Antisera against two peptides, corresponding to different domains of the cystic fibrosis gene product CFTR, have been raised and extensively characterized. Both antisera recognize CFTR as a 165-kDa polypeptide in Western analysis of cells transfected with CFTR cDNA as well as in epithelial cell lines. The cell and tissue distribution of CFTR has been studied by immunocytochemistry. CFTR is abundant in epithelial cells, including those lining sweat ducts, small pancreatic ducts, and intestinal crypts. Unexpectedly, the level of CFTR in lung epithelia is relatively low, while it is abundant in the epithelia of kidney tubules. The protein appears to be restricted to the apical, rather than basolateral, regions of epithelial cells and at least a proportion is associated with the plasma membrane. The cell and tissue distributions of CFTR are consistent with a function for this protein as a chloride channel or as a regulator of channel activity.

Enriched preparations of histidine-rich polypeptides (HRPs) and isolated HRP pairs (1-2, 3-4 and 5-6) degrade in the presence of fresh autologous whole saliva to a series of low-molecular-weight cationic peptides (HRPs 6a-c and 7). Analysis of the HRPs during degradation indicates that: HRP 1 is not the parent molecule of the HRPs; the HRP pairs do not convert to each other in a cascade-like sequence in saliva; and the HRPs can be separated into 2 groups consisting of HRPs 1-2 and 3-7. Preparations containing HRPs 1-7, 1-2, and 3-7 were obtained by fractionation and separation on Bio-Rex 70, and tested for aggregating and antibacterial effects against Streptococcus mutans BHT, S. mutans GS-5 and Streptococcus sanguis G9B. HRPs 1-2 had significant aggregating effects on all 3 strains but the other HRPs had little to no agglutinating ability. The HRPs did not inhibit the growth of S. sanguis, and HRPs 1-2 enhanced its growth. No growth enhancement by the HRPs was observed for the 2 S. mutans strains. However, significant bacterial inhibition of the S. mutans strains was noted after incubation with HRPs 3-7. The data suggest that the dissimilar effects of HRPs 1-2 and 3-7 may be of importance in the colonization and growth of S. mutans and S. sanguis in vivo.

The gene responsible for the lethal disorder cystic fibrosis encodes a 1480-amino acid glycoprotein, CFTR. Using polyclonal antibodies directed against separate phosphorylation sites in the pre-nucleotide-binding fold (exon 9) and the R domain (exon 13), we have identified a 165-kDa protein in Xenopus laevis oocytes iniected with recombinant CFER cRNA transcribed from the full-length CFTR plasmid pBQ4.7.A protein of the same mobility was also detected with Western blotting techniques in whole cell extracts of cells that express CFTR mRNA (T84, FHTE, HT-29), including biopsied human nasal and bronchial tissue. Immunodetectable 165-kDa protein was concentrated in the apical membrane fraction of ileal villus tissue. We also report that the 165-kDa protein levels can be modulated pharmacologically, and these levels are appropriately correlated with second-messenger-regulated Cl-efflux. Thus, native or recombinant CFTR can be recognized by these anti-CFTR peptide polyclonal antibodies.Cystic fibrosis (CF) is a lethal autosomal recessive disorder in which abnormal regulation of epithelial Cl-channels is associated with the pathophysiology of the disease. The CF gene has been cloned and sequenced (1) and the amino acid sequence of the putative product (cystic fibrosis transmembrane conductance regulator, or CFTR) has been deduced (2). CFTR contains two membrane domains, each with six potential transmembrane segments, two nucleotide-binding folds (NBFs), and a highly charged cytoplasmic domain (R domain). The most common mutation associated with CF is the deletion of three nucleotides that would encode a phenylalanine at position 508 within the first NBF (exon 10). Both the NBFs and the R domain contain multiple potential phosphorylation sites for protein kinase A and protein kinase C (2). Protein kinases are important physiologic regulators of Cl-secretion, and defective regulation of outwardly rectifying Cl-channels by protein kinases A and C is one defect in CF patients (3, 4). Since it has been suggested that CFTR is either a Cl-channel (5-7) or a regulatory protein closely associated with the channel, we reasoned that one or more phosphorylation sites in CFTR might be important functionally and detectable immunologically.This report describes the identification of CFTR by the use of antibodies raised against peptides containing phosphorylation sites in the CFTR pre-NBF and R domains. We have confirmed that our antibodies detect a 165-kDa protein by heterologous expression in Xenopus laevis oocytes. With this information at hand, we report on the use of immunological and molecular biological techniques to study the expression and modulation of CFTR protein in established cell lines and human tissue biopsy specimens. METHODSProduction of Synthetic Peptide Antigens and Polyclonal Antisera. Peptide 181 (CFTR amino acid residues 415-427, exon 9, pre-NBF) and peptide 169 (residues 724-746, exon 13, R domain) were synthesized by the Applied Biosystems peptide synthesizer of the Johns Hopkins University Medical Sch...

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SUMMARY. One hundred and twenty six strains of streptococci isolated from the bloodstreams of 123 hospital in-patients-55 with endocarditis-and 255 strains isolated from the gingival crevices of 66 volunteers were characterised. Species isolated were Streptococcus mitior, S. sanguis, undifferentiated viridans streptococci, S. salivarius, S. milleri, S. mutans, S. bovis and S. faecalis. There was no significant difference between the distribution of species in blood and in the gingival crevice. S. mitior and S . sanguis were found less frequently in blood than their presence in the gingival crevice would have suggested, whereas S. milleri was found more frequently. This may indicate that S. milleri is more invasive or that it enters the bloodstream from sources other than the gingival crevice.

The ability of physiological amounts of lysozyme to de-chain two serotype c strains of Streptococcus mutans was determined. Both human and hen lysozymes were equally effective in chain breakage of S. mutans DPR and S. mutans DJR. De-chaining did not affect growth of cultures, but resulted in finely dispersed suspensions, at stationary phase, which were visibly different from untreated cultures. Less than 50 micrograms lysozyme per ml culture medium reduced chain length to virtually all diplococci and single cells, and this chain disruption increased total viable cell count. De-chaining required an active enzyme indicating that a degree of hydrolysis of the peptidoglycan occurred at the septae of the streptococci. De-chained S. mutans did not survive as well as streptococci of normal chain length when incubated under acidic conditions (pH 5.5), but gross cellular lysis was not apparent. The reduced aciduric property of the disrupted chains may have been due to a participation of autolysins or to a lethal triggered by the lysozyme-damaged peptidoglycan. De-chaining may be a mechanism by which lysozyme could regulate the levels of S. mutans in acidogenic plaque samples.

The adhesion to fibrin-platelet clots in vitro of 21 strains of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis (SABE) was measured. The species, in order of greatest adhesion, were Streptococcus faecalis, Streptococcus mutans, Streptococcus milleri, Streptococcus sanguis, dextran-positive Streptococcus mitior, dextran-negative Streptococcus mitior and Streptococcus salivarius. Individual strains within species, however, cannot be assumed to be representative of their species and may exhibit unusually high or low adhesion. Adhesion depended upon both bacterial concentration and period of contact. There was no simple relationship between ability to adhere and liability to cause endocarditis. Formation of dextran did not increase adhesion. The streptococci were more adhesive than strains of Escherichia coli and Neisseria sicca and less adhesive than strains of Staphylococcus aureus and Streptococcus pyogenes.