The multicopy mini-exon-derived RNA (med RNA) locus of Leishmania donovani was enzymatically amplified by the polymerase chain reaction (PCR). The major 180 bp PCR product contained conserved med RNA gene sequences flanking the variable intergenic spacer from the med RNA gene tandem repeat. The oligonucleotide primers cross-reacted with other Leishmania species. In serial dilution experiments, positivity in the PCR assay was observed down to the genomic DNA equivalent of less than a single Leishmania cell. When the major PCR products from Indian L. donovani isolates were cloned and used as probes in dot hybridization analyses, they discriminated between L. donovani and L. amazonensis, L. major and L. infantum under high stringency conditions. DNA from spleen biopsies and blood samples of confirmed kala azar patients was positive, as were two skin biopsies from patients with post-kala azar dermal leishmaniasis (PKDL). These observations demonstrate that PCR amplification of med RNA intergenic spacers is sufficiently sensitive for clinical diagnosis of kala azar and PKDL, and furthermore, that cloned intergenic spacer probes may be useful for identification and classification of L. donovani.
We determined whether hepatic stimulator substance shares its mitogenic specificity for hepatocytes with nonparenchymal epithelial cells in the hepatocyte lineage. Cell lines designated HTC (derived from a rat hepatoma known to respond to hepatic stimulator substance) and FNRL, K-16 and K-22 (derived from rat liver nonparenchymal epithelial cells) were used. After exposure to hepatic stimulator substance, [3H]-thymidine incorporation into DNA was significantly increased (p less than 0.001) in HTC, FNRL and K-16 cells, but not in K-22 cells. Fluorescence-activated cell sorting demonstrated that the mitogenic response to hepatic stimulator substance was associated with a greater proportion of cells entering the S phase. Epidermal growth factor, alone or in combination with hepatic stimulator substance, had no significant mitogenic effect on FNRL cells, but exposure of these cells to transforming growth factor-beta 1 inhibited [3H]-thymidine incorporation into DNA and reduced the proportion of cells in the S and G2/M phases. Simultaneous exposure of FNRL cells to hepatic stimulator substance and transforming growth factor-beta 1 abrogated the inhibitory effect of transforming growth factor-beta 1. Comparison of butyrate-synchronized HTC cells with hepatic stimulator substance-treated HTC cells showed that S-phase progression in these conditions was different, with no intervening cell cycle arrest after treatment with hepatic stimulator substance. Mitogenic stimulation of FNRL and K-16 cells with the liver-specific growth factor hepatic stimulator substance suggests that these cells are of hepatocyte lineage. These results strengthen the evidence for a possible link between hepatocytes and nonparenchymal liver epithelial cells during liver biogenesis and differentiation.
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