Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter.

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single-copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis-regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B-related motifs in the proximal promoter as the sites conferring inducibility and tissue-specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon-gamma responsive sites in this up-regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of -0.6 kb which increases the level of expression by a factor of five.     

Conformational differences between cis and trans proline isomers of a peptide antigen representing the receptor binding domain of Pseudomonas aeruginosa as studied by 1H-nmr

A 17-residue disulfide-bridged peptide (PAK 128–144) corresponding to the C-terminus of Pseudomonas aeruginosa pilin strain K has been studied by one- and two-dimensional nmr techniques. This synthetic immunogen has been found to exist as two distinct conformations in solution, which have been demonstrated to arise as a result of the isomerization of the I¹³⁸-P¹³⁹ amide bond. The two isomers occur in the ratio of 3 : 1 trans to cis at 5°C. Sequential assignments for both forms have been accomplished through the use of nuclear Overhauser enhancement spectroscopy (NOESY) spectra and most side-chain resonances have been assigned using a combination of correlated spectroscopy, total correlated spectroscopy, and NOESY spectra. The presence of the cis isomer, which is considerably more predominant in the oxidized peptide, was confirmed by the observation of the characteristic NOEs between P¹³⁹ and the preceding residue. Further corroboration was given by the disappearance of the cis resonances in the spectrum of the P139A analogue of PAK 128–144. From observation of the differences in the chemical shifts and amide proton temperature coefficients of the two isomers, it is apparent that the two forms differ markedly in their solution conformation. The biological implications of the isomerization are discussed. © 1994 John Wiley & Sons, Inc.     

Unique fimbriae-like structures encoded by sefD of the SEF14 fimbrial gene cluster of Salmonella enteritidis

The SEF14 gene cluster of Salmonella enteritidis was recently shown to contain three genes, sefABC, encoding a unique fimbrin, and proteins homologous to fimbrial chaperones and outer membrane proteins (ushers), respectively. A fourth open reading frame, designated sefD, was found immediately downstream of sefABC and overlapping sefC. The translated protein sequence of sefD was unique, but the composition was similar to that of other bacterial fimbriae. SefD was produced in abundance by wild-type S. enteritidis as shown by Western blot analysis using antibodies raised to affinity-purified, recombinant SefD. Furthermore, unusually long, thin, fimbriae-like structures were evident on S. enteritidis and Escherichia coli by immunoelectron microscopy, but in other bacterial species SefD was expressed as amorphous material. Therefore, in S. enteritidis and E. coli, SefD is the predominant structural subunit of SEF18. The SEF18 fimbriae-like structures were shown to be serologically distinct from the three known S. enteritidis fimbriae SEF14, SEF17 and SEF21. Furthermore, SEF18 was still produced in set A insertion mutants, indicating that SEF14 and SEF18 were structurally distinct. Thus, the SEF14 gene cluster is the first example in the Enterobacteriaceae of a gene cluster that encodes two fimbrin-like proteins, which are assembled into two distinct cell-surface structures, SEF14 and SEF1B. DNA hybridization and Western blot analyses showed that SefD was widely distributed among the Enterobacteriaceae and was present in E. coli, Shigella, Enterobacter, Citrobacter, Erwinia, Hafnia, Klebsiella, Providencia, and Proteus but not in the non-Enterobacteriaceae Gram-negative bacteria Pseudomonas and Aeromonas, or in Gram-positive bacteria Bacillus or Staphylococcus. Immunoelectron microscopy revealed that sefD was also present on the surface of Providencia and Klebsiella but did not appear filamentous. This is the first instance of highly conserved, thin fimbriaelike structers which are ubiquitous among the Enterobacteriaceae.     

Regulation of taurine transport in Ehrlich ascites tumor cells

Summary Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized. Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na, 1Cl, 1taurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the leukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A₂ is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD₄. The swelling-induced taurine efflux is also inhibited by addition of the 5-lipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-lipoxygenase pathway. LTD₄ seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the Cl^– channels, i.e., via a depolarization of the cell membrane.     

Sorption of Heavy Metals to the Filamentous Bacterium Thiothrix Strain A1

A study was undertaken to determine the ability of the filamentous bacterium Thiothrix strain A1 to sorb heavy metals from solution. Cells of Thiothrix strain A1 were harvested, washed, and suspended in solutions of metals. After an equilibration period, biomass was separated from solution and the metal content in acid-digested cells and/or filtrates was determined by atomic absorption spectrophotometry. Sorption of nickel and zinc was very rapid; most of the sorbed metal was bound in less than 10 min. The sorption data for copper fit the Freundlich isotherm, and nickel and zinc data fit biphasic Freundlich isotherms. Sorption of both nickel and zinc was dependent on cell age. Cells harvested 24 h after inoculation sorbed approximately one-half of the amount of metal per gram cell protein than did cells harvested after 48, 72, or 96 h. Calcium and magnesium effectively competed with zinc for binding sites, whereas potassium had only a slight effect on the capacity of cells to sorb zinc. The primary mechanism of metal sorption apparently was ion exchange, because 66 to 75% of nickel or zinc could be desorbed by placing metal-laden cells in a solution of 5 mM CaCl₂. A competition experiment with nickel and zinc indicated that both metals occupied the same sorption sites. The strong chelating agents EDTA and NTA effectively prevented metal uptake, but lactate enhanced the uptake of nickel. Thiothrix strain A1 grown in nickel-containing medium had a relatively low uptake of nickel compared with uptake by resting cells suspended in a simple buffer solution.     

Chromosomal localization of four human zinc finger cDNAs

cDNA clones encoding zinc finger motifs were isolated by screening human placenta and T-cell (Peer) cDNA libraries with zinc finger (ZNF) consensus sequences. Unique cDNA clones were mapped in the human genome by rodent-human somatic cell hybrid analysis and in some cases in situ chromosomal hybridization. ZNF 80 mapped to 3p12-3qter, ZNF 7 was previously mapped to 8q24 and is here shown by in situ hybridization and use of appropriate hybrids to map telomeric to the MYC locus. ZNF 79 mapped to 9q34 centromeric to the ABL gene and between a constitutional chromosomal translocation on the centromeric side and the CML specific ABL translocation on the telomeric side. ZNF77 mapped to 19p while ZNF 78L1 (pT3) mapped to 19q. Chromosome 19 carries many ZNF loci and other genes with zinc finger encoding motifs; the pT3 clone additionally detected a locus designated ZNF 78L2, which mapped to chromosome region 1p, most likely in the region 1p32 where the MYCL and JUN loci map.