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Clinical and genetic characterization of a neurologic disorder resembling Refsum disease in a Norwegian consanguineous family.

Cecropins are a family of small, basic antibacterial polypeptides which can be isolated from pupae of immunized Lepidoptera. They are active against both gram-negative and gram-positive bacteria. We studied a mutant of Escherichia coli, strain SB1004, which is more sensitive to cecropin D than is the parental strain. The mutant was selected as resistant to a host range mutant of a Serratia marcescens phage. When the protein composition of the outer membrane was examined, strain SB1004 and some other phage-resistant mutants were found to be deficient in the OmpC protein. It was concluded that the OmpC protein is the receptor of the phage. Strain SB1004 was found to differ from other ompC mutants in being especially sensitive to hydrophobic antibiotics and to cecropin D. Furthermore, strain SB1004 has a tendency for spontaneous autolysis. A genetic analysis showed the mutations in strain SB1004 and a suppressor mutant to map in the ompC region. The activity of cecropin D against different strains of E. coli was specifically enhanced when divalent cations were absent. No such effect was found with cecropins A and B, which are less hydrophobic than the D form.

We previously showed that in pupae of Hyalophora cecropia, eight hemolymph proteins (P1 through P8) were selectively synthetized after immunization (Faye et al., Infect, Immun. 12:1426-1438, 1975). We also showed that a gross fractionation was obtained by a series of ammonium sulfate precipitations (designed A through D) and that protein P5 was enriched in fraction A. Starting from fraction A, we have now purified protein P5 by using dialysis, isoelectric focusing, and hydroxylapatite chromatography. The final product gave a single band in both gradient gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using the latter method, proteins P5 and P8 were found to be enriched in fraction A, but they were absent in fraction Ac prepared from nonimmunized pupae. Protein P5 was found to have a pI of 6.6 and a molecular weight of 24,000 in sodium dodecyl sulfate and 96,000 in tris (hydroxymethyl) aminomethane-borate, pH 9.0. These data suggest a structure for P5 composed of four subunits of equal size. Protein P5 stimulated the killing of Escherichia coli by hemolymph fractions B and D, but it had neither killing nor phenol oxidase activity of its own.

Abstract: Isolated autosomal dominant hypoparathyroidism is a heterogeneous disorder characterized by parathyroid hormone (PTH) deficiency, hypocalcemia and hyperphosphatemia. The candidate gene approach was used to study a large Norwegian family. The loci for the PTH gene, PTH receptor gene and RET protooncogene were excluded using dinucleotide markers and restriction fragment length polymorphism analysis. Complete cosegregation of this trait was found with the chromosomal region 3q13, using the short tandem repeat markers D3S1267, D3S1269, D3S1303, D3S1518, and RHO. This region contains the candidate locus for the Ca2+-sensing receptor (PCAR1). By single-strand conformation polymorphism (SSCP) analysis of all PCAR1 exons followed by automated sequencing, we identified a C to T transition in exon 2 (cDNA position 452) on the mutant allele in the family. The mutation predicts a substitution of Thr to Met in amino acid position 151 (T151M). A StyI restriction site created by the nucleotide substitution was used to confirm the mutation on all alleles, as well as to exclude it among 100 normal alleles (blood donors). SSCP analysis also identified a novel polymorphism of PCAR1 intron 4 (1609–88t→c) on normal alleles.The T151M mutation is located in the extracellular N-terminal domain of PCAR1, which belongs to the superfamily of G protein-coupled receptors. We suggest that this is a gain-of-function mutation that increases the sensitivity of the receptor to [Ca2+], thereby decreasing the calcium set point.

We have previously shown that pupae of the giant silkmoth Samia cynthia have a humoral antibacterial activity, which was induced by viable, nonpathogenic gram-negative bacteria (H.G. Boman et al., 1974). We show here that this activity was formed simultaneously with a selective incorporation of amino acids into eight polypeptide chains characterized by their electrophoretic behavior. If actinomycin D or cycloheximide were given at an early time, no antibacterial activity was found. If the inhibitors were given at the time of maximum activity, there was no effect with actinomycin D but a rapid decrease of the activity in the case of cycloheximide. The results imply that the messenger ribonucleic acid was stable, but that at least one protein component was turning over. Hemolymph from immunized pupae of another giant silkmoth, Hyalophora cecropia, was fractionated by ammonium sulfate precipitation. This procedure, together with the isotope distribution after co-electrophoresis in polyarylamide gels, was used for comparing the response to injury and to different infections. Almost identical polypeptide patterns were obtained as a response to an infection with either viable Enterobacter cloacae or Bacillus subtilis. These patterns differed both qualitatively and quantitatively from the injury effect created by an injection as such. There was only a low antibacterial activity in each of the four fractions obtained by ammonium sulfate precipitation. However, a combination of three fractions restored a high killing activity. Fractionation of hemolymph from untreated pupae provided evidence for at least one preexisting factor which stimulated the killing of Escherichia coli. The osmotic pressure of the bacteria contributed to the antibacterial activity towards E. coli, but not towards B. subtitlis. The killing of E. coli was inhibited by liped A and, to a lesser extent, by an inhibitor of proteolytic enzymes. The similarities and differences with the mammalian complement system are discussed.

