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M Gorziglia

Publications and source records attributed to M Gorziglia.

67 records · Page 4Linked to original sources
PubMed1987

Development of a defined medium and two-step culturing method for improved exotoxin A yields from Pseudomonas aeruginosa.

A two-step method is described for the production of exotoxin A by Pseudomonas aeruginosa in which a defined growth medium is modified for the toxin production phase. As a result, specific exotoxin A yields comparable to those obtained with complex media were achieved. In the development of this two-step process, several divalent metallic cations (Ca2+, Cu2+, and Mn2+), in addition to iron, were found to inhibit the yield of exotoxin A while Ca2+ and glycerol were found to increase yields. Northern blot analysis of total RNA isolates suggests that these effects on exotoxin A yields are based on events at the transcription level.

ADP Ribose Transferases↗
PubMed1986

Conservation of amino acid sequence of VP8 and cleavage region of 84-kDa outer capsid protein among rotaviruses recovered from asymptomatic neonatal infection.

Within the past few years, rotavirus strains were recovered from four discrete prolonged outbreaks of infection in newborn nurseries in which affected infants failed to develop significant symptoms. The virus strains recovered from each outbreak belonged to a different human rotavirus serotype and thus each of the four human rotavirus serotypes was associated with asymptomatic infection of neonates. Marked conservation of sequence was observed among the fourth genes of the nursery rotavirus strains in a previous study using RNA X RNA hybridization, while a different conserved set of fourth gene sequences was identified among virulent human rotaviruses representing the four known serotypes. In the present study, this sequence dimorphism was further evaluated by comparing the sequence of the region of the fourth gene of virulent and asymptomatic human rotaviruses that codes for the VP8 protein, downstream cleavage sites, and the NH2 terminus of VP5. The corresponding sequences of a simian rotavirus were also determined. The fourth segment (+) strand RNA has a 5' conserved nontranslated sequence of nine nucleotides and encodes a VP8 protein of 240 amino acids in human rotavirus strains and 241 amino acids in simian rotavirus strains. Human and simian rotaviruses exhibit many similarities in this region of their genome, including identical NH2-terminal amino acid sequences, conservation of arginine at the two trypsin cleavage sites, and the position of a cysteine residue. Alignment of amino acid sequences of the VP8 protein, the downstream cleavage region, and the NH2 terminus of VP5 of asymptomatic and virulent human rotavirus strains indicates a high degree of homology (96% or more) among the asymptomatic viruses (serotypes 1, 2, 3, and 4), while homology between asymptomatic strains and virulent viruses is considerably less (68-72%). A high degree of conservation of amino acid sequence (92-97%) is also observed among three of the virulent strains (serotypes 1, 3, and 4). At 48 positions in the protein sequence of VP8, the cleavage region, and the NH2 terminus of VP5, an amino acid is conserved among asymptomatic rotaviruses, while a different amino acid is conserved among virulent rotaviruses. Notably, three of these differences are located within the cleavage region between VP8 and VP5. These findings suggest that the fourth genes of virulent and asymptomatic human rotavirus strains represent two lines of divergent evolution from a common ancestor. Also, it is possible that this sequence dimorphism may be responsible in part for the difference in virulence between these two groups of human rotaviruses.

Amino Acid Sequence↗
PubMed1986

VP7 serotype-specific glycoprotein of OSU porcine rotavirus: coding assignment and gene sequence.

With a reassortant from a cross of human rotavirus DS-1 (serotype 2) and OSU (serotype 5) it was determined that the OSU major neutralization glycoprotein antigen (VP7) was encoded by gene segment 9. A full-sized cloned cDNA copy of the OSU gene 9 was produced and sequenced. Hybridization of such labelled cDNA with the corresponding segment of a reassortant DS-1 X OSU virus confirmed the coding assignment. Comparison of the deduced amino acid sequence of the VP7 of OSU with those previously determined for five other rotavirus strains, representing four distinct serotypes, revealed some hydrophilic regions that exhibited significant homology and other hydrophilic domains with greater amino acid divergence. In one of the latter hydrophilic domains each of the five serotypes had a distinct amino acid substitution at residue 146, suggesting that it may be involved in serotype specificity.

Amino Acid Sequence↗
PubMed1986

Conservation of the fourth gene among rotaviruses recovered from asymptomatic newborn infants and its possible role in attenuation.

