<Emphasis Type="Italic">In Situ</Emphasis> effect of strontium toothpaste on artificially decalcified human enamel

The aim of this study was to measure the strontium levels in surface and subsurface artificially decalcified enamel exposed in situ to a strontium-supplemented toothpaste. Twenty healthy premolar teeth extracted for orthodontic reasons were cut into blocks and demineralized. The samples were cemented to the teeth of six volunteers who brushed with a hydroxyapatite (HAP) and HAP strontium-supplemented toothpaste for 3 and 6 mo. The strontium content in the enamel surface and in the lateral wall of the enamel samples was evaluated using energy-dispersive spectrometry microanalysis. After 3 mo, the strontium content in the enamel surface increased significantly compared to baseline values. On the lateral enamel surfaces, the strontium level was the same in all of the layers after 6 mo and was higher than the level observed after 3 mo. Regular toothbrushing with a strontium-supplemented toothpaste has been found to increase the strontium content in the exposed enamel, which can be an advantage in the prevention of cariogenesis.     

Detection of DNA methylation in eucaryotic cells

The methods of molecular biology allow for analyzing the methylation pattern in the whole genome and in particular genes. We differentiate methylated sequences from unmethylated ones by means of cutting the genomic template with methylation-sensitive restriction enzymes or by sodium bisulfite DNA modification. Chemical modification precedes most quantitative and qualitative PCR techniques: MS-PCR, MS-nested PCR, Real-Time PCR, QAMA, HeavyMethyl, MSHRM. Restriction enzymes, on the other hand, may be used together with PCR or hybridisation methods (Southern blot and microarrays). PCRs are conducted with primers specific for methylated and unmethylated sequences and sometimes, similarly to hybridisation techniques, with specifically labeled probes or dyes intercalating to double-stranded nucleic acids. The most advanced methylation detection techniques (MALDI-TOF MS and HPLC) significantly reduce the amount of biological material used for tests, but they require specialist equipment.     

Crystal structures of ethyl 3-azido-2,3-dideoxy-D-arabino-hexopyranoside anomers

The structures of the title alpha (1) and beta (2) anomers of ethyl 3-azido-2,3-dideoxy-d-arabino-exopyranoside (C(8)H(15)N(3)O(4)) are reported. The single-crystal structures of C(8)H(15)N(3)O(4) were determined by X-ray crystallography at 293K. It has been found that both title compounds crystallize in the orthorhombic space group. In both cases, the unit cell contains four asymmetric molecules. From intensity measurements, it has been shown that each of these molecules adopts a (4)C(1) chair conformation. The packing arrangement in the unit cell displays a stratified structure. Moreover, medium strength O-H...O hydrogen bonds in both crystal lattices can be observed.     

Epimorphosis and repair processes of schistomelic pedipalps and walking appendages in <Emphasis Type="Italic">Tegenaria atrica</Emphasis> (Araneae,Agelenidae)

Experimental studies of regeneration and repair processes of schistomelic walking legs and pedipalps in the early postembryonal stages of Tegenaria atrica were carried out in two parts. In the first part changes in the structure of appendages were produced by exposing the developing embryos to alternating temperatures (14 and 32°C, changing every 12 hours) as teratogenic factor. In the second, principal, part of studies after amputating a given fragment of a larval schistomelic appendage the appearance, then the growth and development of the external regenerates were observed. The completion of epimorphosis of the pedipalps followed at nymph IV stage (after four post-larval moults) that of the walking appendages in most cases at nymph V stage (after five post-larval moults). In control spiders repair processes were noted, which proceeded at a rate comparable to regeneration processes.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified ("free") fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels

Techniques that allow cells to self-assemble into three-dimensional (3-D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular--especially in fields such as stem cell research, tissue engineering, and cancer biology. Unfortunately, caveats involving scale, expense, geometry, and practicality have hindered the widespread adoption of these techniques. We present an easy-to-use, inexpensive, and scalable technology for production of complex-shaped, 3-D microtissues. Various primary cells and immortal cell lines were utilized to demonstrate that this technique is applicable to many cell types and highlight differences in their self-assembly phenomena. When seeded onto micromolded, nonadhesive agarose gels, cells settle into recesses, the architectures of which optimize the requisite cell-to-cell interactions for spontaneous self-assembly. With one pipeting step, we were able to create hundreds of uniform spheroids whose size was determined by seeding density. Multicellular tumor spheroids (MCTS) were assembled or grown from single cells, and their proliferation was quantified using a modified 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. Complex-shaped (e.g., honeycomb) microtissues of homogeneous or mixed cell populations can be easily produced, opening new possibilities for 3-D tissue culture.     

