Replication and transcription of genome complexed with basic proteins

Defensin are 3-4 kDa antimicrobial peptides of which three distinct families have been identified; alpha-defensin, beta-defensins, and insect defensins. Recent investigations have shown that beta-defensins are present in the human airways and may be relevant to the pathogenesis of cystic fibrosis (CF) lung disease. We report here the further characterization of a recently identified mouse beta-defensin gene, Defb1, sometimes referred to as mBD-1, which is homologous to the human airway beta defensin hBD-1. We report that Defb1 is expressed in a variety of tissues including the airways and, similar to hBD-1, is not upregulated by lipopolysaccharide (LPS). Defb1 was found to consist of two small exons separated by a 16-kb intron and cytogenetic, and physical mapping linked it to the alpha defensin gene cluster on mouse Chromosome (Chr) 8. Functional studies demonstrate that, like hBD-1, Defb1 demonstrates a salt-sensitive antimicrobial activity against Pseudomonas aeruginosa. Of relevance to CF lung disease is the fact that neither the hBD-1 nor the mBD-1 peptides are active against Burkholderia cepacia.     

beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice

beta ig-h3 is a novel gene first discovered by differential screening of a cDNA library made from A549 human lung adenocarcinoma cells treated with transforming growth factor-beta 1 (TGF-beta 1). It encodes a 683-amino-acid protein containing a secretory signal sequence and four homologous internal domains. Here we show that treatment of several types of cells, including human melanoma cells, human mammary epithelial cells, human keratinocytes, and human fibroblasts, with TGF-beta resulted in a significant increase in beta ig-h3 RNA. A portion of the beta ig-h3 coding sequence was expressed in bacteria, and antisera against the bacterially produced protein was raised in rabbits. This antisera was used to demonstrate that several cell lines secreted a 68-kD beta IG-H3 protein after treatment with TGF-beta. Transfection of beta IG-H3 expression plasmids into Chinese hamster ovary (CHO) cells led to a marked decrease in the ability of these cells to form tumors in nude mice. The beta IG-H3 protein was purified from media conditioned by recombinant CHO cells, characterized by immunoblotting and protein sequencing and shown to function in an anti-adhesion assay in that it inhibited the attachment of A549, HeLa, and WI-38 cells to plastic in serum-free media. Sequencing of cDNA clones encoding murine beta ig-H3 indicated 90.6% conservation at the amino acid level between the murine and human proteins. Finally, the beta ig-h3 gene was localized to human chromosome 5q31, a region frequently deleted in preleukemic myelodysplasia and leukemia. The corresponding mouse beta ig-h3 gene was mapped to mouse chromosome 13 region B to C1, which confirms a region of conservation on human chromosome 5 and mouse chromosome 13. We suggest that this protein be named p68 beta ig-h3.     

Selective aortic arch perfusion using serial infusions of perflubron emulsion

With the aim of developing foetal gene therapy for cystic fibrosis, we have investigated the possibility of gene targeting to the mouse foetus with two different viral vector systems and at different times of gestation. We report here that recombinant retrovirus producing cells administered into the intra-amniotic cavity of mid- to late-gestation mouse MF1 foetuses survive in the amniotic fluid and are able to engraft to a certain extent in foetal tissues. By production of infectious virus they mediate transduction and beta-galactosidase transgene expression in neighbouring foetal tissues 24 to 72 h following injection. Retrovirus producer cells could, therefore, become a means to overcome the limitations of low retroviral titre, for in vivo foetal gene transfer. To investigate the developmental stage at which transduction of the airways and enteral systems can be obtained we also administered a highly infective first generation adenoviral vector (AdRSV beta gal) into the amniotic cavity of foetal mice between 13 to 16 days post coitus, beta-galactosidase activity was detected between 24 to 120 h after injection. The highest levels of transgene expression were generally observed between 48 to 72 h following injection of the adenoviral vector. We demonstrate that infection of the pulmonary airways is dependent on the developmental stage of the foetus and can be achieved on the 15th day of gestation.     

Immunoglobulin M-capture biotin-streptavidin enzyme-linked immunosorbent assay for detection of antibodies to dengue viruses

Defensins and other antimicrobial peptides act in the innate host defense of epithelial surfaces. Human beta defensin 1 (hBD-1) has recently been shown to be expressed in airway epithelial cells and so has been implicated as a primary component of antibacterial activity in human lung. We attempted to purify these molecules from bronchoalveolar lavage fluid (BALF). Extraction of BALF on SepPak C-18 cartridges, followed by continuous acid-urea polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography yielded one fraction with antibacterial activity associated with factors of < 6.5 kD. N-terminal amino acid sequencing identified these peptides as human neutrophil defensins (HD) 1 through 3. No hBD-1 was detected. Together with lysozyme, it appears that HD-1 through -3 are the most prominent antimicrobial factors in BALF. The contribution of epithelial defensins such as hBD-1 to antibacterial defense of human airway in vivo remains to be elucidated.     

