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Biomedical subjects

R Gajardo

Publications and source records attributed to R Gajardo.

14 recordsLinked to original sources
PubMed2006

Critical factors influencing prion inactivation by sodium hydroxide.

BACKGROUND AND OBJECTIVES: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by aberrantly folded cellular proteins (PrP(Sc); prions) that are generally resistant to conventional pathogen-inactivation techniques. To ensure effective decontamination and inactivation of prions that could be present in source material, we investigated critical factors that influence prion inactivation by NaOH. MATERIALS AND METHODS: A decrease in prion infectivity correlates with the disappearance of the protease-resistant core of PrPSc (PrPRES) observed in biochemical assays. To model prion inactivation, hamster scrapie (strain 263K) brain homogenate (SBH) was incubated for specific periods of time in 0.1 m NaOH at 4 or 18 degrees C, with or without detergent. Neutralized samples were subjected to limited digestion with proteinase K (PK) and then analysed using an endpoint dilution western blot assay and antibody 3F4. Structural changes in prions exposed to NaOH were examined using differential immunoprecipitation. RESULTS: Treatment of SBH with 0.1 m NaOH for 15 min, in the absence of detergent, at 4 and 18 degrees C caused a reduction in the PrP(RES) signal of 3.5 and 4.0 log10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60-min incubation in NaOH further enhanced PrPRES reduction to > or = 4.5 log10 units (i.e. below the limit of detection). NaOH treatment induced conformational changes in PrP that resulted in the exposure of a hidden epitope and enabled prion immunoprecipitation by antibody 3F4. CONCLUSIONS: The use of NaOH can effectively reduce prion levels in an in vitro inactivation assay. After pretreatment of SBH with detergent, NaOH completely eliminates the PrPRES signal. Detergent may liberate lipid membrane-protected PrPSc to improve access to NaOH, which can then inactivate PrPSc by altering its structure. In cases of unidentified exposure to PrPSc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.

Animals↗
PubMed2001

Activation of the interferon-inducible protein kinase PKR by hepatocellular carcinoma derived-hepatitis C virus core protein.

Hepatitis C virus (HCV) is a major etiological agent of chronic liver disease and hepatocellular carcinoma (HCC). We demonstrate herewith that HCV core proteins encoded by sequences isolated from HCC tumor tissues, but not those derived from their non-tumor counterparts in the same liver, co-localise in vitro and in vivo and co-immunoprecipitate with PKR in hepatocytic Huh7 cells. We show that this association in fact augments the autophosphorylation of PKR and the phosphorylation of the translation initiation factor eIF2alpha, which are two markers of PKR activity. The present study therefore identifies a novel model of virus-cell interactions whereby a viral protein, the HCV core, activates PKR activity.

Amino Acid Sequence↗
PubMed2001

Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner.

The impact of hepatitis C virus NS5A protein mutations on interferon alfa (IFN-alpha) signaling pathway, cell proliferation, and viability is an important issue that is still under debate. We have therefore combined transient and stable expression in a human hepatocytic cell line (Huh7) of 3 full-length NS5A sequences, isolated from patients with or without response to IFN-alpha therapy. Expression of all 3 NS5A-reduced IFN-alpha global antiviral activity on both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) replication. We did not show, however, an effect of these 3 NS5A proteins on double-stranded RNA-dependent kinase (PKR) expression and activity as well as colocalization and coimmunoprecipitation between NS5A and PKR. We also failed to show an effect of the 3 NS5A mutants tested on cell proliferation and viability. Overall, our results support an important role of NS5A in controlling IFN-alpha antiviral activity; they show, however, that PKR-independent mechanisms are implicated, at least in liver-derived cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1998

A new continuous epitope of hepatitis A virus.

A new continuous epitope of hepatitis A virus (HAV) was defined in the VP3 protein. Convalescent sera recognised the synthetic peptide 3110-3121 (FWRGDLVFDFQV). The replacement of the arginine, glycine, or aspartic acid at positions 112, 113, or 114, respectively by other amino acids induced the loss of synthetic peptide recognition by human convalescent sera, thereby confirming the presence of an epitope in the original VP3(110-121) sequence. Shorter VP3 peptides such as VP3(110-119). VP3(110-117), and VP3(110-116) and a tandem repeat of VP3(111-116) failed to react with convalescent sera, indicating the importance of the entire peptide in the epitope structure. The maximum inhibition of human convalescent binding to HAV by the VP3(110-121) peptide was around 60%, and 50% inhibition was achieved at a peptide concentration of 2.3-2.4 micrograms/ml. Antibodies generated by this peptide bound to intact HAV and neutralised its infectivity. Antipeptide antibodies inhibited convalescent serum binding to HAV. Monoclonal antibodies H7C27 and MAK-4E7 inhibited completely binding of the antipeptide antibodies to HAV.

