Characterization of the C-terminal region of molecular forms of human milk bile salt-stimulated lipase.

UNLABELLED: Bile salt-stimulated lipase (BSSL) in human milk exists in multiple molecular forms and it has been shown that approximately one-third of lactating mothers secrete two forms. AIM: to determine the structural features of BSSL that may give rise to this heterogeneity. METHODS: Oligosaccharides present in the proline-rich region in the C-terminus of BSSL were investigated using deglycosylating enzymes and lectin affinity probing to determine the origin of the multiple molecular forms. RESULTS: It was found that the variability in the molecular mass of BSSL is due predominantly to glycosylation. The molecular forms contain similar sugar chains; all forms possess the core disaccharide Galbeta1-3GalNAc and beta-D-galactose, fucose linked at alpha1-6 and sialic acid linkage alpha2-3 to galactose. CONCLUSION: The molecular mass difference in the BSSL molecular forms cannot be attributed to the type of carbohydrate moiety in the sugar chains of the N- and O-linked sites suggesting that the differences arise from the extent or quantity of glycosylation. The oligosaccharides in the C-terminal region contain Lewis x and b and, less prominently, Lewis a antigenic structures. Owing to the presence of these blood-group-related antigenic determinants, the C-terminal region of BSSL may have an adhesive function in cell-cell interactions.

The extent of unlicensed and off-label drug use in the paediatric ward of a district general hospital in Northern Ireland.

Children receive many drugs which are either unlicensed or used outside the licensing terms (off-label prescribing). Children are thereby at greater risk of side-effects. We sought to determine non-licensed drug usage in a non-specialised paediatric unit because previous studies had examined only specialised centres. Drug charts were examined prospectively over two months. Items were compared with product licenses and determined as licensed, unlicensed or off-label usage. Seventy four charts (237 prescriptions) were examined in which 183 (77.2%) drug courses were licensed. The commonest drugs were antipyretics and antibiotics. In 32 children, 8 items (3.4%) were unlicensed and 46 items (19.4%) were off-label prescriptions. The unlicensed prescriptions were mostly anti-emetics and pro-motility drugs. The three commonest off-label prescriptions were bronchodilators, antibiotics and laxatives. Non-licensed drug usage is common in non-specialised wards and therefore probably all levels of paediatric healthcare provision. Governments must re-examine paediatric drug licensing.

Mutational analysis of beta-glucanase genes from the plant-pathogenic fungus Cochliobolus carbonum.

Two new beta-glucanase-encoding genes, EXG2 and MLG2, were isolated from the plant-pathogenic fungus Cochliobolus carbonum using polymerase chain reaction based on amino acid sequences from the purified proteins. EXG2 encodes a 46.6-kDa exo-beta1,3-glucanase and is located on the same 3.5-Mb chromosome that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8-kDa mixed-linked (beta1,3-beta1,4) glucanase with low activity against beta1,4-glucan and no activity against beta1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multiple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg1/mlg2) were also constructed by sequential disruption and by crossing. Total mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and exg1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total beta1,3-glucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/exg2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a modest decrease in growth on beta1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.

Psychiatric assessment wards for older adults: a qualitative evaluation of two ward models.

This study was requested to investigate the relative strengths of two different ward arrangements. Both wards were psychiatric assessment wards for people over the age of 65 and both were mixed sex wards. The major focus was to examine if the separation of cognitively impaired (CI) and functional clients on an elderly assessment ward had benefits in terms of client and staff satisfaction. The study involved 192 hours of observation, following four clients on each ward for 24-hours. Results indicate that the split ward and the mixed ward differ qualitatively and that in terms of user and staff satisfaction the split model is preferable. Implications for service development and future research are also discussed.

The Cochliobolus carbonum SNF1 gene is required for cell wall-degrading enzyme expression and virulence on maize.