MHC class II molecules bind indiscriminately self and non-self peptide homologs: effect on the immunogenicity of non-self peptides

Synthetic peptides spanning the entire sequence of both human and mouse beta 2-microglobulin (beta 2M) have been tested for their capacity to bind to three different mouse (I-Ad, I-Ed, and I-Ak) or human (DR1, DR2, and DR5) class II molecules. The results demonstrate that class II molecules do not discriminate between self and non-self peptides. When the immunogenicity of the human beta 2M peptides was measured by their ability to prime H-2d mice for in vitro T cell proliferation, it was found that peptides incapable of binding class II molecules in vitro were also non-immunogenic in vivo. Interestingly, however, several binders, including the human beta 2M peptide 1-16, the best binder in this series to Iad molecules, were found to be non-immunogenic. Since the corresponding mouse beta 2M peptide 1-16 was also capable of binding to Iad molecules, this suggested that lack of responsiveness to the non-self peptide could arise either from central or peripheral tolerance induced by the self homolog. Alternatively, lack of responsiveness could arise from other mechanisms, such as negative selection by other non-homolog sequences or lack of suitable T cell receptor genes. To discriminate between these possibilities, H-2d mice with disrupted beta 2M genes were immunized with the human beta 2M peptide 1-16. This peptide also failed to prime for T cell responsiveness in beta 2M-negative mice, suggesting that a hole in the T cell repertoire for this antigen was not mediated by negative selection or peripheral tolerance induced by self beta 2M peptides.     

Adenovirus-mediated delivery of rhodopsin-promoted bcl-2 results in a delay in photoreceptor cell death in the rd/rd mouse

Gene transfer to retinal cells may provide a means to retard photoreceptor cell death and thus prevent blindness in diseases such as retinitis pigmentosa. We tested the possibility of interfering with apoptotic photoreceptor cell death in the rd mouse through subretinal delivery of a recombinant replication-defective adenovirus containing the human cDNA for bcl-2, Ad.2.5HRPbcl-2. Photoreceptor-specific transgene expression was accomplished through incorporation of the 2.5 kb human rhodopsin upstream fragment (HRP). Ad.2.5HRPbcl-2 was injected alone or in combination with Ad.CMVPDE beta. Ad.CMVPDE beta contains a cDNA encoding the beta subunit of cGMP phosphodiesterase (PDE beta). Recombinant viruses containing lacZ (driven either by the cytomegalovirus (CMV) promoter/enhancer or HRP) and of Ad.CMVPDE beta and vehicle alone were injected in contralateral eyes as control. Injection of Ad.2.5HRPbcl-2 in the rd mouse resulted in histologically detectable rescue lasting 6 weeks after birth. Extent of rescue was not as large as after delivery of wildtype PDE beta, the gene defective in the rd mouse. However, delivery of genes which prevent apoptotic cell death may have broad application to gene therapy of retinal degenerative diseases.     

Regulation of the beta 2 subunit chain of laminin in developing rabbit fetal lung tissue

Laminins are a family of basement membrane-associated heterotrimeric proteins that are important in mediating the growth, migration, and differentiation of a variety of cell types. The beta 2 subunit chain is a component of several laminin isoforms, e.g., laminin-3, laminin-4, laminin-7, and possibly other, as yet uncharacterized laminin isoforms. Utilizing monoclonal antibodies directed against the beta 2 subunit chain of laminin, we detected this protein in fetal, neonatal, and adult lung tissues. The relative amount of laminin beta 2 subunit chain in fetal lung tissue increased as gestation proceeded, reaching its peak around the time of alveolar type II cell differentiation in the rabbit. The laminin beta 2 subunit chain was localized in early gestational age rabbit fetal lung tissue primarily in basement membranes of prealveolar ducts, airways, and smooth muscle cells of airways and arterial blood vessels. A rabbit laminin beta 2 cDNA was generated using RT-PCR and utilized as a probe in northern blot analysis to characterize the levels of laminin beta 2 mRNA in developing rabbit lung tissue. Similar to the pattern of laminin beta 2 protein induction observed in fetal lung tissue, laminin beta 2 mRNA levels were maximal late in gestation. Utilizing a laminin beta 2 chain cRNA probe and in situ hybridization, we detected laminin beta 2 mRNA in the epithelial cells of prealveolar ducts, the alveolar wall, and airways, as well as in connective tissue cells, and the smooth muscle cells of airways and blood vessels in fetal and adult lung tissues. In addition, using an in vitro explant model, we determined that alveolar type II cells are capable of synthesizing laminin beta 2 subunit mRNA and depositing this laminin subunit chain in the basement membrane beneath type II cells. The results of this study are suggestive that the laminin beta 2 chain may be involved in alveolar epithelial cell differentiation.     