In the preceding paper we presented a formula for the composition of lipopolysaccharides (LPS) from Escherichia coli K-12. This formula contains four regions defined from analyses of LPS from four key strains, the parent and mutants which had lost one, two, or three regions of their carbohydrates. Support for the formula was derived from the susceptibility of the key mutants to several bacteriophages. One of these, phage phi W, was found specific for strains which had lost region 4. In this paper we described inactivation in vitro of phage phi W and its host-range mutant phi 5, using LPS devoid of regions 2 to 4. The blocking of phi W was found to require about 0.15 M concentrations of monovalent cations and to be inhibited by low concentrations of calcium and magnesium ions. One particle of phage phi W required 2 times 10-16 g of LPS devoid of region 4 for stoichiometric inactivation. Phage phi 5 was blocked by both heptose-less LPS (devoid of regions 2 to 4) and glucose-less LPS (devoid of regions 3 to 4) but was unaffected by LPS devoid of region 4. LPS from a heptose-less mutant of Salmonella minnesota showed the same inactivation ability as did LPS from heptose-less strains of E. coli K-12. Lipid A was prepared from LPS containing all four regions. Such lipid A was found to inactivate phi 5, whereas both the polysaccharide moiety as well as the intact LPS were without effect. It is suggested that lipid A is part of the receptor site for phage phi 5.

Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.

We have studied the degradation of a set of long peptides (9-30 amino acids) from the nucleoprotein of influenza A. In common for all these peptides is the core sequence NH2-Ser-Arg-Tyr-Trp-Ala-Ile-Arg-Thr-Arg-COOH, NP383-391, known as an antigenic peptide specific for the HLA-B27 class I antigen. We show that this peptide is generated by enriched cytosolic proteasomes of two sizes, 20S and 12S. The 12S proteasome is the precursor, the preproteasome, to the 20S mature proteasome as shown by pulse-chase experiment and is most likely responsible for the proteolytic activity in the 12S region. Cleavage at the N-terminus is distinct and restricted to residue 383, independent of the N-terminal extension of the peptide. The C-terminus is generated via cleavage at three sites. Intermediate and final peptide products were identified by mass spectrometry. Finally, we show that the NP383-391 peptide generated by proteasomes in vitro is functional inasmuch as it possesses the ability to stimulate assembly of in vitro translated HLA-B27 antigens.

Radiolabeled preprocecropin B, with an α-amidated COOH terminus, and preprocecropin A, extended at the COOH terminus by a glycine residue, were synthesized by solid-phase methods. The respective syntheses were interrupted at intervals to allow the preparation of the predicted procecropins A and B as well as three other truncated derivatives of the cecropin A precursors. All the synthetic peptides were purified to near homogeneity by reverse-phase liquid chromatography and their purity was established by analytical high performance liquid chromatography, gel electrophoresis, and amino acid analysis. A dipeptidyl aminopeptidase was purified about 350 times from the hemolymph of cecropia pupae and characterized by its affinity for different substrates and inhibitors. The synthetic prepro peptides were tested for processing by an extract of dog pancreas microsomes and purified leader peptidase from Escherichia coli, with and without partly purified dipeptidyl aminopeptidase, and the two synthetic proforms were also processed with the dipeptidyl aminopeptidase alone. From these experiments we conclude that the signal/leader peptidase cleaves the peptide bond between Ala−5and Ala−4. This cleavage site is further substantiated by radio sequencing of procecropin A isolated after synthesis in a coupled system for in vitrotranscription, translation, and processing. The two procecropins, which are stable to further digestion by the signal peptidase, are further processed by dipeptidyl aminopeptidase which removes, in two steps, the dipeptides Ala-Pro (residues −4 and −3) and Glu-Pro (residues −2 and −1). Although the synthetic peptide with only one dipeptide (Glu-Pro) preceding the mature cecropin sequence could function as a substrate for dipeptidyl aminopeptidase, it could be demonstrated as an intermediate in the enzymatic reaction with the procecropins. Dipeptidyl aminopoptidase did not cleave the procecropin analog when it was preceded by a single alanine residue, i.e.preprocecropin (−5,38). Antibacterial activity was demonstrated for the mature cecropin A-Gly obtained by processing of the synthetic preproprotein.