RNA-RNA hybridization was performed to assess the extent of genetic relatedness among human rotaviruses isolated from children with gastroenteritis and from asymptomatic newborn infants. 32P-labeled single-stranded RNAs produced by in vitro transcription from viral cores of the different strains tested were used as probes in two different hybridization assays: undenatured genomic RNAs were resolved by polyacrylamide gel electrophoresis, denatured in situ, electrophoretically transferred to diazobenzyloxymethyl-paper (Northern blots), and then hybridized to the probes under two different conditions of stringency; and denatured genomic double-stranded RNAs were hybridized to the probes in solution and the hybrids which formed were identified by polyacrylamide gel electrophoresis. When analyzed by Northern blot hybridization at a low level of stringency, all genes from the strains tested cross-hybridized, providing evidence for some sequence homology in each of the corresponding genes. However, when hybridization stringency was increased, a difference in gene 4 sequence was detected between strains recovered from asymptomatic newborn infants ("nursery strains") and strains recovered from infants and young children with diarrhea. Although the nursery strains exhibited serotypic diversity (i.e., each of the four strains tested belonged to a different serotype), the fourth gene appeared to be highly conserved. Similarly, each of the virulent strains tested belonged to a different serotype; nonetheless, there was significant conservation of sequence among the fourth genes of three of these viruses. Significantly, the conserved fourth genes of the nursery strains were distinct from the fourth gene of each of the virulent viruses. These results were confirmed and extended during experiments in which the RNA-RNA hybridization was carried out in solution and the resulting hybrids were analyzed by polyacrylamide gel electrophoresis. Under these conditions, the fourth genes of the nursery strains were closely related to each other but not to the fourth genes of the virulent viruses. Full-length hybrids did not form between the fourth genes from the nursery strains and the corresponding genes from the strains recovered from symptomatic infants and young children.

Diarrhea, Infantile↗
PubMed1985

Biochemical evidence for the oligomeric (possibly trimeric) structure of the major inner capsid polypeptide (45K) of rotaviruses.

The major structural polypeptide of rotaviruses is p45K (VP6), which forms the morphological subunit of the inner capsid. Such subunits show a trimeric structure when examined with the electron microscope. Treatment of single-capsid rotavirus particles with 1.5 M-CaCl2 removes p45K, resulting in the generation of smooth cores. Sucrose density gradient centrifugation analysis of the removed p45K revealed that it has a sedimentation coefficient close to 7.3S, compatible with an oligomeric (possibly trimeric) structure. Polyacrylamide gel electrophoresis under reducing or non-reducing conditions indicated that p45K has intramolecular but not intermolecular disulphide bonds, suggesting that interactions between p45K monomers may be due to some other type of association, such as hydrophobic or charge interactions. Velocity sedimentation of infected cell extracts revealed that native p45K also behaves as an oligomeric protein. Such results were confirmed using p45K partially purified by DEAE-cellulose chromatography. The evidence obtained indicated that all p45K present in the virion is in the oligomeric form, not associated by disulphide bonding, and that most native p45K present in the infected cells is also in the oligomeric form, probably as a consequence of early protein-protein interaction in rotavirus morphogenesis.

Animals↗
PubMed1984

Epidemiological aspects of porcine rotavirus infection in Venezuela.

The prevalence of porcine rotavirus infection in Venezuela was studied from November 1980 to November 1981. Rotaviruses were identified in 21.3 per cent of 286 diarrhoeic samples analysed by electron microscopy and in none of the 48 controls. Rotaviruses were readily detected throughout the year, which may correspond to the absence of seasonal climatic variations in a tropical country such as Venezuela. Rotavirus infection was frequently observed in pigs that were two to six weeks old and was rare in younger animals. The age distribution of rotavirus infection correlated well with the presence of circulating antibodies, as measured by the complement fixation test. Sucking pigs less than two weeks old appeared to be protected from the infection by passive immunity; no antibodies were detected in the two- to three-week-old group, coinciding with an increased susceptibility to rotavirus infection. The antigenic specificity of the rotavirus isolates was confirmed by enzyme-linked immunosorbent assay test, using antibodies directed against the group-specific antigen of human rotavirus. Polyacrylamide gel electrophoresis of viral double-stranded RNA revealed an heterogeneity on the electropherotype of selected samples obtained at different times during the study period.

Age Factors↗
PubMed1983

Molecular cloning of a human rotavirus genome.