The human glycine receptor beta subunit: primary structure, functional characterisation and chromosomal localisation of the human and murine genes

The inhibitory glycine receptor (GlyR) is a pentameric receptor comprised of alpha and beta subunits, of which the beta subunit has not been characterised in humans. A 2106 bp cDNA, isolated from a human hippocampal cDNA library, contained an open reading frame of 497 amino acids which encodes the beta subunit of the human GlyR. The mature human GlyR beta polypeptide displays 99% amino acid identity with the rat GlyR beta subunit and 48% identity with the human GlyR alpha 1 subunit. Neither [3H]strychnine binding nor glycine-gated currents were detected when the human GlyR beta subunit cDNA was expressed in the human embryonic kidney 293 cell line. However, co-expression of the beta subunit cDNA with the alpha 1 subunit cDNA resulted in expression of functional GlyRs which showed a 4-fold reduction in the EC50 values when compared to alpha 1 homomeric GlyRs. Glycine-gated currents of alpha 1/beta GlyRs were 17-fold less sensitive than homomeric alpha 1 GlyRs to the antagonists picrotoxin, picrotoxinin and picrotin, providing clear evidence that heteromeric alpha 1/beta GlyRs were expressed. The beta subunit appears to play a structural rather than ligand binding role in GlyR function. Fluorescence in situ hybridisation was used to localise the gene encoding the human GlyR beta subunit (GLRB) to chromosome 4q32, a position syntenic with mouse chromosome 3. In situ hybridisation using the human GlyR beta subunit cDNA showed that the murine GlyR beta subunit gene (Glrb) maps to the spastic (spa) locus on mouse chromosome 3 at bands E3-F1. This is consistent with the recent finding that a mutation in the murine GlyR beta subunit causes the spa phenotype. It also raises the possibility that mutations in the human beta subunit gene may cause inherited disorders of the startle response.     

Sympathomimetic enantiomers and asthma

Airways of asthma patients can become hyperresponsive to airway spasmogens following regular use of isoprenaline or beta 2-selective sympathomimetics. Hyper-reactivity that results from acute exposure of animals to these drugs is pre-empted by vagal section (a procedure which does not influence spasmolytic efficacy of sympathomimetics), is not diminished by antagonism of beta 2-adrenoceptors and is not associated with loss of responsivity of beta 2-adrenoceptors in the airways. Since activation, modulation, or blockade of beta 2-adrenoceptors does not determine this form of hyperreactivity, the possibility that distomers may induce hyperreactivity must be considered. Ocular and vascular responses to distomers of sympathomimetics have long been recognised and, more recently, comparable observations have been made for the airways. Thus, reactivity of guinea-pig airways to spasmogens was increased following exposure to S-isoprenaline, S-salbutamol, or S-terbutaline and exposure to S-isoprenaline or S-salbutamol can intensify symptoms in asthmatics. Regular exposure to the racemate, especially during or following an allergic reaction, predisposes to expression of hyper-reactivity, which is nullified, acutely, by the eutomer. These observations imply that biological effects of sympathomimetic distomers may contribute to morbidity and mortality in asthma patients.     

The role of Stat4 in species-specific regulation of Th cell development by type I IFNs

Type I IFNs (IFN-alpha/beta), in addition to IL-12, have been shown to play an important role in the differentiation of human, but not mouse, Th cells. We show here that IFN-alpha/beta act directly on human T cells to drive Th1 development, bypassing the need for IL-12-induced signaling, whereas IFN-alpha cannot substitute IL-12 for mouse Th1 development. The molecular basis for this species specificity is that IFN-alpha/beta activate Stat4 in differentiating human, but not mouse, Th cells. Unlike IL-12, which acts only on Th1 cells, IFN-alpha/beta can activate Stat4 not only in human Th1, but also in Th2 cells. However, restimulation of human Th2 lines and clones in the presence of IFN-alpha does not induce the production of IFN-gamma. These results suggest that activation of Stat4, which is necessary for the differentiation of naive T cells into polarized Th1 cells, is not sufficient to induce phenotype reversal of human Th2 cells.     