The production of cell wall-degrading enzymes (wall depolymerases) by plant pathogenic fungi is under catabolite (glucose) repression. In Saccharomyces cerevisiae, the SNF1 gene is required for expression of catabolite-repressed genes when glucose is limiting. An ortholog of SNF1, ccSNF1, was isolated from the maize pathogen Cochliobolus carbonum, and ccsnf1 mutants of HC toxin-producing (Tox2(+)) and HC toxin-nonproducing (Tox2(-)) strains were created by targeted gene replacement. Growth in vitro of the ccsnf1 mutants was reduced by 50 to 95% on complex carbon sources such as xylan, pectin, or purified maize cell walls. Growth on simple sugars was affected, depending on the sugar. Whereas growth on glucose, fructose, or sucrose was normal, growth on galactose, galacturonic acid, maltose, or xylose was somewhat reduced, and growth on arabinose was strongly reduced. Production of HC toxin was normal in the Tox2(+) ccsnf1 mutant, as were conidiation, conidial morphology, conidial germination, and in vitro appressorium formation. Activities of secreted beta-1,3-glucanase, pectinase, and xylanase in culture filtrates of the Tox2(+) ccsnf1 mutant were reduced by 53, 24, and 65%, respectively. mRNA expression was downregulated under conditions that induced the following genes encoding secreted wall-degrading enzymes: XYL1, XYL2, XYL3, XYL4, XYP1, ARF1, MLG1, EXG1, PGN1, and PGX1. The Tox2(+) ccsnf1 mutant was much less virulent on susceptible maize, forming fewer spreading lesions; however, the morphology of the lesions was unchanged. The Tox2(-) ccsnf1 mutant also formed fewer nonspreading lesions, which also retained their normal morphology. The results indicate that ccSNF1 is required for biochemical processes important in pathogenesis by C. carbonum and suggest that penetration is the single most important step at which ccSNF1 is required. The specific biochemical processes controlled by ccSNF1 probably include, but are not necessarily restricted to, the ability to degrade polymers of the plant cell wall and to take up and metabolize the sugars produced.

Herbicide safener-binding protein of maize. Purification, cloning, and expression of an encoding cDNA.

Dichloroacetamide safeners protect maize (Zea mays L.) against injury from chloroacetanilide and thiocarbamate herbicides. Etiolated maize seedlings have a high-affinity cytosolic-binding site for the safener [3H](R,S)-3-dichloroacetyl-2,2,5-trimethyl-1, 3-oxazol-idine ([3H]Saf), and this safener-binding activity (SafBA) is competitively inhibited by the herbicides. The safener-binding protein (SafBP), purified to homogeneity, has a relative molecular weight of 39,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 5.5. Antiserum raised against purified SafBP specifically recognizes a 39-kD protein in etiolated maize and sorghum (Sorghum bicolor L.), which have SafBA, but not in etiolated wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), or Arabidopsis, which lack SafBA. SafBP is most abundant in the coleoptile and scarcest in the leaves, consistent with the distribution of SafBA. SBP1, a cDNA encoding SafBP, was cloned using polymerase chain reaction primers based on purified proteolytic peptides. Extracts of Escherichia coli cells expressing SBP1 have strong [3H]Saf binding, which, like binding to the native maize protein, is competitively inhibited by the safener dichlormid and the herbicides S-ethyl dipropylthiocarbamate, alachlor, and metolachlor. SBP1 is predicted to encode a phenolic O-methyltransferase, but SafBP does not O-methylate catechol or caffeic acid. The acquisition of its encoding gene opens experimental approaches for the evaluation of the role of SafBP in response to the relevant safeners and herbicides.

Targeted mutants of Cochliobolus carbonum lacking the two major extracellular polygalacturonases.

