Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein.

Acyl-CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane enzyme that catalyses the last step of triacylglycerol synthesis from diacylglycerol and acyl-CoA. Here we provide experimental evidence that DGAT is a homotetramer. Although the predicted molecular mass of human DGAT protein is 55 kDa, CHAPS-solubilized recombinant human DGAT was eluted in fractions over 150 kDa on gel-filtration chromatography. Cross-linking of recombinant DGAT in membranes with disuccinimidyl suberate yielded bands corresponding to the dimer (108 kDa) and the tetramer (214 kDa) in SDS/PAGE. Finally, when two differently epitope-tagged forms of DGAT were co-transfected into mammalian cells, they could be co-immunoprecipitated. From a human adipose tissue cDNA library we cloned a cDNA encoding a novel splice variant of DGAT (designated DGATsv) that contained a 77 nt insert of unspliced intron with an in-frame stop codon. This resulted in a truncated form of DGAT that terminated at Arg-387, deleting 101 residues from the C-terminus containing the putative active site. DGATsv was enzymically inactive when transfected in HEK-293E cells but was still able to form dimer and tetramer on cross-linking, indicating that the ability to form tetramers resides in the N-terminal region. When co-expressed in HEK-293E cells, DGATsv did not inhibit the activity of full-length DGAT, suggesting that the subunits of DGAT catalyse triacylglycerol synthesis independently.

Salt-independent abnormality of antimicrobial activity in cystic fibrosis airway surface fluid.

The link between the genetic defect in cystic fibrosis (CF) and the recently described breach in pulmonary host defense has focused on the role of salt and water metabolism in the airways. Using a human bronchial xenograft model we demonstrate a salt-independent abnormality in bacterial killing in CF airway surface fluid (ASF). Biochemical characterization implicates the absence or dysfunction of a molecule critical to the constitution of normal bacterial killing. Our study suggests that CF transmembrane conductance regulator (CFTR) deficiency causes a primary abnormality in the composition of ASF that leads to a salt-independent defect in host defense. Importantly, this defect is corrected by adenovirus-mediated gene transfer of CFTR.

Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide.

Antimicrobial peptides, such as defensins or cathelicidins, are effector substances of the innate immune system and are thought to have antimicrobial properties that contribute to host defense. The evidence that vertebrate antimicrobial peptides contribute to innate immunity in vivo is based on their expression pattern and in vitro activity against microorganisms. The goal of this study was to investigate whether the overexpression of an antimicrobial peptide results in augmented protection against bacterial infection. C57BL/6 mice were given an adenovirus vector containing the cDNA for LL-37/hCAP-18, a human cathelicidin antimicrobial peptide. Mice treated with intratracheal LL-37/hCAP-18 vector had a lower bacterial load and a smaller inflammatory response than did untreated mice following pulmonary challenge with Pseudomonas aeruginosa PAO1. Systemic expression of LL-37/hCAP-18 after intravenous injection of recombinant adenovirus resulted in improved survival rates following intravenous injection of lipopolysaccharide with galactosamine or Escherichia coli CP9. In conclusion, the data demonstrate that expression of an antimicrobial peptide by gene transfer results in augmentation of the innate immune response, providing support for the hypothesis that vertebrate antimicrobial peptides protect against microorganisms in vivo.

Mouse beta-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs.

One component of host defense at mucosal surfaces is epithelium-derived peptides with antimicrobial activity called defensins. We describe in this report the isolation and characterization of a murine homologue of human beta-defensin 2 (hBD-2) called mouse beta-defensin 3 (mBD-3). The predicted amino acid sequence shows the hallmark features of other known epithelial defensins, including the ordered array of six cysteine residues. Analysis of a genomic clone of mBD-3 revealed two exons separated by a 1.7-kb intron. The mBD-3 gene is localized at the proximal portion of chromosome 8, the site where genes for mouse alpha- and beta-defensins are found. Under basal condition, mBD-3 transcripts were detected at low levels in epithelial cells of surface organs, such as the intestine and lung. After instillation of Pseudomonas aeruginosa PAO1 into mouse airways, mBD-3-specific mRNA was upregulated significantly not only in large airways but also in the small bowel and liver. Recombinant mBD-3 peptide, produced from a baculovirus expression system, showed antimicrobial activity against P. aeruginosa PAO1 (MIC of 8 micrograms/ml) and Escherichia coli D31 (MIC of 16 micrograms/ml) in a salt-dependent manner. This study demonstrates that a murine homologue of hBD-2 is present in the respiratory system and other mucosal surfaces. These similarities between murine and human host defense apparatus provide further impetus to evaluate the mouse as a model for studying the human innate host defense system.

Transduction of well-differentiated airway epithelium by recombinant adeno-associated virus is limited by vector entry.

