and integrase (IN) is essential for the generation of the mature RNA dimer conformation. Analysis of HIV-1 mutants that are defective in processing of Pol showed that these mutant virions contained altered dimeric RNA conformation, indicating that the mature RNA dimer conformation in HIV-1 requires the correct proteolytic processing of Pol. The HIV-1 Pol proteins are multimeric in their mature enzymatically active forms; RT forms a heterodimer, and IN appears to form a homotetramer. Using RT and IN multimerization defective mutants, we have found that dimeric RNA from these mutant virions has the same stability and conformation as wild-type RNA dimers, showing that the mature enzymatically active RT and IN proteins are dispensable for the generation of mature RNA dimer conformation. This also indicated that formation of the mature RNA dimer structure occurs prior to RT or IN maturation. We have also investigated the requirement of Pol for RNA dimerization in both Mason-Pfizer monkey virus (M-PMV) and Moloney murine leukemia virus (MoMuLV) and found that in contrast to HIV-1, Pol is dispensable for RNA dimer maturation in M-PMV and MoMuLV, demonstrating that the requirement of Pol in retroviral RNA dimer maturation is not conserved among all retroviruses.

Dimerization of retroviral RNA genomes : an inseparable pair

Many viruses carry more than one segment of nucleic acid into the virion particle, but retroviruses are the only known group of viruses that contain two identical (or nearly identical) copies of the RNA genome within the virion. These RNA genomes are non-covalently joined together through a process known as genomic RNA dimerization. Uniquely, the RNA dimerization of the retroviral genome is of crucial importance for efficient retroviral replication. In this article, our current understanding of the relationship between retroviral genome conformation, dimerization and replication is reviewed.

Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription

The dimerization initiation site (DIS) stem-loop within the HIV-1 RNA genome is vital for the production of infectious virions in T-cell lines but not in primary cells. In comparison to peripheral blood mononuclear cells (PBMCs), which can support the replication of both wild type and HIV-1 DIS RNA mutants, we have found that DIS RNA mutants are up to 100 000-fold less infectious than wild-type HIV-1 in T-cell lines. We have also found that the cell-type-dependent replication of HIV-1 DIS RNA mutants is largely producer cell-dependent, with mutants displaying a greater defect in viral cDNA synthesis when viruses were not derived from PBMCs. While many examples exist of host–pathogen interplays that are mediated via proteins, analogous examples which rely on nucleic acid triggers are limited. Our data provide evidence to illustrate that primary T-lymphocytes rescue, in part, the replication of HIV-1 DIS RNA mutants through mediating the reverse transcription process in a cell-type-dependent manner. Our data also suggest the presence of a host cell factor that acts within the virus producer cells. In addition to providing an example of an RNA-mediated cell-type-dependent block to viral replication, our data also provides evidence which help to resolve the dilemma of how HIV-1 genomes with mismatched DIS sequences can recombine to generate chimeric viral RNA genomes.

An antiviral response directed by PKR phosphorylation of the RNA helicase A

The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)–mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.

RNA interference : more than a research tool in the vertebrates' adaptive immunity

In recent years, RNA silencing, usage of small double stranded RNAs of ~21 – 25 base pairs to regulate gene expression, has emerged as a powerful research tool to dissect the role of unknown host cell factors in this 'post-genomic' era. While the molecular mechanism of RNA silencing has not been precisely defined, the revelation that small RNA molecules are equipped with this regulatory function has transformed our thinking on the role of RNA in many facets of biology, illustrating the complexity and the dynamic interplay of cellular regulation. As plants and invertebrates lack the protein-based adaptive immunity that are found in jawed vertebrates, the ability of RNA silencing to shut down gene expression in a sequence-specific manner offers an explanation of how these organisms counteract pathogen invasions into host cells. It has been proposed that this type of RNA-mediated defence mechanism is an ancient form of immunity to offset the transgene-, transposon- and virus-mediated attack. However, whether 1) RNA silencing is a natural immune response in vertebrates to suppress pathogen invasion; or 2) vertebrate cells have evolved to counteract invasion in a 'RNA silencing' independent manner remains to be determined. A number of recent reports have provided tantalizing clues to support the view that RNA silencing functions as a physiological response to regulate viral infection in vertebrate cells. Amongst these, two manuscripts that are published in recent issues of Science and Immunity, respectively, have provided some of the first direct evidences that RNA silencing is an important component of antiviral defence in vertebrate cells. In addition to demonstrating RNA silencing to be critical to vertebrate innate immunity, these studies also highlight the potential of utilising virus-infection systems as models to refine our understanding on the molecular determinants of RNA silencing in vertebrate cells.