Stabilization of hypoglycosylation in a patient with congenital disorder of glycosylation type Ia.

A follow-up over 7 years on a patient with congenital disorder of glycosylation type Ia showed a significant normalization of hypoglycosylated transferrin. Isoelectric focusing for serum transferrin is a widely used screening method but there could be a limit of detection and the subtle changes can be also overlooked. Re-test with a different method is desirable, especially when the clinical suspicion for congenital disorder of glycosylation is high.

A family of six flagellin genes contributes to the Caulobacter crescentus flagellar filament.

The Caulobacter crescentus flagellar filament is assembled from multiple flagellin proteins that are encoded by six genes. The amino acid sequences of the FljJ and FljL flagellins are divergent from those of the other four flagellins. Since these flagellins are the first to be assembled in the flagellar filament, one or both might have specialized to facilitate the initiation of filament assembly.

The Yersinia enterocolitica pYV virulence plasmid contains multiple intrinsic DNA bends which melt at 37 degrees C.

Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (

The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility.

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.

Temperature-dependent regulation of Yersinia enterocolitica Class III flagellar genes.

Temperature is a key environmental cue for Yersinia enterocolitica as well as for the two other closely related pathogens, Yersinia pestis and Yersinia pseudotuberculosis. Between the range of 30 degrees C and 37 degrees C, Y. enterocolitica phase-varies between motility and plasmid-encoded virulence gene expression. To determine how temperature regulates Y. enterocolitica motility, we have been dissecting the flagellar regulatory hierarchy to determine at which level motility is blocked by elevated temperature (37 degrees C). Here we report the cloning, DNA sequences, and regulation of the two main regulators of Class III flagellar genes, fliA (sigma F) and flgM (anti-sigma F), and a third gene, flgN, which we show is required for filament assembly. Identification of the Y. enterocolitica fliA and flgM genes was accomplished by functional complementation of both S. typhimurium and Y. enterocolitica mutations and by DNA sequence analysis. The Y. enterocolitica fliA gene, encoding the flagellar-specific sigma-factor, sigma F, maps immediately downstream of the three flagellin structural genes. The flgM and flgN genes, encoding anti-sigma F and a gene product required for filament assembly, respectively, map downstream of the invasin (inv) gene but are transcribed in the opposite (convergent) direction. By using Northern blot analyses we show that transcription of both fliA and flgM is immediately arrested when cells are exposed to 37 degrees C, coincident with the timing of virulence gene induction. Unlike S. typhimurium flgM mutants, Y. enterocolitica flgM mutants are fully virulent.

Co-ordinate, temperature-sensitive regulation of the three Yersinia enterocolitica flagellin genes.

Yersinia enterocolitica cells, when cultured at 30 degrees C or below, are flagellated and motile. Cells cultured at 37 degrees C or above lack flagella and are non-motile. To identify flagellin genes that are a target of this temperature-dependent regulation, a library of Y. enterocolitica genomic inserts in a phage lambda vector was probed with the Salmonella typhimurium fliC (flagellin) gene. A DNA fragment subcloned from a recombinant phage which hybridizes with the probe complements a non-motile S. typhimurium fliC-fljB- (flagellin-minus) mutant. DNA sequence analysis shows that Y. enterocolitica contains three tandem flagellin genes, designated fleA, fleB and fleC. All three genes are co-ordinately transcribed at low, but not high, temperature from fliA-dependent (sigma F) promoters. Flagellin transcription arrests rapidly after upshift to 37 degrees C (host temperature). In contrast, flagellin transcription resumes only after several generations when cells cultured at 37 degrees C are downshifted to 28 degrees C.

Thermoregulation in Yersinia enterocolitica is coincident with changes in DNA supercoiling.

Yersinia enterocolitica is a facultative intracellular parasite, displaying the ability to grow saprophytically or invade and persist intracellularly in the mammalian reticuloendothelial system. The transition between such diverse environments requires the co-ordinated regulation of specific sets of genes on both the chromosome and virulence plasmid. Temperature has a profound pleiotropic effect on gene expression and phenotypically promotes alterations in cell morphology, outer-membrane protein synthesis, urease production, lipopolysaccharide synthesis, motility, and synthesis of genes involved in invasion of eukaryotic host cells. By examining thermoregulated flagella biosynthesis, we have determined that motility is repressed at 25 degrees C (permissive temperature) with subinhibitory concentrations of novobiocin. These conditions also induce virulence gene expression suggesting novobiocin addition simulates, at least partially, a high-temperature environment. Furthermore, temperature-shift experiments, using Y. enterocolitica containing pACYC184 as a reporter plasmid, indicate that thermo-induced alterations of DNA supercoiling coincide with temperature-induced phenotypic changes. A class of putative DNA gyrase mutant (novobiocin resistant) likewise demonstrates the 37 degrees C phenotype when cultured at 25 degrees C; it is non-motile, urease negative, calcium growth dependent, and positive for Yop expression. These results support a model implicating DNA topology as a contributing factor of Y. enterocolitica thermoregulation.