Regulation of storage lipid metabolism in developing and germinating lupin (<Emphasis Type="Italic">Lupinus</Emphasis> spp.) seeds

The main storage compound in lupin seeds is protein, whose content can reach up to 45–50 % of dry matter. However, seeds of some lupin species can also contain quite a large amount of storage lipid. The range of lipid content in lupin seeds is from about 6 to about 20 % of dry matter. Storage lipid in developing seeds is synthesized mainly from sugars delivered by mother plants. During seed germination, one of the main end-products of storage lipid breakdown is also sugars. Thus, the sugar level in tissues is considered an important regulatory agent, during both lipid accumulation and lipid breakdown. Generally, in developing legume seeds, there is a strong negative relation between accumulation of storage protein and storage lipid. Results obtained in developing lupin cotyledons cultured in vitro pointed to the possibility of a positive relation between protein and lipid accumulation. Such a positive effect could be caused by nitrate. During lupin seed germination and seedling development, the utilization of storage lipid is enhanced under sugar deficiency conditions in tissues and is controlled at the gene expression level. However, under sugar starvation conditions, autophagy is significantly enhanced, and it can cause disturbances in storage lipid breakdown. The hypothesis of pexophagy, i.e., autophagic degradation of peroxisomes under sugar starvation conditions during lupin seed germination, has been taken into consideration. The flow of lipid-derived carbon skeletons to amino acids was discovered in germinating lupin seeds, and this process is clearly more intense in sucrose-fed embryo axes. At least four alternative or mutually complementary pathways of carbon flow from storage lipid to amino acids in germinating lupin seeds are postulated. The different strategies of storage compound breakdown during lupin seed germination are also discussed.     

Purification of glycomacropeptide from non-dialyzable fraction of sweet whey by hydrophobic interaction chromatography on phenyl-agarose

Non-dialyzable fraction of sweet whey was chromatographed on a column of phenyl-agarose equilibrated with 0.01 M sodium phosphate buffer, pH 6.8 containing 5 M NaCl. Most whey proteins were adsorbed on the column, while the glycomacropeptide (GMP) was not. Amino acid analysis of the GMP fraction showed presence of traces (each < 1 residue/peptide) of arginine, histidine and phenylalanine which are not found in GMP. The estimated yield of GMP fraction was approximately 1.6 g l^-1 of sweet whey.     

A direct spectrophotometric γ-glutamyltransferase inhibitor screening assay targeting the hydrolysis-only mode

γ-Glutamyltransferase (GGT, E.C. 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione. Due to its central role in maintaining mammalian glutathione homeostasis, GGT is now believed to be a valuable drug target for a variety of life-threatening diseases, such as cancer. Unfortunately, however, effective tools for screening GGT inhibitors are still lacking. We report here the synthesis and evaluation of an α-phenylthio-containing glutathione peptide mimic that eliminates thiophenol upon GGT-catalyzed hydrolysis of the γ-glutamyl peptide bond. The concurrent, real-time spectrophotometric quantification of the released thiophenol using Ellman’s reagent creates a GGT assay format that is simple, robust, and highly sensitive. The versatility of the assay has been demonstrated by its application to the kinetic characterization of equine kidney GGT, and enzyme inhibition assays. The ability of the glutathione mimic to behave as an excellent donor substrate (exhibiting Michaelis-Menten kinetics with a K_m of 11.3 ± 0.5 μM and a k_cat of 90.1 ± 0.8 nmol mg⁻¹ min⁻¹), coupled to the assay’s ability to study the hydrolysis-only mode of the GGT-catalyzed reaction, make our approach amenable to high-throughput drug screening platforms.