A click chemistry approach to glycomimetics: Michael addition of 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose to 4-deoxy-1,2-O-isopropylidene-L-glycero-pent-4-enopyranos-3-ulose--a convenient route to novel 4-deoxy-(1-->5)-5-C-thiodisaccharides

The base catalyzed conjugate Michael addition of the 1-thiosugar, 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose, 1, to a new highly reactive enone 4-deoxy-1,2-O-isopropylidene-L-glycero-pent-4-enopyranos-3-ulose, 2, proceeds steroselectively with formation of adduct 3 in 94% yield. Convenient stereoselective reduction of the C-3 keto function of 3 with L-Selectride followed by in situ acetylation produces thiodisaccharide 4 in good 82% yield. Cleavage of the 1,2-O-isopropylidene protecting group with p-toluenesulfonic acid in methanol, followed by de-O-acetylation, produced an inseparable anomeric mixture of methyl 4-deoxy-5-C-(beta-D-glucopyranosyl)-thio-alpha/beta-L-ribo-pyranoside 5 in 72% overall yield. This approach constitutes a new general two-step click chemistry route to the previously unknown class of 4-deoxy-(1-->5)-5-C-thiodisaccharides as stable and biologically important glycomimetics.     

Anchorage-dependent chick embryo fibroblasts synthesise DNA and proliferate when cultured in multilayered sheets suspended among glass fibres.

Chick embryo fibroblasts (CEFs) spontaneously form multicellular and multilayered sheets suspended on the network of glass fibres which are stabilized by fibronectin containing protein deposits located at cell-to-cell contacts. The cells situated within the sheets are surrounded by the neighbouring cells and their mechanical equilibrium is stabilised by intercellular "parabaric" effects. It was found that CEFs in the sheets retain relatively high mitotic activity corresponding to that observed in sparse monolayer cultures. These cells grew up to much higher local density than in confluent and contact-inhibited monolayer cultures and developed an abundance of microfilament bundles that terminated at vinculin-containing protein complexes. The results presented demonstrate that direct contact with solid substratum, cell-to-cell contacts, local cell density, and intercellular exchange of humoral factors are not directly involved in the density-dependent inhibition of growth observed in monolayer cultures. They also support the concepts concerning the role of mechanical equilibrium of cell membrane and sub-membranous cytoskeleton in the regulation of proliferation of non-transformed cells.     

Malate accumulation in different organs of Mesembryanthemum crystallinum L. following age-dependent or salinity-triggered CAM metabolism

Different organs of Mesembryanthemum crystallinum exhibit differing levels of CAM (Crassulacean acid metabolism), identifiable by quantification of nocturnal malate accumulation. Shoots and also basal parts of young leaves were observed to accumulate high concentrations of malate. It was typically found in mature leaves and especially prominent in plants subjected to salt stress. Small amount of nocturnal malate accumulation was found in roots of M. crystallinum plants following age-dependent or salinity-triggered CAM. This is an indication that malate can be also stored in non-photosynthetic tissue. Measurements of catalase activity did not produce evidence of the correlation between activity of this enzyme and the level of malate accumulation in different organs of M. crystallinum although catalase activity also appeared to be dependent on the photoperiod. In all material collected at dusk catalase activity was greater than it was observed in the organs harvested at dawn.     

Thermal and guanidine hydrochloride-induced denaturation of human cystatin C

Wild-type human cystatin C is directly involved in pathological fibrils formation, leading to hemorrhage, dementia and eventually death of people suffering from cerebral amyloid angiopathy. Some studies on cystatin C oligomerization have been already done but some points are still unclear. In order to learn more about this important process, we have investigated thermal and chemical (guanidine hydrochloride-induced) denaturation of human cystatin C. Studies performed using tryptophan fluorescence, calorimetry, circular dichroism and Fourier transform infrared spectroscopy demonstrate that neither chemical nor thermal denaturation of hCC are simple two-state events. One recognized intermediate form was dimeric cystatin C, whose appearance was preceded mainly by changes in the L2 binding loop. The other form occurred only in the chemical denaturation process and was characterized by partially recovered interactions maintaining the protein tertiary structure. Our studies also strongly indicate that the -structural motif of cystatin C is directly implicated in formation of temperature-induced aggregates.Abbreviations Gdn.HCl guanidine hydrochloride - hCC human cystatin C     

Crystal structure of the YffB protein from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> suggests a glutathione-dependent thiol reductase function

Background  

The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein.     

A modular approach to the synthesis of new reagents useful in the chemical synthesis of modified DNA probes: derivatives of 3-(tert-butyldimethylsiloxy)glutaric anhydride as versatile building blocks in the synthesis of new phosphoramidites and modified solid supports

We present a flexible and cost-efficient synthetic strategy for the preparation of a new family of phosphoramidite and solid-support reagents that can introduce a broad range of modifications into DNA probes. The key intermediate material 3 is synthesized using the inexpensive and commercially available 3-(tert-butyldimethylsiloxy)glutaric anhydride 1 and can be used as common starting material for the preparation of new labeling reagents.     

Androgens and FSH affect androgen receptor and aromatase distribution in the porcine ovary

In the present study the authors investigated whether androgens could interact with FSH to induce aromatase and androgen receptor expression in porcine granulosa cells. Dissected whole porcine follicles (small, medium, and large) were incubated for 8 hours in M199 medium supplemented with testosterone (10(-7) M), FSH (100 ng/ml) or both those hormones. After incubation, the follicles were fixed and immunostained to visualise androgen receptor and aromatase. In cultures of granulosa cells isolated from small and large follicles, oestrogen secretion was measured by appropriate RIA. Incubation of follicles with testosterone and FSH increased aromatase immunoreactivity in preantral and early antral (i.e. small) follicles. The immunostaining for androgen receptor was slightly higher in medium follicles, while such hormonal stimulation had no effect on small and large follicles. Moreover, granulosa cells isolated from small follicles cultured with both testosterone and FSH produced more estradiol than control cultures (40 pg vs. 100 pg/10(5) cells). The level was relatively close to that obtained in the culture of control granulosa cells isolated from large preovulatory follicles (105 pg/10(5) cells). These results indicate that testosterone acts synergistically with FSH to increase aromatase expression in the small porcine follicles.     

Occurrence of bacteria in the mouth from genera of Micrococcus,Kocuria, Nesterenkonia,Kytococcus and Dermacoccus

The aim of the study was to assess the prevalence of different bacteria in the oral cavity. The bacteria were present in the oral cavities of 73 (48.7%) of 150 individuals. Nesterenkonia halobia, the most frequently isolated species, was found in 20 (27%) individuals, Micrococcus luteus in 16 (22%), Kocuria kristinae in 12 (16%), Kocuria varians in 10 (14%), Dermacoccus sedentarius in 9 (12%), Micrococcus lylae in 8 (11%), and Kytococcus nishinomiyaensis in 3 (4%). Mean counts of these microorganisms were relatively low and amounted in log10 CFU/ml saliva for M. luteus 1.87 +/- 0.52, for M. lylae 2.03 +/- 0.39, for N. halobia 2.14 +/- 0.56, for K. kristinae 2.20 +/- 0.69, for K. varians 2.19 +/- 0.67, for K. nishinomiyaensis 1.72 +/- 0.39, and for D. sedentarius 2.27 +/- 0.55. The factor limiting the population sizes of these microorganisms was most probably the antagonistic activity of the bacteria living in oral cavity.