Antibodies, Monoclonal↗
PubMed1997

Astrovirus survival in drinking water.

A method based on infection of CaCo-2 cultured cell monolayers (CC) and reverse transcription-PCR (RT-PCR) was developed for the specific detection of infectious astrovirus. The procedure was validated by titrating poliovirus stocks in parallel in CaCo-2 cells by determining the most probable number of cytopathogenic units and by cell culture and subsequent RT-PCR (CC-RT-PCR). CC-RT-PCR was then employed to measure the persistence of astrovirus suspended in dechlorinated tap water. After 60 days, the decay of astrovirus infectivity was 2 log units at 4 +/- 1 degrees C and 3.2 log units at 20 +/- 1 degrees C, while after 90 days, the titer reduction was 3.3 and 5 log units at 4 +/- 1 degrees C and 20 +/- 1 degrees C, respectively. Astrovirus decay in the presence of free chlorine (FC) was monitored by CC-RT-PCR. Residual infectivity was found after 2 h in the presence of 1 mg of FC/liter. Under these conditions, astrovirus shows a log titer reduction (LTR) or 4, while 0.5 mg of FC/liter induced an LTR of 2.4. The possibility of acquiring data on the survival of fastidious viruses in the environment opens new perspectives on the epidemiology of some significant infections transmitted by the fecal-oral route.

Astroviridae Infections↗
PubMed1997

Two proline residues are essential in the calcium-binding activity of rotavirus VP7 outer capsid protein.

Rotavirus maturation and stability of the outer capsid are calcium-dependent processes. It has been shown previously that the concentration of Ca2+-solubilizing outer capsid proteins from rotavirus particles is dependent on the virus strain. This property of viral particles has been associated with the gene coding for VP7 (gene 9). In this study the correlation between VP7 and resistance to low [Ca2+] was confirmed by analyzing the origin of gene 9 from reassortant viruses prepared under the selective pressure of low [Ca2+]. After chemical mutagenesis, we selected mutant viruses of the bovine strain RF that are more resistant to low [Ca2+]. The genes coding for the VP7 proteins of these independent mutants have been sequenced. Sequence analysis confirmed that these mutants are independent and revealed that all mutant VP7 proteins have proline 75 changed to leucine and have an outer capsid that solubilized at low [Ca2+]. The mutation of proline 279 to serine is found in all but two mutants. The phenotype of mutants having a single proline change can be distinguished from the phenotype of mutants having two proline changes. Sequence analysis showed that position 75 is in a region (amino acids 65 to 78) of great variability and that proline 75 is present in most of the bovine strains. In contrast, proline 279 is in a conserved region and is conserved in all the VP7 sequences in data banks. This region is rich in oxygenated residues that are correctly allocated in the metal-coordinating positions of the Ca2+-binding EF-hand structure pattern, suggesting that this region is important in the Ca2+ binding of VP7.

Amino Acid Sequence↗
PubMed1996

Non isotopic automatable molecular procedures for the detection of enteroviruses.

Five microwell non isotopic hybridization assays, based on colorimetric immunoenzymatic reading, were developed and evaluated for the rapid and automatable detection of enteroviruses. Virus nucleic acids and/or capture probes were covalently bound to microtiter wells, and digoxigenin-11-dUTP was used as label for the detection of hybridized material. Among these procedures, a reverse transcriptase polymerase chain reaction (RT-PCR) hybridization assay was the most sensitive, enabling the detection of 10 MPNCU of poliovirus, and offering detection specificity for other enteroviruses, such as coxsackieviruses and echoviruses. The second most sensitive method was a complementary hybridization assay, simultaneously using three detection probes, one from the 5' end and two from the 3' end of poliovirus genome, offering a sensitivity for poliovirus detection of 5 x 10(3) MPNCU.

Automation↗
PubMed1996

Detection of infectious astroviruses in water.

A method based on the infection of CaCo-2 cells and molecular hybridization with a specific cDNA probe has been developed for the detection of infectious astroviruses in environmental samples. By this procedure wild-type astroviruses have been detected in water from an area where a concurrent gastroenteritis outbreak was reported.

Journal Article↗
PubMed1996

Detection of infectious astroviruses in water.