The filamentous fungus Cochliobolus carbonum produces endo-alpha 1,4-polygalacturonase (endoPG), exo-alpha 1,4-polygalacturonase (exoPG), and pectin methylesterase when grown in culture on pectin. Residual activity in a pgn1 mutant (lacking endoPG) was due to exoPG activity, and the responsible protein has now been purified. After chemical deglycosylation, the molecular mass of the purified protein decreased from greater than 60 to 45 kDa. The gene that encodes exoPG, PGX1, was isolated with PCR primers based on peptide sequences from the protein. The product of PGX1, Pgx1p, has a predicted molecular mass of 48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an exoPG from the saprophytic fungus Aspergillus tubingensis. Strains of C. carbonum mutated in PGX1 were constructed by targeted gene disruption and by gene replacement. Growth of pgx1 mutant strains on pectin was reduced by ca. 20%, and they were still pathogenic on maize. A double pgn1/pgx1 mutant strain was constructed by crossing. The double mutant grew as well as the pgx1 single mutant on pectin and was still pathogenic despite having less than 1% of total wild-type PG activity. Double mutants retained a small amount of PG activity with the same cation-exchange retention time as Pgn1p and also pectin methylesterase and a PG activity associated with the mycelium. Continued growth of the pgn1/pgx1 mutant on pectin could be due to one or more of these residual activities.

Incidence of molecular forms of bile salt-stimulated lipase in preterm and term human milk.

Preterm and term human milk samples obtained at various times after delivery were analyzed for the presence of molecular forms of the human milk enzyme, bile salt-stimulated lipase (BSSL). Thirty-five percent of both the preterm and term milk samples contained two molecular forms of BSSL, of variable molecular mass. The remainder contained only one molecular species of either 115 kD (50%) or 120 kD (15%). The number of molecular forms present was not related to length of lactation, maternal age, gestation, or maternal blood group. The specific activity of BSSL purified from term milk was similar to that purified from preterm milk, and there was no difference in specific activity whether one or two molecular forms were present. This study demonstrates heterogeneity of both molecular mass and molecular forms. We conclude that preterm babies fed their own mother's milk are unlikely to be disadvantaged with respect to fat digestion as BSSL secreted in preterm milk appears to be very similar to that produced in term milk, although we cannot exclude other functional differences.

Characterization of random amplified polymorphic DNA (RAPD) products from Xanthomonas campestris and some comments on the use of RAPD products in phylogenetic analysis.

As part of our research to determine phylogenetic relationships of organisms within the phytobacterial species Xanthomonas campestris, we have examined the use of the random amplified polymorphic DNA (RAPD) technique. The objective of this aspect of our research was to determine if a valid cladistic character analysis could be carried out by direct comparison of RAPD products separated on ethidium bromide-stained agarose gels. RAPD products were amplified from 47 Xanthomonas campestris DNA templates using a single oligonucleotide primer. These RAPD products were compared and variation was characterized by Southern analysis of both RAPD products and genomic DNA of the 47 bacterial strains using two cloned RAPD products as probes. Analysis of the data set revealed that the RAPD products were not necessarily homologous or independent, crucial prerequisites for characters to be analyzed in a cladistic phylogenetic analysis. It has been commonly assumed that RAPD variation occurs due to insertion/deletion events or alterations in the primer binding site. Within our data set, we demonstrate absence phenotypes arising from the apparent absence of corresponding loci and also due to the preferred synthesis of alternative RAPD products from unrelated loci. These different types of variation are a reflection of different types of genotypic variation, and direct examination of RAPD products did not allow us to distinguish by which mechanism a particular absence phenotype arose. Although this may not be important for phenetic analyses, for analyses of homologous characters using a cladistic approach it is critical. We also detected unrelated, co-migrating RAPD products and multiple related RAPD products within reaction mixtures. These could both contribute to errors in estimates of similarity, important in any phylogenetic analysis. All of these characteristics of RAPD products should be taken into consideration when RAPD products are used for phylogenetic comparisons.

Effect of femoral component head size on movement of the two-component hemi-arthroplasty.

