Measurement of tooth extraction forces in upper incisors

A new method, applied for the first time in this research, was used for measurement of tooth extraction forces. The research has been done in a group of 50 examinees to whom the tooth extraction has been done with lower premolar forceps - forceps "13" and in the control group of 54 examinees in whom the tooth extraction has been done with upper incisive forceps - forceps "1". The measurement instrument registered the extraction forces values in both types of forceps. There was no difference in any parameters in these two groups except in used pressure. While using the forceps "13", a significantly lower tooth extraction force was measured than the force measured while using the forceps "1" (p < 0.001). This means that in clinical work we can already apply noticeably less force using the lower premolar forceps for the extraction of the upper incisors (in the moments of rotation up to 70%). These results are meaningful, because they lead to better and improved instrument solutions and working techniques.     

Assessment of the effect of cyclic chalcone analogues on mitochondrial membrane and DNA

The cytotoxic and protective effects of selected synthetic chalcone analogues have been shown in previous studies. We studied their cytotoxic effect on the modification of mitochondrial membrane potential and on DNA. The first spectral information about the methoxy group as well as the dimethylamino substituent in E-2-arylmethylene-1-benzosuberones molecule was obtained by absorption and emission spectra. The cytotoxic effect of both cyclic chalcone analogues on DNA were detected by alkaline single-cell gel electrophoresis. Better fluorescent chalcone analogue E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone was studied further in fresh isolated mitochondria. The decrease of rat liver mitochondria membrane potential (Δψ) was observed by fluorescence emission spectra. For the collapsing of mitochondrial potentials and as the negative control of mitochondrial function the CCCP uncoupler was used. The absorption maximum of the methoxy group was found at a shorter wavelength (λ = 335 nm) than that of the dimethylamino group (λ = 406 nm). The excitation spectra were very similar to the absorption spectra for both molecules but the emission spectra showed a better fluorescence for dimethylamino derivative. After the addition of E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone to the intact mitochondria the decrease of mitochondrial membrane potential Δψ was observed by emisssion fluorescence spectra. Both cyclic chalcone analogues induced DNA damage, which was detected by alkaline comet assay. Mainly the apoptotic cells were detected, but necrotic cells were also present. Similarities in the percentages of DNA migration from the head were observed in both treatment groups. Both benzosuberones, with dimethylamino- and methoxy- substituent, were very active biologically, as shown by DNA results of the comet assay. Due to its better fluorescence properties, only the fluorophore with dimethylamino substituent was selected for further study of the function of rat liver mitochondria. Decline of mitochondrial function as well as mitochondrial DNA damage were evident between experimental and control groups.     

Floral scent and its correlation with AFLP data in Sorbus

Comparisons between floral scent-based and DNA-molecular-based taxonomies are rare, yet such comparisons indicate that scent can provide useful taxonomic information. Here, we correlate the phytochemical differentiation in floral scent to the DNA-molecular-based differentiation in the genus Sorbus. Inflorescence scent patterns of the apomictic and endemic Sorbus latifolia microspecies Sorbus franconica, Sorbus adeana, and Sorbus cordigastensis originated by hybridization as well as their parental taxa Sorbus aria agg. and Sorbus torminalis were investigated with the dynamic headspace method. The scent data (presence/absence of compounds) were used to construct an UPGMA tree, to calculate a similarity matrix, and to correlate them with the published amplified fragment length polymorphism (AFLP) data of the same individuals, populations, and taxa. Flow cytometry was used to estimate the DNA-ploidy level of the taxa. Scent analyses showed a total of 68 substances, among them aromatic compounds, terpenoids, aliphatics, and nitrogen-containing compounds. The scent patterns were taxon-specific, and the number of scent components differed among taxa. The correlations with the published AFLP data on population and individual level are highly significant, indicating that the scent and AFLP data are highly congruent in the plants studied. Scent therefore provides useful taxonomic characters in Sorbus.     

Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.     

Contribution of bradykinin and nitric oxide to AT2 receptor-mediated differentiation in PC12 W cells

We investigated the effect of angiotensin II on intracellular cyclic GMP content and neurite outgrowth as an indicator of cell differentiation in PC12 W cells. Neurite outgrowth was examined by phase-contrast microscopy. Outgrown neurites were classified as small, medium and large, and were expressed as neurites per 100 cells. Angiotensin II (10-7 m) increased the outgrowth of medium and large neurites by mean +/- SEM 20.2 +/- 2.3 and 6.6 +/- 1.4 compared with 1.66 +/- 0.5 and 0.1 +/- 0.06 neurites per 100 cells in control. Cellular cyclic GMP content increased by 50-250% with angiotensin II at concentrations of 10-6-10-4 m. Both blockade of AT2 receptors and of nitric oxide synthase markedly reduced angiotensin II-induced neurite outgrowth and cyclic GMP production. In contrast, B2 receptor blockade had no effect or even increased these angiotensin II effects. Sodium nitroprusside and 8-bromo-cyclic GMP both mimicked the effects of angiotensin II on cell differentiation. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both angiotensin II and 8-bromo-cyclic GMP. Our results demonstrate that angiotensin II can stimulate cell differentiation in PC12 W cells by nitric oxide-related and cyclic GMP-dependent mechanisms. The effects of angiotensin II on cell differentiation and cyclic GMP production were mediated via the AT2 receptor and further enhanced by bradykinin B2 receptor blockade.     

An anti-inflammatory role for V alpha 14 NK T cells in Mycobacterium bovis bacillus Calmette-Guérin-infected mice

The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the Valpha14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection determined an early expansion of Valpha14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1(+) Valpha14 NKT population preferentially producing IFN-gamma predominated at an early stage (day 8), which was substituted by an NK1.1(-) population preferentially producing IL-4 at later stages (day 30). Despite the fact that Valpha14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in Valpha14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that Valpha14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spontaneous production and mRNA expression of TNF-alpha in liver and lungs of Valpha14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-alpha mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for Valpha14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.     

Gluconobacter in biosensors: applications of whole cells and enzymes isolated from gluconobacter and acetobacter to biosensor construction

Bacteria belonging to the genus Acetobacter and Gluconobacter, and enzymes isolated from them, have been extensively used for biosensor construction in the last decade. Bacteria used as a biocatalyst are easy to prepare and use in amperometric biosensors. They contain multiple enzyme activities otherwise not available commercially. The range of compounds analyzable by Gluconobacter biosensors includes: mono- and poly-alcohols, multiple aldoses and ketoses, several disaccharides, triacylglycerols, and complex parameters like utilizable saccharides or biological O₂ demand. Here, the recent trends in Gluconobacter biosensors and current practical applications are summarized. An erratum to this article can be found at     

High-throughput knockout screen in fission yeast

We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/. We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.