The genes of a field isolate of human rotavirus were cloned into pBR322. The strategy used involved polyadenylation of denatured double-stranded (ds) genomic RNA, followed by cDNA synthesis using reverse transcriptase. Oligo(dC) tails were added to the 3' end of the single-stranded cDNA and then separated by alkaline agarose electrophoresis. Sized cDNA of both polarities were allowed to hybridize and inserted into the PstI site of pBR322. Transformations done with sized cDNA always resulted in the selection of hybrid plasmids carrying inserts with a size representative of the original cDNA. Four individual clones were selected for preliminary characterization. One clone has an insert of 1360 base pairs (bp) and corresponds to gene 6. The second clone has an insert of 1140 bp and corresponds to one of the genes in the triplet 7-8-9. The other two genes, with inserts of 780 and 660 bp, were identified as corresponding to dsRNA segments 10 and 11.

Base Sequence↗
PubMed1981

Poly(A) polymerase activity in human rotavirus.

A poly(A) polymerase enzymic activity was found in partially purified preparations of a human rotavirus. The activity was demonstrated using conditions similar to those utilized for the detection of the poly(A) polymerase previously described in reovirus (incubation at 43 degrees C in 70 mM-tris buffer pH 7.5 containing 12 mM-Mn2+). The enzymic activity was associated only with complete, double-shelled particles. Characterization of the poly(A)-containing product of the in vitro reaction by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose in the presence of 7 M-urea, showed that it is composed of oligonucleotides of a chain length similar to, or slightly larger than, those observed in reovirus.

Adenosine Triphosphate↗
PubMed1980

Multiplication of human rotavirus in cultured cells: an electron microscopic study.

Human rotaviruses were capable of efficient multiplication in LLC-MK2 cells when the inoculum was pre-treated with trypsin, centrifuged on to the cell monolayer and the infected cells maintained in a medium containing trypsin. However, not all of the human rotavirus isolates used to infect cells resulted in efficient virus production. The ability of human isolates to multiply in cultured cells was studied by direct observation of virus in the electron microscope, by radioactive labelling with 3H-uridine of the newly synthesized virus and by electron microscopic examination of thin sectioned infected cells. With one of the specimens used (F-617) only 5 to 10% of the cells showed evidence of virus multiplication, with the great majority of the infected cells showing numerous complete (double-capsid) virus particles scattered in the cytoplasm. When cells were inoculated with another human specimen (SIB-I), infected cells were more abundant, reaching a maximum of 60%; however, a variety of particle types, probably representing different subviral structures or different steps of rotavirus morphogenesis, were commonly observed. The presence of these aberrant or incomplete virus structures may represent a manifestation of the defectiveness of this virus and may explain the difficulties encountered in its serial passage.

Animals↗
PubMed1980

Preliminary characterization of virus-like particles in a mosquito (Aedes pseudoscutellaris) cell line (Mos. 61).

Electron microscopic examination of an Aedes pseudoscutellaris mosquito cell line (Mos. 61) revealed the presence of a large number of virus-like particles (VLP) in the cytoplasm of approximately 10% of the cells. These particles have a diameter of 36 nm, do not contain a lipid envelope, and have a buoyant density of 1.40 g/ml in CsCl. VLP contain DNA which appears to be single-stranded by SU nuclease assay. PAGE of the VLP demonstrates the presence of four structural polypeptides with molecular weights of 52K, 70K, 75K and 100K daltons, the smaller one being the majro polypeptide species and accounting for 71% of the the total protein mass. The A. pseudoscutellaris VLP appear to share some, but not all, of the characteristics of the genus Densovirus, family Parvoviridae. Their possible biologic importance and taxonomic location are still unclear.

Aedes↗
PubMedDate not supplied

Prospects for development of a rotavirus vaccine against rotavirus diarrhea in infants and young children.

Major advances have been made in elucidating the etiologic agents of severe infantile diarrhea, and it is clear that rotaviruses are the single most important etiologic agents. Progress in the development of rotavirus vaccine candidates has also moved swiftly with the "Jennerian" approach, in which a related live, attenuated rotavirus strain from a nonhuman host is used as the immunizing antigen. If this strategy is not effective against all rotavirus serotypes, reassortant rotaviruses hold great promise for the development of a multivalent vaccine. Field trials with the "Jennerian" approach vaccines are under way, and phase 1 trials with the reassortants have been initiated.

Animals↗