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Biomedical subjects

E G Kroon

Publications and source records attributed to E G Kroon.

At least 19 recordsLinked to original sources
PubMed2003

The use and misuse of the "impact factor" as a parameter for evaluation of scientific publication quality: a proposal to rationalize its application.

We present a critical analysis of the generalized use of the "impact factor". By means of the Kruskal-Wallis test, it was shown that it is not possible to compare distinct disciplines using the impact factor without adjustments. After assigning the median journal the value of one (1.000), the impact factor value for each journal was calculated by the rule of three. The adjusted values were homogeneous, thus permitting comparison among distinct disciplines.

Bibliometrics↗
PubMed2002

Frequency of p12K and p12R alleles of HTLV Type 1 in HAM/TSP patients and in asymptomatic HTLV type 1 carriers.

HTLV-1 has a complex genome, and contains four open reading frames (ORFs) in the 3' region encoding viral and cellular regulatory proteins. p12 is a small, ORF I-encoded hydrophobic protein, the function of which is not well understood. It has been shown that p12 enhances the E5-transforming ability of bovine papillomavirus; and binds to the 16-kDa subunit of the vacuolar ATPase pump, immature forms of the beta and gamma(c) chains of the interleukin 2 receptor, and the free chain of MHC I. p12 carrying a lysine residue (p12K) at position 88 of its sequence may be rapidly degraded in the cell via proteasome, whereas p12 with an arginine residue (p12R) at the same position is severalfold more stable. These alleles are found in proviral DNA of HTLV-1-infected individuals and it was previously observed that the p12K allele was more frequent in HAM/TSP (HTLV-1-associated myelopathy/tropical spastic paraparesis) patients and was not found at all in asymptomatic carriers, whereas patients with adult T cell leukemia/lymphoma (ATLL) carry the p12R allele. To extend these observations and verify whether the p12K mutation could be used as a marker of progression to HAM/TSP, we analyzed 37 HAM/TSP patients and 40 asymptomatic carriers at different stages of infection. In our cohort, only one HAM/TSP patient carried the p12K phenotype, which accounted for a frequency of 2.7% (1 of 37). We also found, among the 40 asymptomatic HTLV-1 carriers, one who presented the p12K phenotype, contrasting with previous publications. Thus, p12K does not seem to be universally diagnostic for HTLV-1-associated neurological disease. Further screening of HTLV-1-infected individuals in other populations may elucidate this observation.

Adult↗
PubMed2001

A mitogenic signal triggered at an early stage of vaccinia virus infection: implication of MEK/ERK and protein kinase A in virus multiplication.

Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF(-)). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNA-protein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF(-)) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.

Animals↗
PubMed2001

Characterization of ATI, TK and IFN-alpha/betaR genes in the genome of the BeAn 58058 virus, a naturally attenuated wild Orthopoxvirus.

The lack of knowledge about the natural host of Vaccinia virus (VV) along with the description of human infections caused by poxviruses after smallpox eradication has increased the need to characterize poxviruses isolated from the wild. Moreover, in the past years poxviruses have been widely studied as potential vaccination tools, with the discovery of several genes implicated in the evasion of the host immune response involved in virus pathogenesis. Among them, an Interferon (IFN)-binding protein was identified in the supernatant of VV strain WR infected cells coded by the B18R gene. It was shown that many other Orthopoxviruses also encode and express this soluble receptor although some VV strains such as Lister and modified Ankara, which were less reactogenic vaccines, do not. The BeAn 58058 virus (BAV) has been recently characterized and proposed to be an Orthopoxvirus. BAV was also shown to be less virulent in animal models than VV Lister. Here we report the identification of an IFN-alpha/betaR gene in the BAV genome with 99% of sequence identity with the VVWR B18R gene. The identified gene encodes a B18R-like IFN binding protein as demonstrated by its capacity to inhibit the IFN-mediated protection of VERO cells against EMC virus. In order to better characterize the virus we have searched for the A type inclusion body (ATI) gene currently used in the classification of Orthopoxviruses but did not detect it in the BAV genome. We have also sequenced the BAV thymidine kinase (TK) gene, a poxvirus-conserved gene, which, as expected, showed high homology with the TK gene of other poxviruses. Phylogenetic trees were constructed based on sequences of the IFN-alpha/betaR and TK genes from several poxviruses and in both cases BAV was placed in the same cluster as other VV strains. These observations strengthened the hypothesis that this virus is a variant of the VV vaccine used in Brazil. However the explanation for the BAV lack of virulence remains to be discovered.

Amino Acid Sequence↗
PubMed2001

Detection of herpesvirus DNA by the polymerase chain reaction (PCR) in vitreous samples from patients with necrotising retinitis.

