Cell differentiation in the embryonic midgut of the tobacco hornworm, Manduca sexta

While the larval midgut of Manduca sexta has been intensively studied as a model for ion transport, the developmental origins of this organ are poorly understood. In our study we have used light and electron microscopy to investigate the process of midgut epithelial cell differentiation in the embryo. Our studies were confined to the period between 56 and 95 hr of embryonic development (hatching is at 101 hr at 25 degrees C), since preliminary studies indicated that all morphologically visible differentiation of the midgut epithelium occurs during this time. At 56 hr the midgut epithelium is organized into a ragged pseudostratified epithelium. Over the next 10 hr, the embryo molts and the midgut epithelium takes on a distinctive character in which the future goblet and columnar cells can be identified. With further differentiation, closed vesicles in the goblet cells expand and subsequently communicate to the outside by way of a valve. The columnar cells form numerous microvilli on their apical surfaces that extend over the goblet cells. Both cell types form basal folds from a series of plasmalemmal invaginations. Differentiation occurs concurrent with a six-fold elongation of these cells.     

A coupled enzyme assay for measurement of sialidase activity.

A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.     

Blot analyses of glycoconjugates: enzyme-hydrazide--a novel reagent for the detection of aldehydes

A procedure for the general staining of glycoproteins and other glycoconjugates on protein blots has been developed. Aldehydes are formed on the sugars of glycoconjugates by periodate oxidation which then react with hydrazide groups of enzyme-hydrazides, a novel reagent designed for aldehyde detection. The bound enzyme-hydrazide is demonstrated histochemically. The new assay is advantageous over periodic-acid Schiff staining of gels as its reagents and signals are stable and the process is simple and expedient, and provides greater sensitivity.     

Capsule of Escherichia coli K29: ultrastructural preservation and immunoelectron microscopy.

The polysaccharide capsule of Escherichia coli K29 fully surrounds the microorganism and thus occupies an extracellular space ca. 20 times larger in volume than that of the decapsulated cell. Since more than 95% of the capsule consists of water, dehydration for electron microscopy causes the material to collapse. We describe here a method for embedding the capsule in an uncollapsed form. Dehydration of gelatin-enrobed, glutaraldehyde-fixed cells was performed in dimethyl formamide. The cells were embedded in Lowicryl K4M with the "progressive lowering of temperature" method and UV polymerization. In ultrathin sections, the capsule can be identified by its low electron contrast. It occupies a layer 3/4 micron thick thick and shows fibrous strands embedded in a fine granular matrix. The thin strands extend radially from the cell wall and transverse the capsule. The entire capsule domain, as well as the outer membrane, binds specific anticapsular antibody, whereas the periplasmic space and most of the inner membrane lack capsule-specific immunostain.     

Adherence of Clostridium thermocellum to cellulose

The adherence of Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, to its insoluble substrate (cellulose) was studied. The adherence phenomenon was determined to be selective for cellulose. The observed adherence was not significantly affected by various parameters, including salts, pH, temperature, detergents, or soluble sugars. A spontaneous adherence-defective mutant strain (AD2) was isolated from the wild-type strain YS. Antibodies were prepared against the bacterial cell surface and rendered specific to the cellulose-binding factor (CBF) by adsorption to mutant AD2 cells. By using these CBF-specific antibodies, crossed immunoelectrophoresis of cell extracts revealed a single discrete precipitation peak in the parent strain which was absent in the mutant. This difference was accompanied by an alteration in the polypeptide profile whereby sonicates of strain YS contained a 210,000-molecular-weight band which was missing in strain AD2. The CBF antigen could be removed from cell extracts by adsorption to cellulose. A combined gel-overlay--immunoelectrophoretic technique demonstrated that the cellulose-binding properties of the CBF were accompanied by carboxymethylcellulase activity. During the exponential phase of growth, a large part of the CBF antigen and related carboxymethylcellulase activity was associated with the cells of wild-type strain YS. However, the amounts decreased in stationary-phase cells. Cellobiose-grown mutant AD2 cells lacked the cell-associated CBF, but the latter was detected in the extracellular fluid. Increased levels of CBF were observed when cells were grown on cellulose. In addition, mutant AD2 regained cell-associated CBF together with the property of cellulose adherence. The presence of the CBF antigen and related adherence characteristics appeared to be a phenomenon common to other naturally occurring strains of this species.     

