Molecular and biochemical characterization of a CNP-sensitive guanylyl cyclase in bovine tracheal smooth muscle.

Muscarinic activation of bovine tracheal smooth muscle (BTSM) is involved in cyclic guanosine monophosphate (cGMP) production mediated through soluble (sGC) and membrane-bound (mGC) guanylyl cyclases. A muscarinic- and NaCl-sensitive mGC exists in BTSM regulated by muscarinic receptors coupled to G proteins. To identify the mGCs expressed in BTSM, reverse transcriptase/polymerase chain reaction (RT-PCR) from total RNA was performed using degenerate oligonucleotides for amplification of a region conserved among GC catalytic domains. Cloning of amplification products revealed that 76% of all BTSM GC transcripts corresponded to the sGC beta1 subunit and 24% to the B-type (C-type NP 1-22 [CNP]-sensitive) GC receptor. cGMP production by BTSM membrane and soluble fractions confirmed that sGC activity is 3-fold with respect to mGC activity. RT-PCR using specific oligonucleotides revealed that A (atrial NP-sensitive) and C (guanylin-sensitive) mGC subtypes are also expressed in BTSM. Stimulation of basal plasma membrane GC activity by CNP was higher than that by ANP, whereas guanylin showed no effect, indicating that CNP-sensitive guanylyl cyclase (GC-B) is the predominant functional BTSM mGC subtype. Strong adenosine triphosphate inhibition of CNP-stimulated mGC activity supports the finding that the tracheal mGC isoform belongs to the natriuretic peptide-sensitive mGCs. Additionally, CNP was able to reverse the chloride inhibition of BTSM mGC activity, suggesting that this is a novel G protein-coupled GC-B receptor.

Cloning and characterization of the human and Caenorhabditis elegans homologs of the Saccharomyces cerevisiae MSH5 gene.

In Saccharomyces cerevisiae the MSH5 gene encoding a MutS homolog was identified as a gene required for meiotic crossing over. To understand the role of MSH5 in higher eukaryotes, we have identified both the human and the Caenorhabditis elegans MSH5 genes. The human and C. elegans MSH5 predicted amino acid sequences share, respectively, 25.3 and 22.0% identity with the S. cerevisiae MSH5 amino acid sequence. The human MSH5 gene consists of 25 exons and spans at least 12 kb of genomic DNA, while the C. elegans gene comprises 17 exons distributed over at least 5.8 kb. Radiation hybrid mapping studies indicate that the human gene is located at 6p22.3-p21.3. Northern blot analysis demonstrates that human MSH5 is expressed to some extent in all tissues, but that particularly high levels of expression occur in testis, thymus, and other tissues of the immune system. Two-hybrid interaction analysis demonstrates that the human MSH4 and MSH5 proteins interact as observed for S. cerevisiae MSH4 and MSH5.

Cytosolic arylamine N-acetyltransferase (NAT) deficiency in the dog and other canids due to an absence of NAT genes.

The purpose of this study was to determine the molecular basis in the dog for an unusual and absolute deficiency in the activity of cytosolic N-acetyltransferase (NAT), an enzyme important for the metabolism of arylamine and hydrazine compounds. NAT activity towards two NAT substrates, p-aminobenzoic acid and sulfamethazine, was undetectable in dog liver cytosol, despite substrate concentrations ranging from 10 microM to 4 mM and a wide range of incubation times. Similarly, no protein immunoreactive to NAT antibody was evident on western blot analysis of canine liver cytosol. Southern blot analysis of genomic DNA from a total of twenty-five purebred and mixed bred dogs, and eight wild canids, probed with a full-length human NAT2 cDNA, suggested an absence of NAT sequences in all canids. Polymerase chain reaction amplification of genomic DNA using degenerate primers designed to mammalian NAT1 and NAT2 consensus sequences generated products of the expected size in human, mouse, rabbit, and cat DNA, but no NAT products in any dog or wild canids. These results support the conclusion that cytosolic NAT deficiency in the domestic dog is due to a complete absence of NAT genes, and that this defect is shared by other canids.

Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs.

