Solving the problem of how to eat something as big as yourself: Diverse bacterial strategies for degrading polysaccharides

Polysaccharide digestion by bacteria is an important activity in many ecosystems, and a number of bacterial genera can perform this function. Although many papers have been published about the properties of isolated polysaccharide-degrading enzymes, relatively little is known about how intact bacteria degrade polysaccharides. This review summarizes recent findings suggesting that there are at least three different strategies. the most familiar one is the excretion of extracellular polysaccharidases, which diffuse to and degrade nearby polysaccharides. An example of this type of strategy is provided by the plant pathogen,Erwinia spp. A second strategy is to have the enzyme exposed to the extracellular medium but attached to the surface of the cell. Examples of this strategy are provided by the pullulanase system ofKlebsiella oxytoca and the cellulosomes ofClostridium thermocellum. A strategy that could be seen as a combination of the extracellular enzyme strategy and the surface organelle strategy is provided byVibrio harveyi, which attaches to its substrate, chitin, via proteins that appear to be specialized for attachment and produces extracellular enzymes that attack the chitin. A third strategy is to import the polysaccharide, as appears to be done byBacteroides spp. In this instance, the polysaccharide is bound to an outer membrane receptor, then passes into the periplasm where the degradative enzymes are located. The ecological advantages and disadvantages of these systems are discussed, and areas where further research is needed are defined.     

Synthesis and bioassay of isoprenoid 3-alkylthio-1,1,1-trifluoro-2-propanones: Potent,selective inhibitors of juvenile hormone esterase

Four 3-alkylthio-1,1,1-trifluoro-2-propanones with juvenile hormone-like side chains were prepared from citronellol and homogeraniol. These substrates were designed as possible transition-state analogs for the juvenile hormone (JH)-specific esterases present in insects. These four isoprenoid trifluoromethyl ketones were assayed in vitro with JH esterase and general esterases from larvae of the cabbage looper, Trichoplusia ni (Lepidoptera, Noctuidae), and with eel acetylcholinesterase and bovine chymotrypsin. JH esterase inhibition I₅₀ values were in the nanomolar range for all four compounds, while the other esterases had I₅₀'S which were 10³ to 10⁵ higher. The high selectivity of these inhibitors is believed to be due to their similarity in size and functionality to natural JH III. Treatment of T. ni larvae in vivo with solutions of the most active analog, 3-[(E)-4,8-dimethyl-3,7-nonadienylthio]-1,1,1-trifluoro-2-propanone (DNTFP) causes a dose-dependent delay in pupation and a concurrent selective inhibition of JH esterase. These data support the hypothesis that the reduction in in vivo JH titer in larval T. ni is due, in part, to hydrolysis of the hormone by selective esterases. DNTFP appears to be competing with JH for the active site of JH esterase.     

Characteristics of a human epidermal squamous carcinoma cell line at different extracellular calcium concentratrions

A continuous line derived from a human skin squamous cell carcinoma has been grown in media of high, normal and low Ca²⁺ concentrations. The growth rate was unaffected by the Ca²⁺ levels even though morphological changes were observed. Desmosomes were absent at low Ca²⁺ and areas of cell piling were observed at high Ca²⁺. Cell protein staining patterns on polyacrylamide gels were identical for cells grown at the three Ca²⁺ levels. The variations were minor for the glycoproteins reacted with ¹²⁵I-conA. Lactoper-oxidase iodination revealed changes in cell surface proteins, most markedly in the emergence of new proteins at high Ca²⁺.     

Copper and cobalt in African species ofAeolanthus Mart. (Plectranthinae,Labiatae)

Summary Hyperaccumulators (here defined as species containing at least 500 μg/g dryweight of either copper or cobalt in leaves), are of interest for the fields of mineral exploration and phytochemistry. Reported hyperaccumulation of copper inAeolanthus biformifolius and the presence of two other species on copper/cobalt mineralization in Shaba (Za?re) led to a survey of these elements in 49 species of the African genusAeolanthus Mart.A. biformifolius appears to be also a hyperaccumulator of Co (2520 μg/g in leaves, 4300 μg/g in corms). Cobalt levels for most species ofAeolanthus were considerably above typical values for phanerogams and indicate the favorable potential of the genus for further study.     