AIMS: Viral uveitis and retinitis, usually caused by herpesviruses, are common in immunosuppressed patients. The diagnosis of viral anterior uveitis and retinitis is usually clinical. The polymerase chain reaction (PCR) has been used for the diagnosis of some viral infections, especially those caused by herpesviruses. This paper reports the use of PCR in the diagnosis of viral retinitis in vitreous samples from Brazilian patients. METHODS: PCR was used for the diagnosis of necrotising retinitis in vitreous samples from patients from the Hospital São Geraldo, Universidade Federal de Minas Gerais, Brazil. The vitreous samples were collected by paracentesis and stored until analysis. Samples were analysed by PCR using specific primers designed to amplify herpes simplex virus 1 (HSV-1), varicella zoster virus (VZV), or human cytomegalovirus (HCMV). In a case of anterior uveitis, PCR was performed with a sample from the anterior chamber. RESULTS: Herpesvirus DNA was amplified in 11 of 17 samples. HCVM DNA was detected in nine samples but DNA from HSV-1 and VZV were detected only once each. CONCLUSION: These results strongly suggest that PCR could be used for a rapid complementary diagnosis of viral uveitis and retinitis. A prospective study to evaluate the PCR results, clinical evolution, and treatment is imperative to corroborate the real value of PCR in diagnosis and how it could help the clinicians' approach.

AIDS-Related Opportunistic Infections↗
PubMed2000

Heteroduplex mobility assay for rapid, sensitive and specific detection of mycobacteria.

We report an improved method for the detection and identification of mycobacteria using PCR and the heteroduplex mobility shift assay (HMA). The HMA for detection of mycobacteria was based on the microheterogeneity within the DNA coding sequences for 16S rRNA. A remarkable shift between single-stranded, heteroduplex and homoduplex bands in PAGE was observed among the Mycobacterium spp. tested. The Mycobacteria HMA (MHMA) of amplified PCR products from mycobacteria DNA coding for 16S rDNA derived from culture showed a specific heteroduplexes formed among different Mycobacterium species. Other bacterium species were distinguished from Mycobaterium due to slow migrating heteroduplexes mobility bands observed when M. bovis (BCG), M. avium, or M. fortuitum were used as a standard. The specific heteroduplexes were detected when as little as 1 etag of DNA template was used, although better results were obtained with 5 etag and when PCR products of sample test and mycobacterium standard were mixed at a ratio of 1.8. To correctly evaluate the feasibility of using MHMA to detect and identify mycobacteria, 15 clinical sample patients were tested. All MTB-positive clinical samples were identified by MHMA as well as the negative samples. In addition, MHMA will, in principle, be applicable to the detection and classification of any microorganism showing differences within the 16S rRNA as well as to the identification of new and unrecognized bacterial species.

Base Sequence↗
PubMed2000

Comparison of virus isolation and various polymerase chain reaction methods in the diagnosis of mucocutaneous herpesvirus infection.

We compared two polymerase chain reaction (PCR) assays (simple and multiplex) and viral isolation to detect herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) in 15 clinical specimens from 13 patients with mucocutaneous herpetic infections. HSV-1 or VZV DNA was detected in 13 specimens by simple PCRs (HSV-1 or VZV PCR) and in 12 specimens by multiplex PCR. On the other hand, viral isolation was positive for 9 specimens only. The PCR protocols used in this study are not only more sensitive and faster than the traditional viral isolation and conventional PCR protocols but also can distinguish rapidly HSV-1 from VZV. We propose the PCRs described here for rapid and precise identification of etiological agents of mucocutaneous herpetic infections.

Adolescent↗
PubMed1999

Protein domains involved in nuclear transport of Fos.

The protein encoded by the proto-oncogene c-fos is constitutively nuclear in most cell types analyzed. It has a predicted molecular weight of about 55 kDa. Proteins with a molecular weight above 40 kDa cannot enter the nucleus passively. Our interest was to study which regions in the protein are involved in the nuclear transport. We prepared a series of deletions and point mutations of the protein and cloned the mutated genes into a eukaryotic expression vector. Cos-1 cells were used to express the mutants transiently. Using indirect immunofluorescence we studied the subcellular localization, analyzing the percentage of cells containing the protein in the nucleus, the cytoplasm, or both locations. Our studies showed that the Fos protein contains several regions which can act independently to translocate the protein into the nucleus.

Animals↗
PubMed1999

Recovering cDNA bands from differential display RT-PCR gels using a transparency film mask.

