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IDENTIFICAZIONE DI TRE NUOVI PEPTIDI SALIVARI BASICI RICCHI DI PROLINA CARATTERIZZATI DA ATTIVITÀ ANTI- HIV-1
Full text linkAlthough important progress has been achieved in preventing new HIV infections and in lowering the annual number of AIDS-related deaths, the number of people living with HIV ineluctably increases. As well known, actual antiretroviral therapies are unfortunately characterized by very marked side effects and the drug resistance problem continues to be a daily issue; so the necessity to extend the list of the new anti-HIV drugs remain a constant priority. The existence of HIV latent reservoirs is one the major obstacle to eradicate the virus from human body hence to successfully treat the HIV infection. These reservoirs are extremely stable, so that it seems rather unrealistic to definitely succeed in the eradication of the virus by using the current therapeutic regimens. Consequently, the identification of new antiretroviral drugs, able to eradicate HIV from its reservoirs, has became a pressing priority.
Unlike other mucosal area of the body, the oral cavity appears to be an extremely uncommon transmission route for HIV [13]. In addition to the distinct oral mucosal architecture and cellular constituents, oral fluids, unlike other mucosal secretions, are rarely a vehicle for HIV infection. One reason for this apparent paradox is the presence of endogenous mucosal antiviral factors, including neutralizing antibodies, secretory leukocyte protease inhibitor (SLPI), antiviral peptides such as defensins and cystatins, glycoproteins including thrombospondin and lactoferrin, and complement components [14]. In 2001 Robinovitch MR demonstrated the presence in human parotid saliva of specific basic proline-rich proteins possessing significant anti-HIV-1 activity independent of that attributable to SLPI or TSP-1.
In this thesis are presented for the first time tree salivary proline-rich basic peptides showing the surprising and unexpected ability to inhibit HIV-1 in vitro replication without exerting cytotoxic effects on human PBMC. In addiction, these peptides demonstrate to be potent inducers of the viral replication in the ex vivo assays. This aspect, which would seem contradictory, represents a very innovative effect, considering the latent viral reservoirs problem. In conclusion, these peptides could target viral reservoirs and eradicate HIV from human body by firstly inducing the replication of latent virus in cells and then inhibiting it.
Moreover, the peptides here analized even show antifungal activity, particularly useful to treat the opportunistic infections AIDS associated
Complex pattern of HTLV-2 splicing and expression in PBMCs from infected patients
No full textHTLV express multiple gene products from the same coding region by employing different strategies, including alternative splicing. Our investigations were focused on the analysis of the levels of expression of different HTLV-2 transcripts, to test their temporal expression at different stages of viral cycle and their quantitation by real time RT-PCR, in infected cell lines and in primary cultures of PBMCs from infected subjects.
An early transcription of tax/rex regulatory mRNA was observed, followed by a gradual and steady increase of gag/pol and env structural transcripts and of other accessory mRNAs. We identified a novel 3’ splice acceptor site, used by both HTLV-2 A and B subtypes to generate alternative doubly spliced mRNAs within the pX region and also a novel env isoform preferentially expressed in 2B subtypes and behaving as a late gene. Among the singly spliced mRNAs coding for other accessory proteins, the p28, p22/p20-II isoform was found to be highly expressed as compared to its alternative p28, p22/p20-I form. The APH-2 transcript from the negative strand behaved as a late gene in stably infected cells, while in ex-vivo PBMCs its kinetics appeared to be variable so that a clear pattern of expression was not yet assessed.
In conclusion, the temporal transcription of different HTLV-2 transcripts follows a distinct expression pattern: tax/rex is the first mRNA to be expressed, thus indicating that it is necessary at the beginning of the infection cycle to transactivate and regulate viral and cellular transcripts, while gag/pol and env structural genes are expressed later in the viral cycle
Time course of Human-T cell Leukemia Virus type 2 (HTLV-2) gene expression in PBMCs from infected patients
No full textThe analysis of HTLV expression during the infection process is essential to understand the influence of viral gene products in proliferation, cell cycle and signalling. Very little information has so far been obtained on the quantification and timing of HTLV-2 viral transcription, therefore the aim of the study was to further investigate the kinetics of different HTLV-2 transcripts.
The time course of transcription of the viral mRNAs, tax/rex, gag/pol, env, p28- p22/p20 rex-1 and -2, p10/p11 and p?, was evaluated using splice-junction-specific primers to quantify all HTLV-2 transcripts by real time RT-PCR. To this end, PBMCs from HTLV-2B infected patients were cultured with IL-2 and mRNA analysed at different time points: 0, 2, 4, 8, 21 and 48 hours. The results obtained showed an early transcription of tax/rex, followed by p28- p22/p20 rex-2 and by a gradual and steady increase of gag/pol and p28- p22/p20 rex-1. The level of expression of gag/pol and p28- p22/p20 rex-2 was about 103-104 fold higher than tax/rex and p28- p22/p20 rex-1.
These results indicate that the expression of different HTLV-2 genes follows a distinct timing in PBMCs isolated from infected patients. The regulatory transcripts are the first to be expressed whereas structural ones are expressed at a later time point. Moreover, the level of expression of accessory genes was significantly lower than that of structural viral genes.
In conclusion, distinct patterns of expression of HTLV-2 were found in infected PBMCs thus indicating that the control of gene transcription is highly regulated both in its kinetics and expression
Complex pattern of HTLV-2 temporal expression in infected cell lines and patient PBMCs and identification of a novel splicing acceptor site
No full textBackground: Similarly to other retroviruses, HTLV-2 expresses multiple gene products from the same coding region by means of different strategies, including a complex pattern of alternative splicing.