Management of asthma based on their peak expiratory flow rate monitoring

SCN1B, the human gene encoding the beta1-subunit of the voltage-gated sodium channel has previously been cloned and mapped to Chr 19q13.1. The sequence of the homologous mouse gene, Scn1b, has now been determined from cDNA. The mouse gene is highly conserved, encoding a predicted protein with 99%, 98% and 96% amino acid identity to the rat, rabbit, and human homologs, respectively. DNA sequence conservation is also striking in the 3' untranslated region which shows 67% and 98% to human and rat, respectively. Unlike the human and rat homologs, high expression of mRNA from the mouse gene is confined to adult skeletal muscle and brain, and is not observed in heart. As Scnlb maps to Chr 7, in close genetic proximity to the quivering gene (qv), the coding region of Scnlb was also cloned from a qvJ/qvJ homozygous mouse and assessed as a candidate for the site of this genetic defect. Comparison of qv and wild-type cDNAs showed no changes in the predicted amino acid sequence that could cause the qv phenotype. However, three silent polymorphisms in the DNA coding region indicate that Scn1b is close to qv, and is within a region of genetic identity with DBA/2J, the inbred background on which the qvJ allele arose.     

Escherichia coli genes expressed preferentially in an aquatic environment

We isolated genomic clones that contain the 5'-flanking region of the mouse activin beta A subunit gene. The nucleotide sequence determination of the 5'-flanking region of the gene and the comparison of that with the reported mouse cDNA structure identified the putative 5' regulatory region, a novel first exon and a part of the first intron of the gene within this region. The putative 5' regulatory region of the mouse activin beta A subunit gene directed the expression of CAT gene in transfected HT1080 cells. Successive deletions of this region demonstrated a 400-bp region that exerts a strong positive effect on promoter activity of the mouse activin beta A subunit gene.     

Sucrose-specific signalling represses translation of the Arabidopsis ATB2 bZIP transcription factor gene

Immunomodulation of an ongoing autoimmune disease can be achieved by inhibitory cytokines or cytokine inhibitors such as TNF antagonists, delivery by gene therapy. The aim of this study was to design and test plasmid and retrovirus vectors expressing the mouse IFN beta gene and a chimeric protein containing the extracellular domain of human p55 TNF receptor linked to a murine Ig. These vectors were transiently expressed in COS-7 cells and permanently in amphotropic packaging cell lines or ABH mouse immortalized fibroblasts. Expression levels were assessed by ELISA. Western blotting and biological activity. In order to achieve tissue-specific expression in the CNS, the IFN beta gene was cloned and expressed under the control of the rat NSE promoter. We evaluated these constructs by direct intracranial injections of DNA-liposome complexes during the induction phase of experimental allergic encephalomyelitis, a murine model of multiple sclerosis, with therapeutic benefit.     

Surgical treatment of obstructive jaundice

Genetically engineered structural variants of human beta2-microglobulin (beta2m) were produced by sequence exchange with mouse beta2m for the purpose of examining species-specific antigenic determinant expression. For aggregate mapping, mouse and human beta2m, which differ by 30% in their primary sequence of 99 amino acids, were prepared as chimeric (human X mouse) molecules and expressed in the FO-1 beta2m-null human melanoma cell line. A chimera containing residues 1-69 from human beta2m (and residues 70-99 from mouse beta2m) induced expression of the epitopes defined by the anti-beta2m monoclonal antibodies (mAb) BBM.1, NAMB-1, and L368; the reverse chimera did not, although HLA class I heavy chain was evident on the cell surface as determined with the TP25.99 mAb. For fine dissection of the epitopes defined by these mAbs, site-directed mutants of beta2m were prepared by replacement of individual amino acids in human beta2m with the dimorphic residue from mouse beta2m. Substitutions were made at each divergent residue between positions 1 and 66 and, as controls for COOH-terminal modification, a series of residues between positions 75 and 94. Replacement of amino acids 38, 44, and 45, but not 16 other dimorphic residues in the linear stretch from residue 1 to residue 66, resulted in the loss of, or gross reduction in, binding by mAbs BBM.1 and NAMB-1. A reduction in binding was also observed for mAb L368. These data provide strong evidence that the antigenic epitopes defined by these mAb map to a region including S3 and its adjacent intra-beta-strand turn of the three-stranded beta-pleated sheet of beta2m. The mapping of these epitopes is consistent with their accessibility in the assembled major histocompatibility complex class I molecule and indicates that the region from amino acid 38 to 45 is an important structural feature in the "foreignness" of human and mouse beta2m.     

Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells

It has been reported that exhaled nitric oxide levels are reduced in cystic fibrosis (CF) patients. We have examined the inducible isoform of nitric oxide synthase (iNOS) in the airways by immunostaining and found that iNOS is constitutively expressed in the airway epithelia of non-CF mouse and human tissues but essentially absent in the epithelium of CF airways. We explored potential consequences of lost iNOS expression and found that iNOS inhibition significantly increases mouse nasal trans-epithelial potential difference, and hindered the ability of excised mouse lungs to prevent growth of Pseudomonas aeruginosa. The absence of continuous nitric oxide production in epithelial cells of CF airways may play a role in two CF-associated characteristics: hyperabsorption of sodium and susceptibility to bacterial infections.