Effect of oxyquinoline ointment on diaper dermatitis.

The effectiveness of oxyquinoline ointment for diaper dermatitis was tested in a randomized double-blind trial. Compared to a combined control treatment group using Desitin or A & D ointment, use of oxyquinoline ointment significantly improved rash.

[Promotion of hair growth with thiocyanate in guinea pigs]. / Förderung der Haarentwicklung durch Thiocyanat beim Meerschweinchen.

The advancement of growth of albino guinea pig hair has been induced by sodium thiocyanate through oral application (32 mg/kg) and more distinctly through balneological use (0.3, 5 resp. 10 g/l). The density of the hair has been increased at 6-38%, the longitudinal growth advanced at 2-14% and the phases of follicle cycle have been changed on behalf of anagenic hair at 11-39%.

Does cold burnishing gutta-percha create a better apical seal?

This study investigated the seal created by cold burnishing the gutta-percha exposed after apical root resection of endodontically treated teeth. Sixty single-rooted extracted human teeth with a single straight canal were divided into four experimental groups of 15 teeth each. In two of the experimental groups the canals were instrumented and obturated well with laterally condensed gutta-percha and sealer. The remaining two groups were instrumented and poorly obturated with laterally condensed gutta-percha and sealer. The apical 2 mm of all the teeth were then resected and the effect of cold burnishing the exposed gutta-percha was investigated using a linear dye penetration technique. Under the condition of this study, cold burnishing gutta-percha after apical root resection of a well-obturated root canal resulted in a poorer apical seal than if no burnishing were performed. Cold burnishing the exposed gutta-percha after apical root resection of poorly obturated root canals improved the apical seal as compared with nonburnished poorly obturated canals.

Role of the 25-, 27-, and 29-kilodalton flagellins in Caulobacter crescentus cell motility: method for construction of deletion and Tn5 insertion mutants by gene replacement.

Caulobacter crescentus incorporates two distinct, but related proteins into the polar flagellar filament: a 27-kilodalton (kDa) flagellin is assembled proximal to the hook and a 25-kDa flagellin forms the distal end of the filament. These two proteins and a third, related flagellin protein of 29 kDa are encoded by three tandem genes (alpha-flagellin cluster) in the flaEY gene cluster (S.A. Minnich and A. Newton, Proc. Natl. Acad. Sci. USA 84: 1142-1146, 1987). Since point mutations in flagellin genes had not been isolated their requirement for flagellum function and fla gene expression was not known. To address these questions, we developed a gene replacement protocol that uses cloned flagellin genes mutagenized by either Tn5 transposons in vivo or the replacement of specific DNA fragments in vitro by the antibiotic resistance omega cassette. Analysis of gene replacement mutants constructed by this procedure led to several conclusions. (i) Mutations in any of the three flagellin genes do not cause complete loss of motility. (ii) Tn5 insertions in the 27-kDa flagellin gene and a deletion mutant of this gene do not synthesize the 27-kDa flagellin, but they do synthesize wild-type levels of the 25-kDa flagellin, which implies that the 27-kDa flagellin is not required for expression and assembly of the 25-kDa flagellin; these mutants show slightly impaired motility on swarm plates. (iii) Mutant PC7810, which is deleted for the three flagellin genes in the flaEY cluster, does not synthesize the 27- or 29-kDa flagellin, and it is significantly more impaired for motility on swarm plates than mutants with defects in only the 27-kDa flagellin gene. The synthesis of essentially normal levels of 25-kDa flagellin by strain PC7810 confirms that additional copies of the 25-kDa flagellin map outside the flaEY cluster (beta-flagellin cluster) and that these flagellin genes are active. Thus, while the 29- and 27-kDa flagellins are not absolutely essential for motility in C. crescentus, their assembly into the flagellar structure is necessary for normal flagellar function.