Volume 62, no. 5, p. 1811, column 2, line 2: Reference "(16)" should read "(15)." Page 1812, column 1, line 17: "Willcocks and Carter (15)" should read "Willcocks et al. (14)." [This corrects the article on p. 1811 in vol. 62.].

Journal Article↗
PubMed1996

The concentration of Ca2+ that solubilizes outer capsid proteins from rotavirus particles is dependent on the strain.

It has been previously shown that rotavirus maturation and stability of the outer capsid are calcium-dependent processes. More recently, it has been hypothesized that penetration of the cell membrane is also affected by conformational changes of the capsid induced by Ca2+. In this study, we determined quantitatively the critical concentration of calcium ion that leads to solubilization of the outer capsid proteins VP4 and VP7. Since this critical concentration is below or close to trace levels of Ca2+, we have used buffered solutions based on ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca-EGTA. This method allowed us to show a very high variability of the free [Ca2+] needed to stabilize, at room temperature, the outer capsid of several rotavirus strains. This concentration is about 600 nM for the two bovine strains tested (RF and UK), 100 nM for the porcine strain OSU, and only 10 to 20 nM for the simian strain SA11. Titration of viral infectivity after incubation in buffer of defined [Ca2+] confirmed that the loss of infectivity occurs at different [Ca2+] for these three strains. For the bovine strain, the cleavage of VP4 by trypsin has no significant effect on the [Ca2+] that solubilizes outer shell proteins. The outer layer (VP7) of virus-like particles (VLP) made of recombinant proteins VP2, VP6, and VP7 (VLP2/6/7) was also solubilized by lowering the [Ca2+]. The critical concentration of Ca2+ needed to solubilize VP7 from VLP2/6/7 made of protein from the bovine strain is close to the concentration needed for the corresponding virus. Genetic analysis of this phenotype in a set of reassortant viruses from two parental strains having the phenotypes of strains OSU (porcine) and UK (bovine) confirmed that this property of viral particles is probably associated with the gene coding for VP7. The analysis of VLP by reverse genetics might allow the identification of the region(s) essential for calcium binding.

Animals↗
PubMed1995

Genotyping of rotaviruses isolated from sewage.

Rotaviruses from environmental samples have been genotyped by a seminested reverse transcription PCR assay with serotype-specific primers derived from variable regions of gene 9, which produce different characteristic segment sizes for serotypes 1 to 4. The method enabled the detection and identification of type 1, 2, and 3 group A rotaviruses in sewage.

Genotype↗
PubMedDate not supplied

Adsorption-elution with negatively and positively-charged glass powder for the concentration of hepatitis A virus from water.

Two methods based on virus adsorption and elution from glass powder were developed for the concentration of hepatitis A virus (HAV) from large volumes of water. The cytopathogenic pHM-175 strain of HAV was used to test these procedures in tap water, fresh water, sea water and raw sewage. HAV was quantitated by a plaque assay in the FRhK-4 cell line. HAV was concentrated by glass powder adsorption-elution from 20-liter samples with satisfactory efficiencies in all types of water: 100% for tap water, 80% for freshwater, 75% for sea water and 61% for sewage. The charge of glass powder was modified by polyethylenimine treatment to avoid the need to pretreat the sample. Concentration efficiencies of HAV in 20-1 samples through adsorption to and elution from positively-charged glass powder were 100% for tap water, 94% for sea water, and 61% for fresh water and sewage. Both methods were used for the detection of wild-type HAV in raw sewage. Wild-type HAV in concentrated sewage samples was detected by molecular hybridization with a digoxigenin-labelled cDNA probe.

Adsorption↗
PubMedDate not supplied

[Blood pressure measurement with Doppler in normal newborn infants and infants].

Systolic blood pressure (SBP) was measured by Doppler method in an aleatory sample of 251 healthy children from south-east Santiago Chile (131 females and 120 males) which were divided by age in five groups: 0 to 28 days (n = 5) 1 to 5 months (n = 48), 6 to 11 months (n = 48), 12 to 17 months (n = 46) and 18 to 24 months (n = 45). Great dispersion of SBP values occurred among newborn infants (range 85 to 125) in comparison with groups 1 to 5 months (105 to 130), 6 to 11 months (118 to 130). Fifty and 95 percentiles were, respectively, 107 to 125; 116 to 130; 121 to 130; 118 to 130 and 120 to 130. These values were greater than those obtained by sphygmomanometry in a similar matched group of children.

Age Factors↗