Hepatocyte nuclear factor-3beta limits cellular diversity in the developing respiratory epithelium and alters lung morphogenesis in vivo.

Hepatocyte nuclear factor-3beta (HNF-3beta), a nuclear protein of the winged helix family of transcription factors, is known to play a critical role in the formation of the embryonic node, notochord, and foregut endoderm. HNF-3beta influences the expression of a number of target genes in the respiratory epithelium, activating transcription of thyroid transcription factor-1, surfactant protein-B and clara cell secretory protein. In order to discern the role of HNF-3beta in differentiation and gene expression in the lung, HNF-3beta was expressed in developing respiratory epithelial cells of transgenic mice, under the control of the human surfactant protein C gene promoter. Pulmonary abnormalities were observed in the lungs of fetal mice bearing the HNF-3beta transgene. Differentiation of distal respiratory epithelial cells was arrested in the early pseudoglandular stage. Branching morphogenesis and vasculogenesis were markedly disrupted in association with decreased E-cadherin and vascular endothelial growth factor expression. HNF-3beta limits cellular diversity of developing respiratory epithelium and alters lung morphogenesis in vivo, suggesting that precise temporal-spatial regulation of HNF-3beta expression is critical for respiratory epithelial cell differentiation and lung morphogenesis.

Arrested lung morphogenesis in transgenic mice bearing an SP-C-TGF-beta 1 chimeric gene.

Transforming growth factor-beta 1 (TGF-beta 1) influences the morphogenesis of many organs, regulating cell growth, differentiation, gene expression, extracellular matrix deposition, and angiogenesis. In order to assess the effects of TGF-beta 1 on lung development in vivo, transgenic mice were generated bearing a chimeric gene composed of human surfactant protein C (SP-C) gene promoter and the porcine TGF-beta 1 cDNA mutated to ensure constitutive activation of the TGF-beta 1 peptide. Because of the perinatal loss related to the SP-C-TGF-beta 1 transgene, embryos bearing the transgene were obtained on Days 16 and 18.5 of gestation. TGF-beta 1 was selectively expressed in respiratory epithelial cells of the transgenic embryos. Body weight, length, and lung size were not altered in the transgenic embryos; however, lung morphogenesis of Day 18.5 transgenic mice was arrested in a late pseudoglandular stage of development, while that of their nontransgenic littermates was typical of the saccular stage. Lungs of transgenic mice on Day 16 contained fewer acinar buds than those of nontransgenic littermates. At both ages, epithelial cell differentiation, assessed by the expression of Clara cell secretory protein2 and pro-SP-C, was inhibited. While collagen III deposition was not affected by the transgene, collagen I expression was persistent in terminal airways of fd 18.5 transgenic lungs. The distribution of alpha-smooth muscle actin was markedly altered, being detected in the mesenchyme surrounding the distal leading edges of epithelial tubules in the SP-C-TGF-beta 1 transgenic mice. Expression of TGF-beta 1 in the developing respiratory epithelium of transgenic mice arrested lung sacculation and epithelial cell differentiation in vivo, supporting the role of TGF-beta family members in lung morphogenesis and differentiation.

Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). A potential animal model of CF, the CFTR-/- mouse, has had limited utility because most mice die from intestinal obstruction during the first month of life. Human CFTR (hCFTR) was expressed in CFTR-/- mice under the control of the rat intestinal fatty acid-binding protein gene promoter. The mice survived and showed functional correction of ileal goblet cell and crypt cell hyperplasia and cyclic adenosine monophosphate-stimulated chloride secretion. These results support the concept that transfer of the hCFTR gene may be a useful strategy for correcting physiologic defects in patients with CF.

Human Mn-superoxide dismutase in pulmonary epithelial cells of transgenic mice confers protection from oxygen injury.