The electrophoretic mobility of gram-negative and gram-positive bacteria: an electrokinetic analysis.

The electrophoretic mobility (EPM) of a variety of Gram-negative and Gram-positive bacteria was measured with a Penkem S3000 analyser. Under standard growth conditions and neutral pH all cells displayed a negative EPM. The polysaccharide capsules of Escherichia coli strains K1, K5, K29 and K30 generated the highest EPM; to a lesser and varying degree O-antigens with charged groups and core lipopolysaccharides also contribute to the net EPM. Very little negative EPM was measured in suspension cultures of the gliding bacterium Cytophaga U67. No difference in the EPM was observed between rapidly growing and stationary-phase E. coli B. De-energization of the cell membranes by carbonyl cyanide m-chlorophenylhydrazone (CCCP) did not affect the EPM of wild-type and deep rough mutants of E. coli; and the EPM of Cytophaga U67 and Acholeplasma laidlawii remained unaltered by CCCP when measured in their respective growth media. Extrusion of filamentous bacteriophage f1 from cells of its host, E. coli A95, caused a shift to a higher negative EPM. We also measured a variety of Gram-positive strains, all of which displayed different EPMs. When membrane fractions of E. coli were adsorbed to latex spheres, characteristic differences between the EPM of beads coated with either inner or outer membrane were observed. The results suggest that the rapid EPM analysis is a useful tool to study the net electric charge of microorganisms and to examine changes of surface properties during interaction of cells with viruses, proteins (antibody) and charged antibiotics.     

Genetic variation,breeding system evolution,and conservation of the narrow sand dune endemic Stellaria arenicola and the widespread S. longipes (Caryophyllaceae)

Genetic variation was examined by electrophoresis in 14 populations of Stellaria arenicola, an endemic of the Athabasca sand dunes in northern Saskatchewan, Canada, and seven populations of S. longipes, its progenitor. Three of the 5. longipes populations were sympatric with the endemic. Populations of the endemic were found to have fewer alleles per polymorphic locus (2.21 vs. 2.37), fewer polymorphic loci (29.9 vs. 33.8), and lower genetic diversity (0.087 vs. 0.107) than populations of the progenitor. Genetic identities for all pairs of populations were high (0.932 to 1.000). The endemic had one novel allele and shared ten alleles with progenitor populations from the sand dunes that were not found in other populations of S. longipes. Populations of both species were found to partition most of their genetic variation within populations. An investigation of the multilocus outcrossing rates revealed that S. arenicola had higher rates of selling and biparental inbreeding than S. longipes. This study suggests that partial genetic isolation through a shift in the breeding system, in addition to previously reported strong directional selection, has been important in the sympatric evolution of the endemic S. arenicola. The close genetic relationship between populations of S. arenicola and S. longipes found on the Athabasca sand dunes supports the suggestion that the endemic evolved while sympatric to the gene pool of the progenitor species that is found presently in the region.     

Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH, NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8-9 U mg^-1 protein (µmoles mg^-1 min^-1) for NADPH and 3-4 U mg^-1 protein for NADH formation with reduced methylviologen (MV⁺⁺) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg^-1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.

The K_m-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.

Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg^-1 protein of an NADP⁺-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV⁺⁺ regeneration. The regeneration of MV⁺⁺ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg^-1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)⁺ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.

Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (D_s-(+)-isocitrate), L_s-(-)-isocitrate and 6-phosphogluconate.