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C D Sifri

Publications and source records attributed to C D Sifri.

8 recordsLinked to original sources
PubMed2001

A simple model host for identifying Gram-positive virulence factors.

We demonstrate the use of the nematode Caenorhabditis elegans as a facile and inexpensive model host for several Gram-positive human bacterial pathogens. Enterococcus faecalis, Streptococcus pneumoniae, and Staphylococcus aureus, but not Bacillus subtilis, Enterococcus faecium, or Streptococcus pyogenes, kill adult C. elegans. Focusing our studies on the enterococcal species, we found that both E. faecalis and E. faecium kill C. elegans eggs and hatchlings, although only E. faecalis kills the adults. In the case of adults, a low inoculum of E. faecalis grows to a high titer in the C. elegans intestine, resulting in a persistent infection that cannot be eradicated by prolonged feeding on E. faecium. Interestingly, a high titer of E. faecium also accumulates in the nematode gut, but does not affect the longevity of the worms. Two E. faecalis virulence-related factors that play an important role in mammalian models of infection, fsr, a putative quorum-sensing system, and cytolysin, are also important for nematode killing. We exploit the apparent parallels between Gram-positive infection in simple and more complex organisms by using the nematode to identify an E. faecalis virulence factor, ScrB, which is relevant to mammalian pathogenesis.

Animals↗
PubMed2000

Pyogenic liver abscesses.

Pyogenic liver abscess is a classic clinical entity whose presentation and management have evolved significantly with the advent of potent antimicrobials and the availability of improved diagnostic imaging. The classic triad of fever, upper right quadrant pain or fullness, and jaundice resulting from advanced pylephlebitis is now seldom seen. Despite these changes, pyogenic liver abscess remains an important clinical entity for which prompt recognition and treatment are essential to achieve a favorable outcome. This article discusses the presentation and diagnosis of and current therapy for liver abscesses.

Anti-Bacterial Agents↗
PubMed1995

Transfection of Plasmodium falciparum within human red blood cells.

Plasmodium falciparum malaria parasites within human red blood cells (RBCs) have been successfully transfected to produce chloramphenicol acetyltransferase (CAT). Electroporation of parasitized RBCs was used to introduce plasmids that have CAT-encoding DNA flanked by 5' and 3' untranslated sequences of the P. falciparum hsp86, hrp3, and hrp2 genes. These flanking sequences were required for expression as their excision abolished CAT activity in transfected parasites. Transfection signals from native CAT-encoding DNA compared well with those from a synthetic DNA sequence adapted to the P. falciparum major codon bias, demonstrating effective expression of the bacterial sequence despite its use of rare P. falciparum codons. Transfected ring-stage parasites produced CAT signals at least as strong as transfected schizont-stage parasites even though ring stages are surrounded by more RBC cytoplasm than schizonts. The transfection of erythrocyte-stage P. falciparum parasites advances our ability to pursue genetic analysis of this major pathogen.

Amino Acid Sequence↗
PubMed1994

Amplification of the inosinate dehydrogenase gene in Trypanosoma brucei gambiense due to an increase in chromosome copy number.

Drug resistance is a significant impediment to the therapy of African sleeping sickness in humans. To evaluate molecular mechanisms that contribute to drug resistance in African trypanosomes, a clonal strain of Trypanosoma brucei gambiense, MPA10, was selected in medium containing mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase (IMPDH) activity. IMPDH activity in MPA10 cells was approximately 6-fold higher than that of wild type parental cells, although the enzymes in both strains were equally sensitive to inhibition by MPA. To evaluate the mechanism of IMPDH overexpression in MPA10 cells, the gene encoding IMPDH (impdh) was isolated from a T.b. brucei library by cross-hybridization to the Leishmania donovani impdh. Sequence analysis indicated that the T. brucei IMPDH was 76% identical with the L. donovani IMPDH. The T. brucei impdh hybridized to a 2.7-kb transcript that was expressed at approximately 10-fold greater levels in the MPA10 cells, and this impdh overexpression could be ascribed to an approximately 10-fold amplification of the impdh copy number. Pulsed field gel electrophoresis revealed that the extra impdh copies in MPA10 cells were localized to an approximately 6.0-Mb chromosome that comigrated with the wild type chromosome encompassing impdh. The amplification of impdh could be ascribed to an increased copy number of this 6.0-Mb chromosome, and a predicted augmented DNA content in MPA10 cells was confirmed by flow cytometry. This is the first demonstration that DNA amplification can serve as a molecular mechanism by which T. brucei become resistant to cytotoxic drugs, and the amplification of the 6.0-Mb chromosome represents a novel mechanism of drug resistance in parasitic protozoa.

Animals↗
PubMed1994

Cloning and sequence analysis of a Drosophila melanogaster cDNA encoding IMP dehydrogenase.

A cDNA encoding the entire Drosophila melanogaster IMP dehydrogenase (IMPDH) protein was isolated and sequenced. Translation of the impdh cDNA nucleotide sequence indicated that the Drosophila IMPDH exhibited 48% and 65% amino acid identity to the Leishmania donovani and human isoform II counterparts, respectively. Northern analysis revealed that expression of the 2.4 kb impdh transcript was equivalent in adult Drosophila head and body and that impdh expression was developmentally regulated. In situ hybridization of the cloned impdh cDNA probe to Drosophila salivary gland chromosomes indicated that the impdh gene is located at position 9E1-4 on the X chromosome.

Amino Acid Sequence↗
PubMed1993

Molecular characterization of the ldmdr1 multidrug resistance gene from Leishmania donovani.

The ldmdr1 gene that confers resistance to multiple structurally dissimilar hydrophobic drugs in Leishmania donovani has been isolated within a 5.4-kb XmaI fragment from a genomic library of L. donovani DNA and its protein coding region sequenced. The longest open reading frame within ldmdr1 encodes a 146.5-kDa protein of 1341 amino acids, designated LDMDR1. The primary structure and predicted membrane topology of LDMDR1 indicates that it is a member of the P-glycoprotein superfamily with the greatest homology to the mammalian multidrug resistance P-glycoproteins. A 2.3-kb SalI fragment derived from a second ldmdr1 allele was also cloned from the L. donovani library. Nucleotide sequence analysis of a portion of the SalI insert revealed 5 single base differences from its counterpart within the 5.4-kb XmaI fragment, one of which created a PvuI restriction site polymorphism. Southern blots of PvuI-digested DNA divulged that the amplified ldmdr1 gene copies in a multidrug-resistant L. donovani strain were all derived from the single ldmdr1 allele whose protein coding segment was sequenced in its entirety.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene.

Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