[Group Training of Patients with Chronic Lung Diseases under Outpatient Conditions - Recommendations of the Working Group Lung Sports in Germany and the German Airways League]. / Ambulanter Lungensport und körperliches Training bei Patienten mit Atemwegs- und Lungenkrankheiten.

To improve acceptance and use of physical training by patients with chronic lung diseases, recommendations for performing lung exercises on an outpatient basis in a group setting are given by experts in physical training, sports therapists and pulmonologists. The evidence-based positive effects of physical training were analyzed for asthma , COPD, interstitial lung diseases, cystic fibrosis, lung carcinoma, and pulmonary hypertension. The requirements for lung exercises in outpatient groups as well as compensation by care providers were given on the basis of legal regulations. Furthermore, the main items of the training units as well as supervision by specially trained group leaders in relation to the severity of the underlying lung disease are described. Finally, aspects of safety of the participating patients are discussed, including the prevention of infection with corona-2-virus.

Development of adenovirus serotype 35 as a gene transfer vector.

While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral vectors designed for gene transfer have been generated in the adenovirus serotype 5 (Ad5) backbone. Viral infections caused by Ad5 are endemic in most human populations and the majority of humans carry preexisting humoral and/or cellular immunity to Ad5 which may severely limit the use of Ad5-based vectors for gene therapy applications. To circumvent this preexisting Ad5 immunity, we have identified Ad35 as an alternative adenoviral serotype to which the majority of humans do not have neutralizing antibodies. Importantly, Ad35 can be grown to high titers with a low particle-to-PFU ratio. As a prerequisite for the development of Ad35 for use as a gene transfer vector, a genome organization map was constructed using the available Ad35 sequence information, and E1a-deficient Ad35 vectors encoding marker genes were generated. Ad35 biodistribution in mice was assessed following intravenous administration and compared with that of Ad5. Extremely low levels of Ad35 were detected in all organs evaluated, including liver, lung, spleen, and bone marrow, while Ad5 displayed high transduction of these organs. Due to the lack of Ad35 liver tropism, minimal hepatotoxicity was observed in mice treated with Ad35. Furthermore, the half-life of Ad35 in mouse blood was found to be two to three times longer than that of Ad5. These data suggest that either mice do not express the Ad35 cell surface receptor or that Ad35 does not efficiently transduce mouse cells in vivo following systemic delivery. Therefore, to begin to elucidate the Ad35 cell entry mechanisms, in vitro competition studies were performed. These data demonstrated that Ad35 cell entry is CAR independent, and may involve protein(s) expressed on most human cells.

DNA binding by single HMG box model proteins.

The HMG1/2 family is a large group of proteins that share a conserved sequence of approximately 80 amino acids rich in basic, aromatic and proline side chains, referred to as an HMG box. Previous studies show that HMG boxes can bind to DNA in a structure-specific manner. To define the basis for DNA recognition by HMG boxes, we characterize the interaction of two model HMG boxes, one a structure-specific box, rHMGb from the rat HMG1 protein, the other a sequence-specific box, Rox1 from yeast, with oligodeoxynucleotide substrates. Both proteins interact with single-stranded oligonucleotides in this study to form 1:1 complexes. The stoichiometry of binding of rHMGb to duplex or branched DNAs differs: for a 16mer duplex we find a weak 2:1 complex, while a 4:1 protein:DNA complex is detected with a four-way DNA junction of 16mers in the presence of Mg(2+). In the case of the sequence-specific Rox1 protein we find tight 1:1 and 2:1 complexes with its cognate duplex sequence and again a 4:1 complex with four-way branched DNA. If the DNA branching is reduced to three arms, both proteins form 3:1 complexes. We believe that these multimeric complexes are relevant for HMG1/2 proteins in vivo, since Mg(2+) is present in the nucleus and these proteins are expressed at a very high level.

The isolation and characterization of missense mutants in the general repressor protein Ssn6 of Saccharomyces cerevisiae.

