Kinetic analysis of chloride efflux from normal and cystic fibrosis fibroblasts

Chloride permeability in 9 cystic fibrosis- and 11 normal-skin fibroblast lines has been investigated. Chloride efflux, under steady-state conditions, involves two intracellular compartments characterized by slow- and fast-rate constants of efflux. We show here that the fast rate constant in cystic fibrosis cells is reduced by 25% in comparison with controls. The data presented support recent studies indicating that isolated sweat glands and respiratory epithelia of patients suffering from cystic fibrosis have an unusual low permeability to chloride ions compared to control epithelia. It is concluded that variation in chloride transport can successfully be studied in cultured fibroblasts, which are not directly involved in the pathology of the disease.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes

The nucleotide sequences of the Escherichia coli genome between the glycogen biosynthetic genes glgB and glgC, and 1170 bp of DNA which follows glgA have been determined. The region between glgB and glgC contains an open reading frame (ORF) of 1521 bp which we call glgX. This ORF is capable of coding for an M_r 56 684 protein. The deduced amino acid (aa) sequence for the putative product shows significant similarity to the E. coli glycogen branching enzyme, and to several different glucan hydrolases and transferases. The regions of sequence similarity include residues which have been reported to be involved in substrate binding and catalysis by taka-amylase. This suggests that the proposed product may catalyze hydrolysis or glycosyltransferase reactions. The cloned region which follows glgA contains an incomplete ORF (1149 bp), glgY, which appears to encode 383 aa of the N terminus of glycogen phosphorylase, based upon sequence similarity with the enzyme from rabbit muscle (47% identical aa residues) and with maltodextrin phosphorylase from E. coli (37% identical aa residues). Results suggest that neither ORF is required for glycogen biosynthesis. The localization of glycogen biosynthetic and degradative genes together in a cluster may facilitate the regulation of these systems in vivo.     

The role of monocytes in rheumatoid synovitis

In non-specific and rheumatoid synovitis, the use of specific monoclonal antibodies against antigenic determinants of cells of the immune system showed that the characteristic changes of rheumatoid synovitis are located in the synovial internal layers. The monocytes were OKM1, OKM5, S100, OKDR positive, while the subintimal monocytes in non-specific synovitis were OKDR negative. We suggest that, in rheumatoid synovitis, the previously activated monocytes are transported by the bloodstream and pass through the so-called "sinovial barrier" to arrive in the subintimal layers ready to interact with T helper lymphocytes and initiate the immune response mechanisms responsible for lesions in rheumatoid synovitis.     

The muskrat in biomedical research

Muskrats are aquatic rodents of moderate size which are plentiful throughout North America, but are not used commonly in the laboratory. Recently, we tested the feasibility of muskrats as experimental models and have found them to be acquired and cared for easily in conventional laboratory animal facilities. Some of their natural characteristics and diseases are described. The husbandry techniques that we used are presented and form a base for the preparation of future guidelines for the maintenance and use of feral animals in research. The results of some initial experiments testing the muskrat's utility for investigations of cardiorespiratory control mechanisms also are presented. Our data show that even anesthetized muskrats possess brisk and dramatic cardiovascular and respiratory reflexes. Our findings that their brains possess the cytoarchitectural and myeloarchitectural features comparable to other mammals, combined with their relative uniformity in size, has allowed us to locate specific neuronal loci stereotaxically. We suggest that the muskrat be considered as an experimental animal model for studies of the neural control of cardiorespiratory systems.     

Deletion ΔF508 and haplotype analysis of CFTR gene region in Slovak CF patients

Summary Analysis of a sample of 50 unrelated cystic fibrosis (CF) patients and 46 nuclear families from Slovakia (Czechoslovakia) by the polymerase chain reaction and Southern hybridization revealed that the proportion of the F508 mutation was 58% in this population, and that the frequency of the B (i.e., KM19/XV2c [1–2]) haplotype was increased in both F508 and nonF508 CF chromosomes (98% and 46%, respectively). These results support the view that the trans-European gradient of the F508 frequency is of a geographical rather than of an ethnic origin, and that in Slavonic populations, there exists an as yet unidentified but frequent CF mutation other than F508, associated with the B haplotype.     

Familial porphyria cutanea tarda: hybridization analysis of the uroporphyrinogen decarboxylase locus

Familial porphyria cutanea tarda (PCT) results from a deficiency of uroporphyrinogen decarboxylase (URO-D) activity. Hybridization analysis of genomic DNA from unrelated normal individuals and PCT pedigree members failed to detect any major deletions, rearrangements or restriction fragment length polymorphisms at the URO-D locus.     

Recombinations between IRP and cystic fibrosis.

A candidate gene for cystic fibrosis was recently isolated by selective cloning of HpaII-tiny-fragment islands; it maps considerably closer to CF than does MET or D7S8 (pJ3.11), and DNA polymorphisms from this region are in marked disequilibrium with CF. cDNA cloning has shown that this protein has a growth factor-like structure and shows homology to the murine and human proto-oncogene int-1; it is designated IRP (int-1-related protein). DNA sequences from the IRP locus that recognize RFLPs are proving to be highly informative for prenatal diagnosis. We report five crossovers that have been identified which occur either within the IRP locus or between IRP and CF; these recombinants demonstrate that CF maps between the DNA markers D7S8 and KM.19.     

IFN-gamma facilitates NGF-induced neuronal differentiation in PC12 cells

Natural or recombinant murine interferon-gamma causes a reversible arrest of proliferation of PC12 cells. Treatment with other antimitotics (AraC, colchicine, mitomycin C, hydroxyurea) or removal of serum, on the contrary, leads to mitotic arrest followed by cell death. IFN-gamma-treated PC12 cells respond more rapidly to NGF in terms of speed of neuronal outgrowth. On the other hand, NGF potentiates the action of IFN-gamma in stimulating the enzyme 2',5'-A synthetase which shifts from an average of 4.4-fold stimulation at 48 h with IFN-gamma alone to increments varying between 5- and 18-fold when PC12 cells are treated for 48 h with IFN-gamma and NGF. NGF alone, on the contrary, does not exert any detectable effect on this enzyme. From the findings we propose the use of a combined treatment of PC12 cells with NGF and IFN-gamma for a more rapid induction of neuronal differentiation.