Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants

To test the hypothesis that human milk fucosyloligosaccharides are part of an innate immune system, we addressed whether their expression (1) depends on maternal genotype and (2) protects breastfed infants from pathogens. Thus the relationship between maternal Lewis blood group type and milk oligosaccharide expression and between variable oligosaccharide expression and risk of diarrhea in their infants was studied in a cohort of 93 Mexican breastfeeding mother-infant pairs. Milk of the 67 Le(a-b+) mothers contained more LNF-II (Le(a)) and 3-FL (Le(x)) (oligosaccharides whose fucose is exclusively alpha 1,3- or alpha 1,4-linked) than milk from the 24 Le(a-b-) mothers; milk from Le(a-b-) mothers contained more LNF-I (H-1) and 2'-FL (H-2), whose fucose is exclusively alpha 1,2-linked. The pattern of oligosaccharides varied among milk samples; in each milk sample, the pattern was summarized as a ratio of 2-linked to non-2-linked fucosyloligosaccharides. Milks with the highest ratios were produced primarily by Le(a-b-) mothers; those with the lowest ratios were produced exclusively by Le(a-b+) mothers (p<0.001). Thus maternal genetic polymorphisms expressed as Lewis blood group types are expressed in milk as varied fucosyloligosaccharide ratios. The four infants who developed diarrhea associated with stable toxin of Escherichia coli were consuming milk with lower ratios (4.4 +/- 0.8 [SE]) than the remaining infants (8.5 +/- 0.8; p<0.001). Furthermore, the 27 infants who developed moderate to severe diarrhea of any cause were consuming milk with lower ratios (6.1 +/- 0.9) than the 26 who remained healthy (10.5 +/- 1.9; p = 0.042). Thus, milk with higher 2-linked to non-2-linked fucosyloligosaccharide ratios affords greater protection against infant diarrhea. We conclude that specific oligosaccharides constitute a major element of an innate immune system of human milk.     

Efficient Transformation of <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> by Electroporation and Conjugation with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.     

Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose

An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.     

Nuclear ploidy is contingent on the microtubular cycle responsible for plant cytokinesis

Summary. Division of the plant cell relies on the preprophase band of microtubules (PPB)-phragmoplast system. Cells of onion (Allium cepa L.) root meristems were rendered binucleate by preventing the consolidation of cell plate formation in telophase with 5mM caffeine. These binucleates developed either a single PPB around one of their two nuclei or two PPBs, one per nucleus, in the prophase of the ensuing mitosis. Prophase cells developing one single PPB were shorter in length (42.3±4.1µm) than those developing 2 PPBs (49.8±4.1µm), and interphase duration was inversely related to cell length. Cells whose length was less than or equal to 42µm, i.e., which had not even reached the mean size of the small binucleates in prophase, were followed throughout mitosis. In metaphase, they always assembled two mitotic spindles (one per nucleus). However, the cells that had assembled a single PPB also developed a single phragmoplast in telophase, leading to polyploidization. As these meristematic cells were not wide enough to accommodate the midzones of both mitotic spindles in any single plane transversal to the cell elongation axis, the spindles tilted until their midzones formed a continuum where the single common phragmoplast assembled. Its position was thereby uncoupled from that of the preceding PPB. Subsequently, the chromosomes from two different half-spindles were included, by a common nuclear envelope, in a single tetraploid nucleus. Finally, the cytokinetic plate segregated the two tetraploid nuclei formed at each side of the phragmoplast into two independent sister cells.Correspondence and reprints: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.     

Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.     

Interleukin-1 beta (IL-1beta) induces tumor necrosis factor alpha (TNF-alpha) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation

Interleukin-1 alpha (IL-1alpha) and beta (IL-1beta) are well known factors that stimulate hematopoiesis, nevertheless there are reports that show that they can also inhibit this activity. While both IL-1alpha and IL-1beta induce the expression of hematopoietic cytokines, such as growth factors and their receptors on myeloid cells, helping thus to regulate hematopoiesis, it is not known if their inhibitory activity is also mediated through the induction of other specific cytokines. In this work we show that recombinant human IL-1beta (rhIL-1beta) inhibits the proliferation of a mouse IL-3-dependent myeloid multipotent cell line (32D cl3), without inducing its differentiation. We show that rhIL-1beta induces in 32D cl3 cells the expression of the tumor necrosis factor alpha (TNF-alpha) gene, a well known growth inhibitor, and that the rhIL-1beta growth inhibition property on 32D cl3 cells is partially due to this secreted TNF-alpha, hinting thus that the inhibition of hematopoiesis by IL-1 is mediated through other induced cytokines.     

Purification and Characterization of an Extracellular Non-Aspartyl Acid Protease (pumAe) from <Emphasis Type="Italic">Ustilago maydis</Emphasis>

The proteinase pumAe was purified to homogeneity from haploid U. maydis FB1 growing on acid mineral medium. The purification procedure consisted of ammonium sulfate fractionation and gel filtration chromatography, resulting in a 7.7% recovery and a 15.1-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 72 kDa and 74 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 4.0 and at 45°C toward hemoglobin, and the pI was determined to be 5.5. The effects of six protease inhibitors on pumAe were tested, and no inhibitory effect was observed. The pure enzyme degraded gelatin and albumin, but casein and collagen were not degraded. The K_m value was 3.5 M, and the V_max value was 11430 mol h^–1 mg^–1 for Suc-R-P-F-H-L-L-V-Y-MCA.     

Novel excitatory Conus peptides define a new conotoxin superfamily

A new class of Conus peptides, the I-superfamily of conotoxins, has been characterized using biochemical, electrophysiological and molecular genetic methods. Peptides in this superfamily have a novel pattern of eight Cys residues. Five peptides that elicited excitatory symptomatology, r11a, r11b, r11c, r11d and r11e, were purified from Conus radiatus venom; four were tested on amphibian peripheral axons and shown to elicit repetitive action potentials, consistent with being members of the 'lightning-strike cabal' of toxins that effect instant immobilization of fish prey. A parallel analysis of Conus cDNA clones revealed a new class of conotoxin genes that was particularly enriched (with 18 identified paralogues) in a Conus radiatus venom duct library; several C. radiatus clones encoded the excitatory peptides directly characterized from venom. The remarkable diversity of related I-superfamily peptides within a single Conus species is unprecedented. When combined with the excitatory effects observed on peripheral circuitry, this unexpected diversity suggests a corresponding molecular complexity of the targeted signaling components in peripheral axons; the I-conotoxin superfamily should provide a rich lode of pharmacological tools for dissecting and understanding these. Thus, the I-superfamily conotoxins promise to provide a significant new technology platform for dissecting the molecular components of axons.