A set of positively regulated flagellar gene promoters in Caulobacter crescentus with sequence homology to the nif gene promoters of Klebsiella pneumoniae.

The study reported here describes nuclease S1 mapping of the in-vivo transcription start sites of transcription units I and III of the hook gene cluster of Caulobacter crescentus. We show that transcription units I and II of this flagellar (fla) gene cluster, which have divergent promoters with transcription start sites separated by 218 nucleotides, are under positive transcriptional control by genes in transcription unit III. The promoters of transcription units I, II, and III were compared with flagellin gene promoters P25, P27 and P29 recently identified in C. crescentus. Promoters PII, P25, and P27, which are under positive regulation by transcription units III to V have strongly conserved sequence elements at -13 and -24 with the consensus sequence (C/T)TGGC(C/G)C-N5-TTGC. The -13, -24 sequence elements are not well conserved in promoter PI, but the promoter does contain a copy of the -13 and -24 consensus sequence 23 base-pairs upstream (PI). The C. crescentus fla gene promoters are not homologous to the canonical Escherichia coli -10, -35 promoter sequence, but they are very similar to the -12, -24 nif gene promoter sequence reported for Klebsiella pneumoniae and Rhizobium sp. The four positively regulated fla gene promoters examined here also share a third conserved element designated II-1, with the consensus sequence C-C-CGGC--AAA--GC-G, located at approximately -100. We speculate that the conserved sequence elements mapping at -13, -24 and -100 are cis-acting regulatory elements required for the transcription and periodic regulation of these fla genes in the C. crescentus cell cycle.

Promoter mapping and cell cycle regulation of flagellin gene transcription in Caulobacter crescentus.

Caulobacter crescentus contains a 25- and a 27-kDa flagellin, which are assembled into the flagellar filament, and a 29-kDa flagellin, which is related in sequence but is of unknown function. We have used DNA sequence analysis and nuclease S1 assays to map the in vivo transcription start sites of the three flagellin genes and to study their regulation. These experiments lead to several conclusions. First, copies of the 29-, 25-, and 27-kDa flagellin genes are organized in a tandem array in the flaEY gene cluster of C. crescentus. Second, flagellin genes are under transcriptional control and each gene is expressed with a characteristic periodicity in the cell cycle. Third, flagellin gene promoters contain conserved nucleotide sequence elements at -13, -24, and -100 that are homologous to the fla genes in the hook gene cluster. The -13 and -24 sequences conform to a fla gene promoter consensus sequence (C/TTGGCC/GC-N5-TTGC) that is similar in sequence to the -12, -24 consensus sequence of the Klebsiella pneumonia nif gene promoters. Fourth, the sequence element at approximately -100 in the 25- and the 27-kDa flagellin genes is homologous to a 19-base-pair sequence [designated previously as II-1; see Chen, L.-S., Mullin, D. M. & Newton, A. (1986) Proc. Natl. Acad. Sci. USA 83, 2860-2864]at -101 in the promoter of transcription unit II of the hook gene cluster; the two flagellin genes, like the fla genes examined in the hook gene cluster that contain the -100 element, are under positive control by transcription unit III of the hook gene cluster. This result supports a model in which the timing of fla gene transcription in the C. crescentus cell cycle is determined in part by a cascade of trans-acting regulatory gene products.

Enzyme immunoassay: binding of Salmonella antigens to activated microtiter plates.

A heat extract prepared from radiolabeled Salmonella cells was used to determine if covalent binding to activated surface of polystyrene plates would improve antigen retention thus contributing to increase sensitivity in an enzyme immunoassay for Salmonella antigen. The effect of treatment with ethylchloroformate on the retention of antigens passively absorbed to polyvinylchloride and polystyrene plates was also investigated. Chemically modified plates retained more radiolabeled antigens after washing than did untreated plates in which the antigens had been physically adsorbed. However, improvement of assay sensitivity depended on the type of plate used for covalent binding of antigen. N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), was found to be potentially useful for mediation of covalent binding of antigens to activated plates.

Regulation of protoxin synthesis in Bacillus thuringiensis.