The limitations of adeno-associated virus (AAV)-mediated vectors for lung-directed gene transfer were investigated by using differentiated human respiratory epithelium in air-liquid interface cultures. Transduction efficiency was high in undifferentiated cells and was enhanced in well-differentiated cells after basolateral application of the vector or after apical application following disruption of tight junctions or pretreatment of the cultures with glycosidases. These results indicate that transduction of airway epithelia by AAV vectors is limited by entry and reinforce the importance of a physical barrier on the airway surface.

Transfer of a cathelicidin peptide antibiotic gene restores bacterial killing in a cystic fibrosis xenograft model.

Recent studies suggest that the gene defect in cystic fibrosis (CF) leads to a breach in innate immunity. We describe a novel genetic strategy for reversing the CF-specific defect of antimicrobial activity by transferring a gene encoding a secreted cathelicidin peptide antibiotic into the airway epithelium grown in a human bronchial xenograft model. The airway surface fluid (ASF) from CF xenografts failed to kill Pseudomonas aeruginosa or Staphylococcus aureus. Partial reconstitution of CF transmembrane conductance regulator expression after adenovirus-mediated gene transfer restored the antimicrobial activity of ASF from CF xenografts to normal levels. Exposure of CF xenografts to an adenovirus expressing the human cathelicidin LL-37/hCAP-18 increased levels of this peptide in the ASF three- to fourfold above the normal concentrations, which were equivalent in ASF from CF and normal xenografts before gene transfer. The increase of LL-37 was sufficient to restore bacterial killing to normal levels. The data presented describe an alternative genetic approach to the treatment of CF based on enhanced expression of an endogenous antimicrobial peptide and provide strong evidence that expression of antimicrobial peptides indeed protects against bacterial infection.

Human corneal cells and other fibroblasts can stimulate the appearance of herpes simplex virus from quiescently infected PC12 cells.

A two-cell system for the stimulation of herpes simplex virus type 1 (HSV-1) from an in vitro model of long-term (quiescent) infection is described. Rat pheochromocytoma (PC12) cells differentiated with nerve growth factor were infected with HSV-1 strain 17. Little, if any, cytotoxicity was observed, and a quiescent infection was established. The long-term infection was characterized by the absence of all detectable virus in the culture medium and little, if any, detectable early or late viral-gene expression as determined by reverse transcriptase PCR analysis. The presence of HSV-1 DNA was determined by PCR analysis. This showed that approximately 180 viral genomes were present in limiting dilutions where as few as 16 cells were examined. The viral DNA was infectious, since cocultivation with human corneal fibroblasts (HCF) or human corneal epithelial cells (HCE) resulted in recovery of virus from most, if not all, clusters of PC12 cells. Following cocultivation, viral antigens appeared first on PC12 cells and then on neighboring inducing cells, as determined by immunofluorescent staining, demonstrating that de novo viral protein synthesis first occurred in the long-term-infected PC12 cells. Interestingly, the ability to induce HSV varied among the cell lines tested. For example, monkey kidney CV-1 cells and human hepatoblastoma HepG2 cells, but not mouse neuroblastoma cells or undifferentiated PC12 cells, mediated stimulation. This work thus shows that (i) quiescent HSV infections can be maintained in PC12 cells in vitro, (ii) HSV can be induced from cells which do not accumulate significant levels of latency-associated transcripts, and (iii) the activation of HSV gene expression can be induced via neighboring cells. The ability of adjacent cells to stimulate HSV gene expression in neuron-like cells represents a novel area of study. The mechanism(s) whereby HCF, HCE, and HepG2 and CV-1 cells communicate with PC12 cells and stimulate viral replication, as well as how this system compares with other in vitro models of long-term infection, is discussed.

A 437-base-pair deletion at the beginning of the latency-associated transcript promoter significantly reduced adrenergically induced herpes simplex virus type 1 ocular reactivation in latently infected rabbits.

In this study we used a herpes simplex virus type 1 (HSV-1) deletion mutant to identify a segment of the genome necessary for epinephrine-induced reactivation in the rabbit eye model of herpetic recurrent disease. In HSV-1 latently infected neural tissue, the only abundant viral products are the latency-associated transcripts (LATs). At least one promoter of LAT has been identified, and mutations in the LAT domain have been used to investigate HSV-1 reactivation. We used an ocular rabbit model of epinephrine-induced HSV-1 reactivation to study the effects of deleting a 437-bp region beginning 796 bp upstream of the LAT CAP site. Specifically, the 437-bp deletion is located between genomic positions 118006 and 118443 of the parent 17Syn+, and the construct is designated 17 delta S/N. This region also controls a portion of the genome encoding two transcripts (1.1 and 1.8 kb) from the LAT domain. A rescuant, 17 delta S/N-Res, was constructed from 17 delta S/N. Following ocular infection, all three viruses produced similar acute dendritic lesions in rabbits. Five weeks after infection, rabbits received transcorneal iontophoresis of epinephrine. The parent, 17Syn+, and the rescuant, 17 delta S/N-Res, underwent a high frequency of HSV-1 ocular reactivation as determined by recovery of infectious virus in the tear film. Rabbits infected with 17 delta S/N had a significantly lower frequency of ocular reactivation. Analysis of the trigeminal ganglia from all three groups of latently infected rabbits revealed (i) similar amounts of HSV DNA (genomic equivalents), (ii) accumulation of 2.0- and 1.45-kb LATs, and (iii) explant reactivation at the same high frequency. Therefore, these studies indicate that the 437-bp deleted region in 17 delta S/N is essential for epinephrine-induced reactivation and could implicate the 1.1- and 1.8-kb transcripts in the mechanisms controlling HSV-1 reactivation.