OBJECTIVES: To use exon 7-specific genomic polymerase chain reaction (PCR) products to identify the genotypes of normal, affected, and carrier female dogs in pedigrees segregating Golden Retriever muscular dystrophy (GRMD), and to confirm the concordant segregation of the mutation in all carrier and affected dogs presently available. DESIGN: The GRMD mutation is found in the consensus splice acceptor site in intron 6 of the canine dystrophin gene. PCR cycle-sequencing and restriction fragment length polymorphism/PCR were used for determination of the pattern of segregation of the point mutation which causes GRMD. ANIMALS: Normal, clinically affected, and obligate carrier dogs in pedigrees of GRMD. PROCEDURE: DNA from blood was amplified, using PCR and primers that bracket all of exon 7 of the canine dystrophin gene as well as 100 base pairs of intron on either side. PCR products were either cycle-sequenced directly or submitted to a second round of PCR, using 1 of the original primers coupled with a mutagenic restriction fragment length polymorphism-primer, which thus creates an artificial restriction site. Digestion with Stu I detected the normal allele. To detect the affected allele, Sau96 I was used to digest the 310-base pair exon 7 genomic fragment directly. CONCLUSIONS: Simple, clear diagnosis of carrier status was possible using these methods. This mutation is passed through all carrier and affected dogs in both United States GRMD colonies and the colony in Australia. CLINICAL RELEVANCE: Rapid, accurate diagnosis of carrier and affected dogs will enhance study of this homologue of Duchenne muscular dystrophy.

Deletion of the dystrophin muscle promoter in feline muscular dystrophy.

We have characterized the mutation in a feline model of DMD that selectively eliminates expression of the muscle and Purkinje neuronal dystrophin isoforms. The cortical neuronal isoform was expressed at a detectable level in skeletal muscle in the absence of the muscle promoter and levels of PCR products representing cortical neuronal-type transcripts in dystrophic muscle were comparable to those of normal feline skeletal muscle. Although localized at the sarcolemma, cortical neuronal dystrophin apparently failed to protect skeletal muscle. Neuronal transcripts could not be amplified from feline heart, indicating that these promoters are not active in this tissue in the cat.

Canine X-linked muscular dystrophy as an animal model of Duchenne muscular dystrophy: a review.

Canine X-linked muscular dystrophy is a spontaneously occurring, progressive, degenerative myopathy of dogs that is clinically and pathologically similar to Duchenne muscular dystrophy in man. The molecular basis for the disease has been shown to be a lack of dystrophin, the protein product of the Duchenne muscular dystrophy gene. Breeding colonies of dystrophic dogs have been established. This report reviews the findings of genetic, clinical, pathologic, molecular biologic, and immunocytochemical studies of the canine model, and compares the features of the canine disease to those of Duchenne dystrophy in man.

Mosaic expression of dystrophin in carriers of canine X-linked muscular dystrophy.

The expression of dystrophin was examined immunocytochemically in skeletal and cardiac muscle of canine carriers of X-linked muscular dystrophy, a model of Duchenne muscular dystrophy. In skeletal muscle, the expression of dystrophin was heterogeneous, individual fibers being strongly positive, negative, or intermediate in staining. Some fibers expressed dystrophin discontinuously along their length. Between 4 and 24 weeks of age, the expression of dystrophin in skeletal muscle of carriers became more uniform and the number of negative fibers declined. In the heart dystrophin was also expressed in a mosaic pattern, individual cells being either fully positive or negative by immunocytochemistry. There was no apparent change in the pattern of expression between 12 weeks and 4 years of age. These results indicate that in the mosaic skeletal muscle fibers of carriers dystrophin is initially expressed in a heterogeneous pattern and that, in time, it becomes more uniformly distributed. This change could be explained by recruitment of competent satellite cells or by gradual diffusion of the protein.

The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs.

Duchenne muscular dystrophy (DMD) is the most common and the most severe of the muscular dystrophies in man. It is inherited as an X-linked recessive trait and is characterized by ongoing necrosis of skeletal muscle fibres with regeneration and eventually fibrosis and fatty infiltration. Although the gene and gene product which are defective in DMD have recently been identified, the pathogenesis of the disease is still poorly understood. A myopathy has been described in the dog which has been shown to be inherited as an X-linked trait and which is therefore a potential model of the human disease. We have studied the phenotypic expression of the disease, canine X-linked muscular dystrophy (CXMD), and have examined the molecular relationship between it and DMD. We report here that dogs with CXMD faithfully mimic the phenotype of Duchenne muscular dystrophy and that they lack the Duchenne gene transcript and its protein product, dystrophin.

Comparative studies on three isolates of Breda virus of calves.

Three isolates of Breda virus of calves were compared morphologically and antigenically. The isolates demonstrated similar morphology and shared common antigens, as determined by enzyme-linked immunosorbent assay and immunoelectron microscopy. On the basis of results of the hemagglutination-inhibition test, enzyme-linked immunosorbent assay, and immunoelectron microscopy, the 3 isolates were further subdivided into 2 serotypes: serotype 1 (Breda virus 1) represented by the Iowa isolate 1; and serotype 2 (Breda virus 2), by the Ohio isolate and the Iowa isolate 2. The 3 isolates caused diarrhea in gnotobiotic calves.