The movement of 75 two-component hemi-arthroplasties, implanted following displaced subcapital fracture of the femoral neck, were examined radiologically in equal numbers of Hastings hips (22 mm), 22-mm Bi-articular hips and 32-mm Bi-articular hips. A classification of the movement of the two-component hemi-arthroplasty was devised. The 22-mm Bi-articular hips showed predominantly intraprosthetic movement compared with the 32-mm Bi-articular hips, where movement was mainly extraprosthetic, thus confirming in vivo the Charnley low friction principle. True bipolar movement was found predominantly in the Hastings hips. In selecting a two-component hemi-arthroplasty, the prosthesis of choice is therefore one with a 22-mm rather than a 32-mm femoral head.

The cyclic peptide synthetase catalyzing HC-toxin production in the filamentous fungus Cochliobolus carbonum is encoded by a 15.7-kilobase open reading frame.

Race 1 of Cochliobolus carbonum, a fungal plant pathogen, owes its exceptional virulence on certain genotypes of maize to the production of HC-toxin, a cyclic tetrapeptide. Production of HC-toxin is controlled by a single known gene, TOX2. Race 1, but not races that do not make HC-toxin, contains two copies of a 22-kilobase (kb) region of chromosomal DNA that is required for HC-toxin biosynthesis and hence virulence. We have sequenced this 22-kb region and here show that it contains an open reading frame of 15.7 kb that encodes a multifunctional cyclic peptide synthetase of potential M(r)574,620. This gene, called HTS1, apparently contains no introns. The predicted gene product, HC-toxin synthetase (HTS), contains four amino acid-binding (adenylate-forming) domains that are highly similar to those found in other cyclic peptide synthetases and other adenylate-binding enzymes. The DNA sequence encodes tryptic peptides derived from two HC-toxin biosynthetic enzymes, HC-toxin synthetase 1 (HTS-1) and HC-toxin synthetase 2 (HTS-2), indicating that these two enzymes exist in vivo as part of a single polypeptide. Consistent with this, in some enzyme preparations antibodies against the enzyme HTS-2, which was originally purified as a protein with a subunit M(r) of 160,000, recognize a protein with an estimated subunit M(r) greater than 480,000.

Amygdala kindling potentiates seizure-stimulated immediate-early gene expression in rat cerebral cortex.

Kindling induces long-term adaptations in neuronal function that lead to a decreased threshold for induction of seizures. In the present study, the influence of amygdala kindling on levels of mRNA for the immediate-early genes (IEGs) c-fos, c-jun, and NGF1-A were examined both before and after an acute electroconvulsive seizure (ECS). Although amygdala kindling did not significantly influence resting levels of c-fos mRNA in cerebral cortex, ECS-stimulated levels of c-fos mRNA (examined 45 min after ECS) were approximately twofold greater in the cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was dependent on the time course of kindling, as ECS-stimulated levels of c-fos mRNA were not significantly increased in stage 2 kindled animals. ECS-stimulated levels of c-jun and NGF1-A mRNA were also significantly increased in cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was long-lasting because an acute ECS stimulus significantly elevated levels of c-fos and c-jun mRNA in the cerebral cortex of animals that were kindled 5 months previously. In contrast to these effects in cerebral cortex, kindling did not influence ECS-stimulated levels of c-fos mRNA in hippocampus. Finally, immunohistochemical studies revealed lamina-specific changes in the cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of amygdala kindling on the in vivo release of GABA and 5-HT in the dorsal raphe nucleus in freely moving rats.