Evaluation of the tumor-initiating activity of 4-, 5-, 6-, and 7-fluorobenzo[b]fluoranthene in mouse skin.

Benzo[b]fluoranthene and 4-, 5-, 6-, and 7-fluorobenzo[b]fluoranthene were evaluated for tumor-initiating activity in mouse skin. These fluorinated benzo[b]fluoranthene derivatives were assayed at doses of 400, 120, 40, and 10 nmol per mouse. Similar tumorigenic activity was observed for benzo[b]fluoranthene and 5-fluorobenzo[b]fluoranthene. While 4-fluorobenzo[b]fluoranthene did produce a significant tumorigenic response at each dose assayed, substantially fewer tumors per mouse were observed compared to benzo[b]fluoranthene at initiator doses at or above 120 nmol. Only moderate tumorigenic activity was observed for 6- and 7-fluorobenzo[b]fluoranthene. Both of these fluorinated derivatives were significantly less tumorigenic (P < 0.05) than 4-fluorobenzo[b]fluoranthene when administered at initiator doses at or below 120 nmol. These results were unanticipated in view of data which indicate that metabolism of trans-9,10-dihydro-9,10-dihydroxybenzo[b]-fluoranthene to trans-9,10-dihydro-5,9,10-trihydroxybenzo[b]fluoranthene represents a principal activation mechanism of benzo[b]fluoranthene in mouse skin. The potential of fluorine substitution not only to inhibit metabolism, but also to alter the genotoxic activity of those metabolites which do form could explain the tumorigenic activity observed with these fluorinated derivatives of benzo[b]fluoranthene. These data suggest caution in the interpretation of results based exclusively upon the assumption that the only influence of fluorine substitution is inhibition of the formation of specific metabolites.

Exceptional tumor-initiating activity of 4-fluorobenzo[j]-fluoranthene on mouse skin: comparison with benzo[j]-fluoranthene, 10-fluoro-benzo[j]fluoranthene, benzo[a]pyrene, dibenzo[a,l]pyrene and 7,12-dimethylbenz[a]anthracene.

Exceptional tumorigenic potency was observed with 4-fluorobenzo[j]fuoranthene (4-fluoroB[j]F) relative to benzo[j]fluoranthene (B[j]F) and 10-fluorobenzo[j]fluoranthene (10-fluoroB[j]F) in a mouse skin initiation promotion bioassay. Comparison of the tumorigenic response obtained at total initiating doses of 50, 100, and 1000 nmol firmly established the greater tumorigenic potency of 4-fluoroB[j]F. B[j]F produced a significant tumorigenic response only at total initiating doses of 100 and 1000 nmol per mouse. 10-FluoroB[j]F produced a significant tumorigenic response only at the highest initiating dose, 1000 nmol per mouse. In contrast, 4-fluoroB[j]F produced a significant tumorigenic response at all three doses. At a total initiating dose of 50 nmol, a 90% incidence of tumor-bearing mice with an average of 3.05 tumors per mouse was observed with 4-fluoroB[j]F. A second initiation promotion bioassay was performed to establish the tumorigenic potency of 4-fluoroB[j]F relative to benzo[a]-pyrene (B[a]P), 7,12-dimethylbenz[a]anthracene (DMBA), and dibenzo[a,l]pyrene (DB[a,l]P). 4-FluoroB[j]F did exhibit significant tumor-initiating activity at doses of 10 and 25 nmol per mouse, inducing a 45 and 60% incidence of tumor-bearing mice with an average of 0.75 and 1.65 tumors per mouse, respectively. While B[a]P was not tumorigenic at these doses, DMBA and DB[a,l]P exhibited significant tumorigenic activity at doses of 1, 4, 10, and 25 nmol per mouse. DB[a,l]P induced a 95% incidence of tumor-bearing mice with an average of 5.0 tumors per mouse at a total initiator dose of 1 nmol. DMBA at this dose produced an 85% incidence of tumor-bearing mice with an average of 1.30 tumors per mouse. The results of these initiation promotion bioassays clearly demonstrate that 4-fluoroB[j]F is significantly more active than B[j]F, 10-fluoroB[j]F and B[a]P and less active than either DMBA or DB[a,l]P as a tumor initiator on mouse skin.