Production of hyaluronic Acid by a streptococcal strain in batch culture

A simple method for the production and preparation of hyaluronic acid (HA) is described. Three media were tested, which all supported relatively good yields of cells, but only in one of them were appreciable amounts of HA formed. In this medium, HA was produced during the logarithmic growth phase and showed a molecular weight of 7.3 × 10⁵. HA prepared from 24-hr cultures of the same organism had a molecular weight of only 2.6 × 10⁵. The kinetics of HA production in batch culture were studied, as well as of cell production and glucose consumption.     

Inhibition of common fouling organisms by marine bacterial isolates ith special reference to the role of pigmented bacteria

Two questions of relevance to the establishment of marine biofouling communities were addressed, viz (1) what is the frequency with which bacterial strains isolated from living and inanimate surfaces in the marine environment show inhibitory activity against the settlement of common fouling organisms, and (2) is the antifouling bacterium, D2, an inhabitant of different marine waters, and how unique is this bacterium, in its mode of action against different target organisms? With respect to the first question, ninety three marine bacteria isolated from various rock surfaces from the marine environment were tested against larvae of Balanus amphitrite and spores of Ulva lactuca. Settlement assays against the diatom Amphora sp. were also performed on 10 of these strains. Nine bacterial isolates were shown to be inhibitory against larval settlement and eight of these strains were also inhibitory against algal spores. Altogether 16 strains were inhibitory against the settlement of algal spores while none of the bacterial strains inhibited diatom settlement. With respect to the second question, D2, a dark green pigmented bacterium, isolated from an adult tunicate off the Swedish west coast, has been found to be a very effective inhibitor against common fouling organisms. In order to see if this bacterium can be found in other marine waters, bacteria from living surfaces of marine plants and animals from waters around Sydney, Australia, were isolated and screened for inhibitory activity against barnacle larvae. Seventy four percent of the 23 plant isolates were shown to be inhibitory against larval settlement while only 30% of the 23 isolates from marine animals reduced settlement. Twenty two of the isolates from different seaweeds were dark pigmented and 20 of these strains inhibited settlement of barnacle larvae and algal spores. Three of the strains showed the same phenotypic expression as D2, and the results indicate that these strains may be D2 or closely related strains, suggesting that D2 may be a common inhabitant in the marine environment.     

PilC of pathogenic Neisseria is associated with the bacterial cell surface

Adherence of pathogenic Neisseria to target host cells is mediated by pili. PilC1 and PilC2 are two high-molecular-weight proteins involved in pilus assembly and cellular adherence functions of the pili. Inactivation of pilC1 or pilC2 in N. meningitidis resulted in clones that expressed the same number of pili as the parent, contained no alterations in pilE and showed no detectable differences in PilE glycosylation. However, the PilC2+ pilC1- mutant showed much reduced adherence to target cells, indicating that production of PilC1 is essential for pilus-mediated adherence. To study further the functional differences between the meningococcal pilC genes, we determined the complete nucleotide sequence of pilC1 and pilC2 of N. meningitidis. Alignment of six PilC sequences demonstrated that PilC is composed of both conserved and variable regions. By immunogold labelling of bacterial sections we showed that PilC is present in the membranes of both piliated and non-piliated bacteria. Further, we demonstrated that PilC is associated with the bacterial cell surface.     

An improved methodology for the quantification of uronic acid units in xylans and other polysaccharides

Uronic acids can be quantified either by a colorimetric determination after treatment with concentrated sulfuric acid and carbazole or by gas chromatography after methanolysis and subsequent acetylation. Both methods suffer from incomplete hydrolysis, an unavoidable degradation of the products to be analysed, and an inability to separate and quantify different types of uronic acids. In the present work, the fundamental chemistry involved in the two methods has been evaluated, and some modifications to increase their accuracy are suggested. By combining the two methods, a complete quantification of all individual types of uronic acids present in a sample can be achieved.     