Our laboratory has previously reported a significant subsensitivity to iontophoretically applied GABA (gamma-aminobutyric acid) in dorsal raphe neurons of amygdala-kindled rats. This subsensitivity was selective for GABA and persisted at least 3 months after the last kindled seizure. In the present series of experiments, we explored mechanisms by which kindling could result in persistent GABA sensitivity changes, using in vivo microdialysis to quantitate neurotransmitter [including GABA and 5-hydroxytryptamine (5-HT)] release in the dorsal raphe nucleus of awake, unrestrained amygdala-kindled rats. Depolarization-induced release of GABA is markedly increased in the dorsal raphe nucleus in amygdala-kindled animals. This change in depolarization-induced GABA release appeared to be graded, dependent upon the stage to which the animal is kindled. Thus GABA release is increased in animals kindled to Stage 2 and even greater in animals kindled to Stage 5 seizures. The change in GABA release is also selective, since no consistent change in the release of other putative amino acid neurotransmitters or 5-HT was observed in these same animals. We hypothesize that this increase in depolarization-induced release of GABA in the amygdala-kindled animal underlies the development of subsensitivity to GABA in dorsal raphe neurons.

Analysis of DNA sequences transcribed at high levels in Bradyrhizobium japonicum bacteroids but not necessary for symbiotic effectiveness.

Five regions of the Bradyrhizobium japonicum genome, which are transcribed at high levels in nitrogen-fixing soybean (Glycine max) nodules, were identified. None of these regions contained previously identified genes (e.g., nif, nod, and fix genes) that are known to be essential for development of functional nitrogen-fixing nodules. To assess the role of these regions in the development of the B. japonicum-soybean symbiosis, we cloned and used them to construct B. japonicum strains, in which large DNA segments (2.0-6.8 kilobases) containing the highly transcribed regions were deleted. The deletion strains were examined for symbiotic effectiveness and were found to be indistinguishable from the wild-type strain. Transcription of the cloned regions under a variety of physiological conditions and in several defined mutant B. japonicum strains was also examined. The transcriptional start sites for one pair of divergent transcripts were determined; the promoters do not contain any of the conserved sequences found in B. japonicum genes involved in symbiosis or nitrogen metabolism.

A cyclic peptide synthetase gene required for pathogenicity of the fungus Cochliobolus carbonum on maize.

Specificity in many plant-pathogen interactions is determined by single genes in pathogen and host. The single locus for host-selective pathogenicity (TOX2) in the fungus Cochliobolus carbonum governs production of a cyclic tetrapeptide named HC-toxin. We have isolated a chromosomal region, 22 kilobases (kb) long, that contains a 15.7-kb open reading frame (HTS1) encoding a multifunctional cyclic peptide synthetase. The 22-kb chromosomal region is duplicated in toxin-producing isolates of the fungus but is completely absent from the genomes of toxin-nonproducing isolates. Mutants of the fungus with disruptions in both copies of HTS1, at either of two different sites within HTS1, were engineered by DNA-mediated transformation. Disruption of both copies at either site resulted in loss of ability to produce HC-toxin and loss of host-selective pathogenicity, but the mutants displayed different biochemical phenotypes depending on the site of disruption. The results demonstrate that TOX2 encodes, at least in part, a large, multifunctional biosynthetic enzyme and that the evolution of host range in C. carbonum involved the insertion or deletion of a large piece of chromosomal DNA.

Isolation of Bradyrhizobium japonicum DNA sequences that are transcribed at high levels in bacteroids.

DNA sequences have been isolated that are expressed at high levels in bacteroids, the differentiated form of the soybean microsymbiont, Bradyrhizobium japonicum. Random-primed cDNA was synthesized using total RNA isolated from purified B. japonicum bacteroids or from cells grown in culture. When used directly to screen bacteriophage lambda libraries, these cDNA probes produced a high background hybridization signal due to sequence similarity between B. japonicum and E. coli ribosomal DNA (rDNA) operons. To reduce this background signal, the rDNA operon of B. japonicum was cloned and the rDNA plasmid DNA used in subtractive hybridization with the cDNA probes and as a competitor in hybridization solutions. This method greatly reduced the background signal in screening of genomic libraries and thus permitted the identification of twelve unique recombinant phage which contained sequences that are expressed at higher levels in B. japonicum bacteroids than in cells grown in culture.