Transcriptional and physiological adaptation to defective protein-O-mannosylation in Candida albicans.

Five Pmt isoforms O-mannosylate secretory proteins in Candida albicans. Comparisons of genome-wide transcript patterns of each pmt mutant revealed commonly downregulated genes involved in glycolysis and glycerol production. Increased phosphorylation of the Cek1p- but not the Mkc1p-MAP kinase, as well as increased transcript levels for some stress-related genes were detected in the pmt1 strain but not in the other pmt mutants. The transcriptomal pattern after short-term inhibition of Pmt1p activity confirmed stress responses, but did not indicate an alteration of glycolytic flow. Short- but not long-term adaptation to Pmt1p inhibition required signalling components Cek1p, Mkc1p, Efg1p and Tpk1p. Cna1p (calcineurin) but not its downstream effectors Crz1p and Crz2p was generally essential to allow growth during Pmt1p inhibition; accordingly, cyclosporin A strongly inhibited growth of the pmt1 mutant. The lack of Pmt isoforms influenced transcript levels for the remaining isoforms both positively and negatively, suggesting complex cross-regulation among PMT genes. These results confirm individual functions of Pmt isoforms but suggest a common biphasic adaptation response to Pmt deficiency. While known signalling pathways modulate adaptation for a short-term, long-term adaptation requires calcineurin, adjustments of remaining Pmt activities and of glycolytic flow.

Oligo- and polysaccharides exhibit a structure-dependent bioactivity on human keratinocytes in vitro.

In traditional medicine, a variety of plants with high carbohydrate contents were used for dermatological therapies. Contemporary investigations confirmed exogenous carbohydrates as biologically active. The recent study describes the characterization of oligo- and polysaccharides from medicinal herbs and evaluation of composite-dependent physiological activity of carbohydrates on human keratinocytes in vitro. Polysaccharide isolation was followed by size- and charge fractionation. Identification of monosaccharide components was performed by GLC/MS. Primary human keratinocytes (NHK) and cells of the cell line HaCaT were used for investigation of carbohydrate action on cellular proliferation (BrdU-uptake), differentiation specific enzymes (involucrin), cell viability (MTT-reduction) and cytotoxicity. Incubation of keratinocytes with a purified beta-glucan from Reed mace seeds resulted in an improved proliferation followed by an increased differentiation after contact inhibition. Fucosylated oligo- and polysaccharides of human milk and Sea weed induced involucrin expression as maker for early differentiation without an increase in proliferation. Cell viability and proliferation of keratinocytes were enhanced by an arabinogalactan of Kaki fruits. Okra fruit rhamnogalacturonans increased cell proliferation. Heart sease pectin-like polysaccharides reduced the proliferation significantly but improved the cell viability. These results led assume that the carbohydrates of traditional used herbs play a part in their efficacy.

Glycosylated compounds from okra inhibit adhesion of Helicobacter pylori to human gastric mucosa.

In Asian medicine the fruit of the okra plant, Abelmoschus esculentus (L.) Moench., is used as a mucilaginous food additive against gastric irritative and inflammative diseases. To find a rational basis for its use against these diseases, several crude and purified carbohydrate-containing fractions from immature okra fruits were isolated and analyzed, and their effects against Helicobacter pylori in an in situ adhesion model on sections of human gastric mucosa were determined. Pretreatment of the bacteria with a fresh juice preparation inhibited the bacterial adhesion almost completely. Lyophilization and reconstitution of an extract solution led to a reduction of this effect. A crude polysaccharide (RPS) isolated from the fresh juice by ethanolic precipitation showed strong inhibitory effects. Further fractionation of RPS revealed a purified, highly acidic subfraction (AF III) with high antiadhesive qualities. Carbohydrate analysis revealed the presence of rhamnogalacturonans with a considerable amount of glucuronic acid, whereas other inactive subfractions contained little glucuronic acid or were glucuronic acid-free. After heat denaturation of the fresh juice or protein precipitation with 5% TCA the antiadhesive activity of the fresh extract was reduced, indicating that besides polysaccharides, protein fractions also exhibited antiadhesive properties. SDS-PAGE analysis of the precipitate revealed several bands of glycosylated proteins between 25 and 37 kDa that were almost diminished in the nonactive supernatant. Preincubations of gastric tissue with any of the active fractions did not lead to reduced bacterial binding. The antiadhesive activity is therefore due to the blocking capacity of specific Helicobacter surface receptors that coordinate the interaction between host and bacterium. Neither of the active fractions showed inhibitory effects on bacterial growth in vitro. The antiadhesive qualities of okra were assumed to be due to a combination of glycoproteins and highly acidic sugar compounds making up a complex three-dimensional structure that is fully developed only in the fresh juice of the fruit.