Growth and survival trade-offs and outbreeding depression in rainbow trout (Oncorhynchus mykiss)

The purpose of this study was to examine, using a rainbow trout (Oncorhynchus mykiss) model system, the fitness consequences of three generations of introgression of genotypes adapted to two different environments (culture and nature). The experiments also isolated the influence of competitive interactions and risk of predation on the relative growth and survival of the wild and backcrossed lines. Line crosses representing fast-growing pure domestic (D), slow-growing pure wild (W), domestic x wild hybrids (F1), F1 x wild backcrosses (B1), and B1 x wild backcrosses (B2) were generated and reared under (1) culture conditions, (2) seminatural conditions with competition among genotypes, and (3) seminatural conditions under risk of predation. Survival of the fry in a seminatural environment with competition fit an additive model of gene action with the domestic fish having the highest survival and the wild fish the lowest, but under risk of predation outbreeding depression was suggested by low survival of the B2 lines. Evidence of a trade-off in growth and survival under risk of predation along with observations of genetically determined behavioral differences among the strains may provide some explanation for the observed differences in survival among the strains. This information is relevant to improving our evolutionary understanding of the interaction among genomes, and the influence of environment, during hybridization events. Results from this experiment indicate that alteration of phenotype likely played a prominent role in the reduced fitness experienced by progeny produced after three generations of introgression, supporting the theory that disruption of genotypes selected for adaptation to local conditions may be a primary cause of outbreeding depression in species such as salmon.     

HDAC inhibition amplifies gap junction communication in neural progenitors: potential for cell-mediated enzyme prodrug therapy

Enzyme prodrug therapy using neural progenitor cells (NPCs) as delivery vehicles has been applied in animal models of gliomas and relies on gap junction communication (GJC) between delivery and target cells. This study investigated the effects of histone deacetylase (HDAC) inhibitors on GJC for the purpose of facilitating transfer of therapeutic molecules from recombinant NPCs. We studied a novel immortalized midbrain cell line, NGC-407 of embryonic human origin having neural precursor characteristics, as a potential delivery vehicle. The expression of gap junction protein connexin 43 (Cx43) was analyzed by western blot and immunocytochemistry. While Cx43 levels were decreased in untreated differentiating NGC-407 cells, the HDAC inhibitor 4-phenylbutyrate (4-PB) increased Cx43 expression along with increased membranous deposition in both proliferating and differentiating cells. Simultaneously, Ser 279/282-phosphorylated form of Cx43 was declined in both culture conditions by 4-PB. The 4-PB effect in NGC-407 cells was verified by using HNSC.100 human neural progenitors and Trichostatin A. Improved functional GJC is of imperative importance for therapeutic strategies involving intercellular transport of low molecular-weight compounds. We show here an enhancement by 4-PB, of the functional GJC among NGC-407 cells, as well as between NGC-407 and human glioma cells, as indicated by increased fluorescent dye transfer.     

A conserved folding mechanism for PDZ domains

An important question in protein folding is whether the folding mechanism is sequence dependent and conserved for homologous proteins. In this work we compared the kinetic folding mechanism of five postsynaptic density protein-95, disc-large tumor suppressor protein, zonula occludens-1 (PDZ) domains, sharing similar topology but having different primary structures. Investigation of the different proteins under various experimental conditions revealed that the folding kinetics of each member of the PDZ family can be described by a model with two transition states separated by an intermediate. Moreover, the positions of the two transition states along the reaction coordinate (as given by their beta(T)-values) are fairly constant for the five PDZ domains.     

Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase

This protocol describes affinity purification of bacterially expressed, recombinant membrane proteins fused with calmodulin-binding domains. As exemplified by the Escherichia coli nicotinamide nucleotide transhydrogenase, this method allows isolation of the protein fusions in a single chromatography step using elution with the calcium chelating agent EDTA and, unlike purification of His-tagged proteins on nickel chelate, it is not sensitive to the presence of strong reducing agents (e.g., DTT). Our protocol involves disruption of host bacteria by sonication, sedimentation of membranes by differential centrifugation, solubilization of membrane proteins and affinity chromatography on calmodulin-agarose. To achieve maximum purity and yield, the use of a combination of non-ionic and anionic detergents is suggested. Purification takes two working days, with an overnight wash of the column to increase the purity of the product.