Methods: Our investigation has been focused on the analysis of the levels of expression of different HTLV-2 transcripts, to highlight the kinetics of transcription at different stages of virus gene expression and their quantitation, by real time RT-PCR, in infected cell lines and in short term cultures of PBMCs from infected patients.
Results: Results obtained show an early transcription of tax/rex regulatory mRNA, followed by a gradual and steady increase of gag/pol and env structural transcripts and of other accessory mRNAs.Data demostrate that the expression of different HTLV-2 genes follows a distinct timing, both in chronically infected cells and in PBMCs isolated from infected patients. The regulatory transcript tax/rex is the first one to be expressed whereas the structural and accessory mRNAs are expressed at a later time point.
Further studies aimed at better understanding the complex pattern of splicing, allowed us to identify a novel 3’ acceptor site of splicing for the second exon of HTLV-2. This new 3’ acceptor site is capable of expressing an isoform of the singly spliced env mRNA. In addition, it could also be used as an alternative splicing system within the X region for the tax/rex, p10/p11 and p? transcripts.
Conclusions: This study demostrated that the expression of HTLV-2 transcripts is presenting distinct patterns, indicating that the control of viral gene expression is highly regulated both in its kinetics and expression level. Moreover, the use of multiple acceptor sites is likely to play an important role for the preferential expression of specific proteins at different phases of the viral cycle
Analysis of viral transcripts of Human T-cell Leukemia Virus type 2 (HTLV-2) in human T- and B- infected cells and PBMCs from infected subjects
No full textRecent studies carried out on HTLV-1-infected cells have provided information on the relative abundance and timing of expression of different viral transcripts. In the case of HTLV-2, information on the profile and temporal regulation of viral gene expression is still incomplete.
To address this point, we set up a splice-junction-specific real-time RT-PCR to evaluate the quantitative level of individual HTLV-2 transcripts. Results obtained led to the quantitation of all the HTLV-2 mRNAs described so far, namely gag/pol, env, tax/rex, p28,p22/p20rex-1, p28,p22/p20rex-2 and p10/p11. This analysis was carried out first on chronically infected lines: T-cells infected with the HTLV-2A subtype (Mo-T and C344) or B-cells infected with the HTLV-2B subtype (BJAB-Gu cells). Results showed different levels of expression of env and tax/rex transcripts in T- and B-cells. The expression profile and kinetics of expression of the different transcripts was analyzed in PBMC obtained ex vivo from infected individuals and cultured for several hours. Current experiments are also aimed at testing the possible connections between the pattern of HTLV-2 gene expression and the different phases of the cell cycle
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Kinetic analysis of Human T-cell Leukemia Virus type 2 expression in chronically-infected cells and patient PBMCs
Full text linkIntroduction:
The elucidation of the viral gene expression
profile provides useful information in assessing the function of specific viral genes in the process of infection and cellular transformation.
HTLV-2 pattern of mRNAs expression produces three major classes of mRNAs: unspliced genomic mRNA for Gag, protease and
Pol proteins; singly spliced mRNAs encoding
Env and the accessory proteins p28, p22/p20-1
and -2; and a doubly spliced mRNA for the
regulatory proteins Tax, Rex and for the
p10/p11 and p? accessory ones (Ref.1 and Fig.
1). To date, very little information has been
obtained on the temporal regulation of different HTLV-2 transcripts expression in infected cells.
Aim of this study was to investigate the kinetics of gene expression from HTLV-2 infected cell lines and from PBMCs of HTLV-2B infected subjects. The expression profile and kinetics of the different transcripts were analysed by real time RT-PCR using splice-junction-specific primers.
Results:
This approach was used to first determine the
steady-state levels of expression for the different viral transcripts in three different cell lines in log phase of growth . Experiments performed indicated that gag/pol is the most abundant
transcript. The expression level of env was
comparable in the two T-cell lines, Mo-T and
C344, infected by the 2A subtype, and was
considerably higher than in the B-cells infected with HTLV-2B subtype, where p10/p11 and p? transcripts were below the limit of detection.
We next investigated the kinetics of viral
transcripts expression in infected BJAB-Gu cells.
As in the previous experiment, the absolute copy number of gag/pol was the highest over the time period analysed . Among the accessory transcripts, p28,p22/p20-2 was the most abundant while other regulatory and accessory genes were lower. The analysis of fold variation, reported in
g. 4B, indicated that tax/rex and p28, p22/p20-1
showed a biphasic profile with an early peak at 24
hours and a second one at 72 hours, whereas the
transcripts gag/pol, env and p28,p22/p20-2 were
expressed later.The kinetics of gene expression also was
analysed from ex-vivo PBMCs of HTLV-2B
infected subjects. Fig. 5 shows a typical pattern
of expression. Also in this case, among the
mRNAs species, gag/pol was consistently the
most abundant transcript, p28, p22/p20-2 was
approximately 15 fold lower than gag/pol,
followed by tax/rex and p? that were present at
approximately 25 fold lower than the unspliced
mRNA coding for gag/pol . Very low
levels of expression were found for p28,
p22/p20-1, while env and p10/p11 transcripts
were below the limit of detection. In Fig. 5B the
fold variation analysis showed that the first
mRNAs expressed were tax/rex and
p28,p22/p20-1 with a peak at 4 hours followed
by all the other transcripts that showed a later
peak at 24 hours.These results indicate that tax/rex is the earliest
transcript expressed, while the other genes,
coding for accessory and structural proteins, are
expressed in a later phase of the viral cycle.
Conclusions:
The expression of different HTLV-2 genes
follows a distinct timing both in infected cell
lines and PBMCs isolated from infected patients.
The transcript tax/rex is the first to be
expressed, thus indicating that it is necessary at
the beginning of the infection cycle to
transactivate and regulate viral and cellular
transcripts. These results also suggest that the
control of viral gene expression is highly
regulated both in its kinetics and expression
level
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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