No abstract

Upon contact with the eukaryotic cell, Yersinia pseudotuberculosis increased the rate of transcription of virulence genes (yop), as determined by in situ monitoring of light emission from individual bacteria expressing luciferase under the control of the yopE promoter. The microbe-host interaction triggered export of LcrQ, a negative regulator of Yop expression, via the Yop-type III secretion system. The intracellular concentration of LcrQ was thereby lowered, resulting in increased expression of Yops. These results suggest a key role for the type III secretion system of pathogenic bacteria to coordinate secretion with expression of virulence factors after physical contact with the target cell.

The peptide FA-LL-37, previously termed FALL-39, was originally predicted from an ORF of a cDNA clone isolated from a human bone marrow library. This peptide was synthesized and found to have antibacterial activity. We have now characterized and sequenced the complete gene for FA-LL-37, termed FALL39. It is a compact gene of 1963 bp with four exons. Exons 1-3 code for a signal sequence and the cathelin region. Exon 4 contains the information for the mature antibacterial peptide. Our results indicate that FALL39 is the only member of the cathelin gene family present in the human genome. Potential binding sites for acute-phase-response factors are identified in the promoter and in intron 2. A possible role for the cytokine interleukin-6 in the regulation of FA LL39 is discussed. Anti-(FA-LL-37) IgG located the peptide in granulocytes and we isolated the mature peptide from these cells after degranulation. Structural analysis determined the mature peptide to be LL-37. To obtain LL-37 for antibacterial assays, synthetic FA-LL-37 was degraded with dipeptidyl-peptidase I. This analysis showed that mature LL-37 is a potent antibacterial peptide.

To determine the role of actin-ribonucleoprotein complexes in transcription, we set out to identify novel actin-binding proteins associated with RNA polymerase II (Pol II). Using affinity chromatography on fractionated HeLa cells, we found that hnRNP U binds actin through a short amino acid sequence in its C-terminal domain. Post-transcriptional gene silencing of hnRNP U and nuclear microinjections of a short peptide encompassing the hnRNP U actin-binding sequence inhibited BrUTP incorporation in vivo. In living cells, we found that both actin and hnRNP U are associated with the phosphorylated C-terminal domain of Pol II, and antibodies to actin and hnRNP U blocked Pol II-mediated transcription. Taken together, our results indicate that a general actin-based mechanism is implicated in the transcription of most Pol II genes. Actin in complex with hnRNP U may carry out its regulatory role during the initial phases of transcription activation.

We recently isolated from pig intestine and characterized a 31-residue antibacterial peptide named cecropin PI with activity against Escherichia coli and several other Gram-negative bacteria. The isolation involved a number of batch-wise steps followed by several chromatography steps. The continued investigation of these antibacterial peptides has now yielded another antibacterial peptide with high activity against both E. coli and Bacillus megatrrium. Amino acid analysis showed a very high content of proline (49 mol%) and arginine (26 mol%), an intermediate level of phenylalanine and low levels of leucine, tyrosine, isoleucine, and glycine. The primary structure was determined by a combination of Edman degradation, plasma desorption mass spectrometry and C-terminal sequence analysis by carboxypeptidase Y degradation using capillary zone electrophoresis for detection of liberated residues. The calculated molecular mass was 4719.7 Da, which is in excellent agreement with 4719 D a obtained by plasma desorption mass spectrometry. The peptide was named PR-39 (proline-arginine-rich with a size of 39 residues). The lethal concentration of the peptide was determined against six Gram-negative and four Gram-positive strains of bacteria.