The apical lamina of the sea urchin embryo: Major glycoproteins associated with the hyaline layer

The hyaline layer (HL) surrounding the sea urchin blastula appears to dissolve in 1 M glycine. However, after this treatment, there persists over the surfaces of the blastomeres a layer of material, referred to here as the apical lamina (AL), that sloughs off as an adhesive convoluted bag upon gradual dissociation of the embryo. Isolated hyaline layers, referred to as HL-AL complexes, were analyzed by urea-SDS-polyacrylamide gel electrophoresis. A major protein of the HL-AL complex, hyalin, bands or precipitates in the stacking gel. Two other major proteins, both strongly PAS positive, migrate with apparent molecular weights of 175K and 145K daltons. As with intact embryos, the glycine wash removes the hyalin protein from the isolated HL-AL complex, leaving the undissolved AL which consists primarily of the 175K- and 145K-dalton proteins. The embryo's own perivitelline-localized cortical granule peroxidase heavily radioiodinates the proteins of the HL-AL complex, further verifying their apical, extracellular location. Unlike hyalin, the AL proteins do not precipitate with calcium ions. Compared to the entire HL-AL complex, the AL contains a greater percentage of carbohydrate. No sialic acid is associated with the HL-AL complex, but the AL contains some sulfate. In contrast to a published report based on ultrastructural staining, no biochemical evidence was found in this study for the presence of collagen or significant glycosaminoglycan within the HL-AL complex. No developmental differences were observed in AL proteins from 1-hr-old embryos compared to those from blastulae. However, there is evidence suggesting heterogeneity and developmental differences in hyalin. The possible organization of hyalin and the AL proteins into separate layers surrounding the embryo is discussed. The influence of the AL proteins in morphogenesis and cell adhesion is considered, and hypothetical roles attributed to the HL and hyalin are critically questioned.     

Enzymatic synthesis and isolation of thymidine diphosphate-6-deoxy-D-xylo-4-hexulose and thymidine diphosphate-L-rhamnose. Production using cloned gene products and separation by HPLC.

A two-step enzymatic synthesis of dTDP-L-rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rfb gene cluster of Salmonella enterica LT2. The intermediate dTDP-6-deoxy-D-xylo-4-hexulose was isolated after a 1-h reaction, using only dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89%). In a two-step reaction using dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase in the first step, and with NADPH, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase in the second hour of incubation, the dTDP-D-glucose was fully converted to dTDP-L-rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP-L-rhamnose, after protein precipitation, anion-exchange chromatography and desalting by gel chromatography, was 62%, corresponding to more than 150 mg, starting from 250 mg of dTDP-D-glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months. Both dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP-6-deoxy-D-xylo-4-hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier. Furthermore, an HPLC technique is presented for determining the net activity of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase, based on separation of dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.     

Insulin-induced lipoatrophy: evidence for an immune pathogenesis.

Skin biopsy samples from 14 diabetic patients with lipoatrophy at injection sites and from five insulin-treated diabetic patients without such lipoatrophy (controls) were examined by immunofluorescence for the deposition of immunological components. Also sera from 13 of the patients with lipoatrophy and from all of the controls were assayed for insulin-binding capacity. Biopsy samples from the edge of lipoatrophic areas (eight cases) invariably showed abnormal deposition of immunological components in dermal vessel walls, whereas no such deposition was seen in the control samples. Mean serum insulin-binding capacity was 33.1 microgram/l in the patients with lipoatrophy compared with only 4.6 microgram/l in the controls. These findings suggest that insulin-induced lipoatrophy results from the local formation of immune complexes, complement fixation, and release of inflammatory mediators from the cellular infiltrate.