A derivative of Bacillus thuringiensis subsp. kurstaki (HD-1) formed parasporal inclusions at 25 degrees C, but not at 32 degrees C. This strain differed from the parent only in the loss of a 110-megadalton (Md) plasmid, but plasmid and chromosomal copies of protoxin genes were present in both strains. On the basis of temperature shift experiments, the sensitive period appeared to be during midexponential growth, long before the time of protoxin synthesis at 3 to 4 h after the end of exponential growth. The conditional phenotype could be transferred by cell mating to naturally acrystalliferous Bacillus cereus. In all such cases, a 29-Md protoxin -encoding plasmid was transferred, but this plasmid alone was barely sufficient for protoxin synthesis. Protoxin production increased to detectable levels, but well below those of the parental donor strain, by simultaneous transfer of a 44-Md protoxin -encoding plasmid. Transfer of a 5-Md plasmid with the two larger protoxin -coding plasmids resulted in a protoxin synthesis level approaching that of the donor strain. A role for some of the cryptic plasmids of kurstaki in parasporal body formation was implied. In contrast, a closely related B. thuringiensis strain, HD73 , produced crystals at both 25 and 32 degrees C even when the capacity was transferred on a 50-Md plasmid to B. cereus. The amount of protoxin produced in these B. cereus transcipients , however, was somewhat less than that produced in the parental strain HD73 , implying that catabolic differences, gene dosage, or the presence of a chromosomal gene (or a combination of these) may be necessary for maximum production. A regulatory component of the 29-Md plasmid appeared to be trans-acting and dominant since B. cereus transcipients containing the 29-Md plasmid from kurstaki and the 50-Md plasmid from HD73 produced more protoxin at 25 degrees C than at 30 degrees C. Similar results were obtained when protoxin synthetic capacity was transferred from B. thuringiensis subsp. israelensis to the conditional B. thuringiensis subsp. kurstaki strain.

Cloning and localization of the lepidopteran protoxin gene of Bacillus thuringiensis subsp. kurstaki.

Bacillus thuringiensis subsp. kurstaki produces a proteinaceous crystalline inclusion that is toxic for lepidopteran larvae. There are several size classes of plasmids in this organism and the presence of one or more has been correlated with production of this protein, defined as a protoxin. DNA fragments of B. thuringiensis subsp. kurstaki, obtained by EcoRI digestion, were cloned into the vector Charon 4A. Recombinant phage were screened immunologically for the production of protoxin. Cells infected with one phage, C4K6c, produced antigen that was the same size as the protoxin and was toxic to Manduca sexta larvae. A 4.6-kilobase-pair (kbp) EcoRI fragment from C4K6c was subcloned into pBR328 and in both orientations in pHV33. Both Escherichia coli and Bacillus subtilis containing these recombinant plasmids produced antigen that crossreacted with antibody directed against the protoxin. The various sized plasmids of B. thuringiensis were purified and only an EcoRI fragment from the 45-kbp plasmid hybridized to phage C4K6c. One of the pHV33 subclones, pSM36, hybridized to the same size EcoRI/HindIII restriction fragments from plasmid or chromosomal DNA. The cloned EcoRI fragment contained a 0.9-kbp Pvu II fragment that was also present in chromosomal but not in plasmid digests. The original clone was therefore of chromosomal origin, although very similar or identical protoxin genes were present in both the 45-kbp plasmid and the chromosome. Several acrystalliferous nontoxic mutants have been isolated that lacked the 45-kbp plasmid and in some cases all plasmids. All of the mutants contained the chromosomal gene but did not produce protoxin antigen.

Enzyme immunoassay for detection of Salmonellae in foods.

An enzyme immunoassay was developed to detect Salmonella in foods. Indirect test protocols were developed for use with microtitration plates or Gilford microcuvettes. Samples from enrichment cultures were mixed with H-specific immunoglobulin G and allowed to react; unbound antibody was removed by three 5-min centrifugation washes; goat anti-rabbit antibody conjugated to alkaline phosphatase was added and allowed to react; and unbound conjugate was removed by centrifugation washing as before. Salmonella-positive samples were indicated by the production of a chromogenic reaction product after the addition of alkaline phosphatase substrate. The color could be read visually or quantified by absorbance. Ninety-eight food samples were examined to compare the enzyme immunoassay with enrichment serology, immunofluorescence, and the Food and Drug Administration pure culture technique. The enzyme immunoassay was sensitive and specific, and it possessed advantages over methods currently in use. Furthermore, when the enzyme immunoassay was used to screen preenrichment media, the results indicated that it might be decidedly more sensitive than the conventional pure culture technique.