To test directly whether mitochondrial Mn-superoxide dismutase (Mn-SOD) protects the lung epithelium from oxygen-induced injury, transgenic mice were produced in which the expression of human Mn-SOD mRNA was directly by transcriptional elements from the human pulmonary surfactant protein C gene. Human Mn-SOD mRNA was expressed in a lung-specific manner, and increased Mn-SOD protein was detected within mitochondria of alveolar Type II and nonciliated bronchiolar cells of the distal respiratory epithelium of the transgenic mice. The activity of Mn-SOD, but not catalase, CuZn-SOD, or glutathione peroxidase, was increased in lungs of transgenic mice. Transgenic mice were highly protected from lung injury during exposure to 95% oxygen, surviving significantly longer than nontransgenic littermates. Pulmonary pathology demonstrated decreased hemorrhage, hyaline membrane formation, and alveolar and interstitial edema in transgenic animals. The finding that increased Mn-SOD in distal respiratory epithelial cells confers protection from oxygen injury provides a basis for novel therapies to protect lung from injury during oxygen therapy of acute and chronic lung diseases.

Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice.

Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.

Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae.

We have sequenced the structural gene and flanking regions for lanosterol 14 alpha-demethylase (14DM) from Saccharomyces cerevisiae. An open reading frame of 530 codons encodes a 60.7-kDa protein. When this gene is disrupted by integrative transformation, the resulting strain requires ergosterol and, as expected, grows only in the absence of oxygen. The deduced amino acid sequence of 14DM includes a hydrophobic segment near the amino terminus which may be a transmembrane domain. The deduced sequence has been compared with those of eight other eukaryotic P450s, each from a different family within the P450 superfamily. These comparisons indicate that this yeast gene is the first member of a new P450 family, P450LI. The P450, designated P450LIA1, is more closely related to mammalian P450s than to the bacterial P450cam. In fact, both the yeast P450 and several mammalian P450s have equivalent alignment scores when each is compared with the bovine P450scc. Matrix comparisons of the amino acid sequence of this P450 with those of mammalian P450s reveal three conserved regions. The DNA region 5' to the structural 14DM gene includes poly(dA:dT) sequences and a repeating hexamer sequence.

Isolation of a cytochrome P-450 structural gene from Saccharomyces cerevisiae.

We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14 alpha-demethylase. Two plasmids were isolated which transformed yeast to both increased resistance to Kc and increased levels of total P-450. Hybrid-selection and immunoprecipitation experiments showed that these plasmids, pVK1 and pVK2, contained the structural gene for an S. cerevisiae P-450. This conclusion was confirmed by the nucleotide sequence of a portion of pVK2, which revealed an open reading frame encoding a characteristic P-450 heme-binding region.

Aliphatic halogenated hydrocarbons produce volatile Salmonella mutagens.

Production of volatile mutagenic metabolites from 5 halogenated promutagens was examined by a simple modification of the conventional Salmonella/microsome mutagenicity assay. This method incorporates the taping together of 2 agar plates face to face during the initial portion of their incubation at 37 degrees C. By varying the contents of the soft agar in each of the two plates with respect to promutagen, S9 and tester strain cells, mutagenesis due to volatile promutagens and their metabolites could be quantitated separately. Using the taped plate assay, volatile mutagenic metabolites were detected from the promutagens 3-(2-chloroethoxy)-1,2-dichloropropene, the herbicides diallate, triallate and sulfallate, and the flame-retardant tris-(2,3-dibromopropyl) phosphate (Tris-BP). All compounds except Tris-BP were also found to be volatile promutagens. The mutagenic metabolites accounted for 50-80% of the activity of these compounds observed in the standard assay. Morever, our studies suggest that a small, but appreciable percentage of the mutagenic metabolites from all 5 compounds escaped detection in the conventional, untaped assay. Mutagenic activity of the volatile mutagenic metabolites from diallate was quenched by various Salmonella tester strains independent of their responsiveness to diallate mutagenesis. Detection of volatile mutagen formation from diallate was also prevented by cysteine and glutathione, but not by DNA or metyrapone. This taped plate method for the Salmonella assay should facilitate future investigations of the detection, isolation and identification of volatile mutagenic metabolites from other promutagenic compounds or mixtures.