We describe here the use of a transparency film as a mask for recovering bands from differential display reverse transcription-polymerase chain reaction (DDRT-PCR) gels. This method represents a simple and rapid way to isolate the differentially expressed bands from dried and nondried polyacrylamide, radioactive and nonradioactive, denaturing and native DDRT gels. A transparency film is overlaid on the DDRT autoradiogram, and the marks and bands of interest are drawn using a permanent marker. The reproduced band marks are cut out of the transparency sheet with a scalpel to facilitate the recovery of the desired bands. The transparency film mask is then overlaid on the top of the gel and the bands are recovered from the gel. The use of the transparency film mask avoids damage to the autoradiogram and is also extremely useful in DDRT-PCR experiments involving different RNA samples that produce band patterns of different intensities that require many X-ray exposures for different periods of time.

Autoradiography↗
PubMed1999

The genome of cowpox virus contains a gene related to those encoding the epidermal growth factor, transforming growth factor alpha and vaccinia growth factor.

Cowpox virus (CPV) is a member of the Orthopoxvirus genus and has the genetic capacity to encode a multitude of genes that interfere with the host inflammatory and immune response or modulate the physiological state of infected and non-infected cells. Among these CPV factors are receptors homologous to interferon and tumor necrosis factor receptors and also a viral cellular serine-proteinase analog. Here we describe the detection of a CPV gene that encodes a protein homologous to epidermal growth factor, transforming growth factor alpha and poxvirus growth factors, such as the vaccinia growth factor (VGF). The VGF and other poxvirus growth factors are produced early in the infection and are secreted into the medium where they bind to the EGF receptors, generating mytotic responses. The cowpox growth factor (CGF) gene was detected in three copies on the virus genome by PCR, and by northern and southern blot hybridization using VGF nucleotide sequences as primers and probes. The CPV gene has a strong nucleotide and predicted amino acid similarity with VGF, and is also produced early in the infection.

Amino Acid Sequence↗
PubMed1998

Morphological and molecular characterization of the poxvirus BeAn 58058.

BeAn 58058 virus (BAV) was isolated from an Oryzomis rodent in Brazil. BAV was shown to be antigenically related to another poxvirus also isolated in Brazil, the Cotia virus, but it remained ungrouped. Electron microscopy revealed that BAV has a typical poxvirus morphology. The Hind III DNA profile of BAV genome was similar with that of VV WR and Lister, but some differences in the profile were detected. We have also detected the presence of genes homologous to vaccinia virus (VV WR) genes in the genome of BAV. Genes related to vaccinia thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene were found. The patterns of TK and VGF mRNA transcripts described for vaccinia virus infected cells were observed in BAV infected cells. Nucleotide sequence of BAV VGF homologous gene was similar to VV WR VGF sequences. This similarity was further seen when cross-hybridization of total genomes of BAV and VV was done. Polypeptide synthesis of BAV and vaccinia in infected cells also showed similar profiles. The genetic data was used to construct a phylogenetic tree where BAV and VV were placed at the same cluster. Based on our findings we propose that BAV is a vaccinia virus variant.

Amino Acid Sequence↗
PubMed1998

Culture of human amniotic cells: a system to study interferon production.

This study investigated whether primary culture of human amniotic membrane cells (PCHAM) could be used as an in vitro model system for the study of interferon (IFN) production. PCHAM cells infected with Newcastle disease virus (NDV) produced the two antigenic types of IFN, previously shown in a amniotic membrane cells (HAM) system. PCHAM IFN was detected as early as 2 h after NDV infection and was composed by two antigenically distinct fractions, one neutralized with anti-HuIFN beta antibody and another that is not related to IFN beta, -alpha and -gamma. These fractions correspond respectively to 80 and 20 per cent of the IFN produced 4 h after virus induction, 55 and 45 per cent of the IFN produced from 4 to 12 h and 67 and 33 per cent of the IFN produced 12 h after virus induction. A cDNA library, established from PCHAM with or without NDV infection, was screened for IFN alpha and -beta using specific primers. The PCR product, amplified by IFN beta primers, was cloned, sequenced and expressed in Escherichia coli M15. The sequences of several cloned cDNAs were identical to HuIFN beta gene and the antiviral activity of the expressed protein was neutralized only by antiHuIFN-beta antibody. The other IFN fraction not neutralized by polyclonal antibodies anti-IFN beta, -alpha and -gamma is now being studied.

Amnion↗
PubMed1998

HIV-1 detection and subtyping by PCR and heteroduplex mobility assay in blood donors: can these tests help to elucidate conflicting serological results?

Testing blood donors for the human immunodeficiency viruses (HIV-1 and 2), requires serological tests that are frequently inconclusive. Peripheral blood mononuclear cells of 50 blood donors with the screening test (ELISA) reactive to HIV-1, but with indeterminate results in the first Western Blot (WB) performed, were submitted to the polymerase chain reaction (PCR) and heteroduplex mobility assay (HMA), a nonsequencing method that can distinguish between HIV-1 subtypes (A to I). PCR amplification of HIV-1 env gene regions has been obtained in 12 (24.0%) samples. HMA of the amplified DNA showed that 12 belonged to HIV-1 B subtype and one to F subtype. PCR testing of the amplified DNA helped to elucidate doubtful serological results and HMA proved to be a relatively simple and rapid way of subtyping HIV-1 for epidemiological purposes.