Cecropin D was synthesized by solid-phase methods and shown to be homogeneous and of correct composition and molecular weight. It was indistinguishable from natural cecropin D and constitutes a structure proof for this peptide. Several analogs of cecropin D were synthesized and used to draw conclusions about the structural features contributing to antibacterial activity. They included [Lys1]cecropin D, [Gin3, Leu4] cecropin D, and cecropin D-(9–37). It was concluded that a strongly basic NH2-terminal segment is a prerequisite for antibacterial activity. A hybrid analog cecropin A-(1–11) D-(12–37) was designed and predicted to have enhanced potency. It was found to be 5 to 55 times as active as cecropin D against six of the bacteria tested and was slightly more active than cecropin A. However, against Bacillus subtilisBs11 the analog was 6 times more active than cecropin A.

Pig small intestine was used as starting material for a batchwise isolation of a peptide fraction enriched in antibacterial activities against Escherichia coli (anti-Ec factor) and against Bacillus megaterium (anti-Bm factor). Separation and further purification were by different types of chromatography. Sequence analysis showed the anti-Bm factor to be apparently similar to vasoactive intestinal peptide. The anti-Ec factor was found to have a 31-residue sequence that was cecropin-like. It was named cecropin P1 and its structure was confirmed by solid-phase synthesis. Synthetic cecropin P1 with and without C-terminal amide was assayed on eight different bacteria. Mobility comparison between synthetic and natural cecropin P1 indicates that the natural peptide has a free C-terminal carboxyl group.

Mutants of Bacillus thuringiensis lacking either beta-exotoxin or gamma-endotoxin were compared for their virulence using pupae of a giant silk moth. Known doses of viable log-phase bacteria were injected, and the response was followed as the number of viable bacteria in the hemolymph. The results obtained imply that, in the system used, neither the beta-exotoxin nor the gamma-endotoxin and the sporeforming ability are of importance for virulence. Results with sterile culture filtrate from B. thuringiensis have given evidence for the production of two inhibitors, A and B, which interfere with the humoral defense system in pupae of Hyalophora cecropia. Inhibitor A, which blocked the lysis of Escherichia coli,was precipitated by trichloroacetic acid and sensitive to heating. Inhibitor B, which blocked the killing of Bacillus cereus, was soluble in trichloroacetic acid and resistant to 90 degrees C for 5 min. Both inhibitors are believed to contribute to the insecticidal nature of B. thuringiensis.

Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance. A key step was the induction of competence by treatment of the bacteria with 50 mM Tris hydrochloride buffer (pH 8.9) containing 30% sucrose. Transformation frequency was strongly influenced by culture density during the uptake of DNA and required the presence of polyethylene glycol. Growth in a minimal medium supplemented with Casamino Acids gave 35 times more transformants than growth in a rich medium. The highest frequencies were obtained with covalently closed circular DNA. With all parameters optimized, the frequency was 10(-3) transformants per viable cell or 10(4) transformants per microgram of DNA. Cells previously frozen were also used as recipients in transformation experiments; such cells gave frequencies similar to those obtained with freshly grown cells. The procedure was optimized for B. thuringiensis subsp. gelechiae, but B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis, and B. thuringiensis subsp. israelensis were also transformed. Compared with protoplast transformation, our method is much faster and 3 orders of magnitude more efficient per microgram of added DNA.

Starting from a crystal-negative parental strain of Bacillus thuringiensis, we isolated certain bacteriophage-resistant mutants which showed decreased virulence in pupae of the cecropia moth (Hyalophora cecropia). These strains (class I mutants) were highly pleiotropic and showed resistance to seven or eight different phages, sensitivity to methicillin, and loss of flagella. They were also more sensitive to cecropia immune hemolymph in vitro. In addition, the export of at least three proteins was reduced. Revertants (class II mutants) were sensitive to phages, virulent, and resistant to penicillin derivatives. One class II mutant was a complete revertant in all properties examined. The other class II mutant was an incomplete revertant still susceptible to immune hemolymph and with repressed export of proteins. Virulence was not coupled to phage resistance as such or to lack of flagella because other mutants affected in these properties were virulent. Other factors which could be excluded as causes of virulence were production of extracellular protease and hemolysin.