Surveillance of gentamicin-resistant gram-negative bacilli in a general hospital.

Aerobic gram-negative bacilli isolated from clinical specimens from 1 January to 31 December 1976 were tested for gentamicin and tobramycin resistance by standardized disk testing. For Pseudomonas isolates, gentamicin resistance was 17.1% and tobramycin resistance was 2.8%. For other gram-negative bacilli, gentamicin resistance was 5.5% and tobramycin resistance was 5.4%. Seventy-four patients from whom gentamicin-resistant organisms were isolated from 1 January to 30 June 1976 were studied prospectively. These patients were elderly, had serious underlying diseases, and had received prior antibiotic therapy. Eleven patients carried gentamicin-resistant organisms at the time of transfer to our hospital from community hospitals or nursing homes. Of the 82 isolates from these 74 patients, 52 were from the urine. Pseudomonas was found most frequently (32 isolates), followed by Klebsiella (15 isolates), Enterobacter (10 isolates), Serratia (10 isolates), and Proteus (9 isolates). Only 3 of 32 Pseudomonas isolates caused symptomatic infection, while 16 of 50 other gram-negative bacilli were responsible for symptomatic infection. Although amikacin was the most active drug against gentamicin-resistant gram-negative bacilli and had not been used in our hospital at the time of this study, 25% of Pseudomonas and 18% of all gram-negative bacilli showed resistance to this aminoglycoside.

Superiority of MacConkey's agar over salmonella-shigella agar for isolation of Shigella dysenteriae type 1.

The efficiency of MacConkey's agar in the isolation of various types of Shigella was compared with that of salmonella-shigella (SS) agar during an extensive 18-month outbreak of disease caused by Shigella. In all, 1,580 isolates of Shigella were obtained from 12,307 rectal swab and fecal samples of patients with diarrhea and their contacts by direct plating onto MacConkey's and SS agars. Shigella dysenteriae type 1 and Shigella flexneri constituted 55 percent and 33 percent of all isolates, respectively, with a smaller number of Shigella boydii and Shigella sonnei. MacConkey's agar was superior to SS agar in the detection of S. dysenteriae type 1; 83 percent of the isolates were detectable on MacConkey's agar, compared with 40 percent on SS agar. In contrast, 84 percent of S. flexneri isolates were detectable on SS agar, compared with 51 percent on MacConkey's agar alone. These findings confirm that, for the culture of fecal specimens thought to contain S. dysenteriae type 1, one of the media used should be noninhibitory. Shigella was isolated at a consistently and significantly higher rate from fecal samples than from rectal swabs.

Clinical studies of the use of bacteriophage in the treatment of cholera.

This study reports the use of bacteriophage prepared in the USSR in the treatment of cholera. Patients with acute cholera were rehydrated with a standard intravenous solution and were then given a bacteriophage preparation in addition to maintenance intravenous therapy. The titre of the phage preparations was between 10(8) and 10(9) pfu/ml. Bacteriophage was given by mouth (25 ml for adults and 20 ml for children) for 3 days; in addition, some patients were also given an intramuscular injection (20 ml) of phage on the first day in hospital. For comparison, other groups of patients were given a standard tetracycline regimen or a placebo preparation. Daily vibrio and phage counts were made on stool samples from all patients and the vibrio strains isolated from each patient were tested for sensitivity to the phage preparation.The criteria used to evaluate the various therapies were duration of diarrhoea, volume of stool, and duration of vibrio excretion.The results of the study demonstrate that, in the doses used, the therapeutic effect of bacteriophage, if any, was markedly inferior to that of tetracycline and that in the current state of our knowledge bacteriophage, as used in this study, has no place in the treatment of cholera.