Adult↗
PubMed1996

Distinct antigenic subtypes of human beta interferon can be distinguished by neutralization.

Different molecular configurations of human beta interferon were titrated with the standard reference antiserum of the National Institutes of Health (NIH) which had been prepared with natural beta fibroblast interferon in order to determine to what extent differences in these configurations would influence the neutralization of the antiviral action of interferon. Neutralization tests were carried out in Vero cells by diluting both interferon and antiserum. Encephalomyocarditis virus was employed as challenge virus. The neutralization titer was considered to have been reached when the effect of eight units of interferon was reduced to one. Two natural beta interferons prepared from fibroblasts and from amniotic membranes gave similar high titers. However, titers were reduced five-fold with recombinant interferons expressed in Escherichia coli, which do not contain carbohydrate, one with the natural sequence and a mutant with a single amino acid substitution (cysteine for serine). The NIH antiserum did not neutralize the effect of a protein fraction from amniotic membranes antigenically different from the human alpha, beta or gamma interferons but having the biological activity of interferon. We conclude that the carbohydrate moieties of human beta interferons are essential for their recognition by the NIH antiserum and that antibodies specific for human recombinant beta interferon, which does not contain carbohydrate, are needed.

Amniotic Fluid↗
PubMed1991

Partial characterization of human amniotic membrane interferon.

1. The molecular weight and the electrofocusing profile of human amniotic membrane interferon (IFN-AM) were determined. 2. When submitted to gel filtration, IFN-AM showed a single 26-28 kDa component; in polyacrylamide gel electrophoresis one component of 19,500. 3. In electrofocusing, IFN-AM displayed a heterogeneous profile with 5 to 7 peaks, but different from human alpha or beta IFNs. This heterogeneity was reduced by previous treatment of IFN-AM with neuraminidase. 4. IFN-AM is a sialoglycoprotein similar to human beta IFN in terms of antigenicity but different from it in electrofocusing profile.

Chromatography, Gel↗
PubMed1989

Antigenic characterization of human interferon derived from amniotic membranes induced by virus.

The presence of protein(s) with interferon (IFN)-like activity in culture fluid from human amniotic membranes induced by viruses has been described by different groups. However, the antigenic structure of this protein is controversial. Here we report the presence of IFN activity in supernatants of human amniotic membranes induced by Sendai virus. The major component responsible for this antiviral activity seems to be the classical IFN-beta. However, we were able to demonstrate the presence of a protein fraction with antiviral activity that does not bind to an affinity column for IFN-beta. The antiviral activity of this unbound fraction cannot be neutralized by antibodies to IFN-alpha, -beta, gamma, or by a mixture of them. We called this unbound fraction IFN-AM. We also report the development of a monoclonal antibody that does not neutralize the antiviral activity of IFN-alpha or IFN-beta but reduces the antiviral activity of a partially purified preparation of Sendai virus-induced amniotic membrane supernatant. These observations suggest that the IFN-AM (the unbound fraction that lacks reactivity with antibodies against known IFNs) contains a unique antigenic determinant that is not present, or, if so, is not located at the functional domain of IFN-alpha, -beta, or -gamma.

Amnion↗
PubMedDate not supplied

The low proliferation rates of human amniotic cells are neither associated to deregulated proto-oncogenes' expression nor to the effect of IFN alpha 2.

Primary cultures of human amniotic membrane (PCHAM) cells display very low proliferation rates while their doubling times vary between 150 h and 210 h even after mitogenic stimuli. However, the pattern of proto-oncogenes (c-fos, c-myc and c-jun) expression in these cells, upon serum restimulation, resembled that of cell lines that display shorter population doubling times. Serum stimulation of quiescent PCHAM cells promoted a rapid and transient c-fos mRNA expression, which was detected within 10 min, reached maximal levels at 30 min and decreased to undetectable levels 2-3 h later. The levels of c-myc or c-jun mRNA increased within 10 min after serum restimulation, peaked at 3 h and decreased to intermediate levels thereafter. We also present evidence showing that IFN alpha 2 treatment of PCHAM cells had no effect on their population doubling times nor in c-fas, c-myc, or c-jun mRNA expression, under conditions in which induction of IFN-stimulated genes, such as 2'-5' oligo-adenylate synthetase (OAS) and 6-16 was observed. We conclude that the growth constraints observed with this cells are not directly associated with a negative cellular growth regulation exerted by IFN alpha 2, nor due to a deregulated proto-oncogenes' expression.

Amnion↗