Ssn6, a TPR repeat-containing protein, associates with the Tup1 protein to form a general transcriptional repression complex in Saccharomyces cerevisiae. As part of a genetic analysis of this complex, we targeted mutations to the TPR repeat-coding region of the SSN6 gene, and applied selection for constitutive expression of the hypoxic gene ANB1. All but one of the resulting mutants failed to express full-length Ssn6 protein, indicating that they harbored deletion, frameshift, or nonsense mutations. The one missense mutation encoded a protein with three amino acid substitutions, and the combination was required for the mutant phenotype. One mutation, a proline substitution for a serine at codon 51, was used in a second round of mutagenesis in which six further multiple-substitution alleles were obtained. These were separated into their component mutations, and again, all but one of the single substitutions displayed the wild-type phenotype. The single and multiple mutants were characterized in terms of their effects on the repression of the glucose-repressible SUC2 gene and the a mating-type gene STE2. The mutant Ssn6 proteins were also tested for their ability to associate with Tup1. The S5 P mutation, despite its lack of a mutant phenotype, had lost the ability to fully associate with Tupl in vitro. In general, those single substitutions that fell within the first two TPR repeats impaired Tup1-associating activity, while the two that fell in TPR repeats five and eight retained this activity. Overexpression of TUP1 partially suppressed the mutant phenotype in only some of the multiple mutants. The results are discussed in terms of the current models of Ssn6 function and the structure of TPR repeats.

Approaches to the study of Rox1 repression of the hypoxic genes in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a facultative aerobe that responds to changes in oxygen tension by changing patterns of gene expression. One set of genes that responds to this environmental cue is the hypoxic genes. Oxygen levels are sensed by changes in heme biosynthesis, which controls the transcription of the ROX1 gene, encoding a protein that binds to the regulatory region of each hypoxic gene to repress transcription. Several experimental molecular and genetic approaches are described here to study Rox1 repression. Derepression of the hypoxic genes is rapid, and one model for such a response requires that Rox1 have a short half-life. This was demonstrated to be the case by immunoblotting using a c-myc epitope-tagged protein. Rox1 repression is mediated through the general repressors Ssn6 and Tup1. To explore possible interactions among these proteins, all three were expressed and partially purified using a baculovirus expression system and histidine-tagged proteins. The effect of Ssn6 and Tup1 on the formation of Rox1-DNA complexes was explored using these purified proteins by both electrophoretic mobility shift and DNase I protection assays. We found that Rox1 DNA-binding activity decayed rapidly and that Ssn6 could stabilize and restore lost activity. Finally, genetic selections are described for the isolation of loss-of-function mutations in Rox1. Also, schemes are proposed for the reversion of such mutations. These selections have been extended to genetic analyses of the TUP1 and SSN6 genes.

Rox3 and Rts1 function in the global stress response pathway in baker's yeast.

Yeast respond to a variety of stresses through a global stress response that is mediated by a number of signal transduction pathways and the cis-acting STRE DNA sequence. The CYC7 gene, encoding iso-2-cytochrome c, has been demonstrated to respond to heat shock, glucose starvation, approach-to-stationary phase, and, as we demonstrate here, to osmotic stress. This response was delayed in a the hog1-delta 1 strain implicating the Hog1 mitogen-activated protein kinase cascade, a known component of the global stress response. Deletion analysis of the CYC7 regulatory region suggested that three STRE elements were each capable of inducing the stress response. Mutations in the ROX3 gene prevented CYC7 RNA accumulation during heat shock and osmotic stress. ROX3 RNA levels were shown to be induced by stress through a novel regulatory element. A selection for high-copy suppressors of a ROX3 temperature-sensitive allele resulted in the isolation of RTS1, encoding a protein with homology to the B' regulatory subunit of protein phosphatase 2A0. Deletion of RTS1 caused temperature and osmotic sensitivity and increased accumulation of CYC7 RNA under all conditions. Over-expression of this gene caused increased CYC7 RNA accumulation in rox3 mutants but not in wild-type cells.

Nucleotide sequence of the genes encoding the canine herpesvirus gB, gC and gD homologues.

The nucleotide sequence of the genes encoding the canine herpesvirus (CHV) gB, gC and gD homologues was determined. These genes are predicted to encode polypeptides of 879, 459 and 345 amino acids, respectively. Comparison of the predicted amino acid sequences of CHV gB, gC and gD with the homologous sequences from other herpesviruses indicates that CHV is an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties. Alignment of the homologous gB, gC and gD amino acid sequences indicates that most of the cysteine residues are conserved, suggesting that these glycoproteins possess similar tertiary structures. The nucleotide sequence of the open reading frame downstream from the CHV gC gene was also determined. The predicted amino acid sequence of this putative polypeptide appears to be homologous to a family of proteins encoded downstream from the gC gene in most, although not all, alphaherpesviruses.