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.     

Melanosomes transported by myosin-V in Xenopus melanophores perform slow 35 nm steps

We studied the motion of pigment organelles driven by myosin-V in Xenopus melanophores using a tracking technique with precision of 2 nm. The organelle trajectories showed occasional steps with a distribution centered at 35 nm and a standard deviation of 13 nm, in agreement with the step size of myosin-V determined in vitro. In contrast, trajectories of melanosomes in cells expressing a dominant negative form of myosin-V did not show steps. The step duration was in the range 20-80 ms, slower than what it would be expected from in vitro results. We speculate that the cytoplasm high viscosity may affect significantly the melanosomes' motion.     

New mechanisms of biological effects of electromagnetic fields

ATP production in mitochondria depends on the nuclear spin and magnetic moment of Mg²⁺ ion in creatine kinase and ATPase. Consequently, the enzymatic synthesis of ATP is an ion-radical process and depends on the external magnetic field and microwave fields that control the spin states of ion-radical pairs and influence the ATP synthesis. The chemical mechanism of ATP synthesis and the origin of biological effects of electromagnetic (microwave) fields are discussed.     

Putidaredoxin-to-cytochrome P450cam electron transfer: differences between the two reductive steps required for catalysis

Cytochrome P450cam (P450cam) is the terminal monooxygenase in a three-component camphor-hydroxylating system from Pseudomonas putida. The reaction cycle requires two distinct electron transfer (ET) processes from the [2Fe-2S] containing putidaredoxin (Pdx) to P450cam. Even though the mechanism of interaction and ET between the two proteins has been under investigation for over 30 years, the second reductive step and the effector role of Pdx are not fully understood. We utilized mutagenesis, kinetic, and computer modeling approaches to better understand differences between the two Pdx-to-P450cam ET events. Our results indicate that interacting residues and the ET pathways in the complexes formed between reduced Pdx (Pdx(r)) and the ferric and ferrous dioxygen-bound forms of P450cam (oxy-P450cam) are different. Pdx Asp38 and Trp106 were found to be key players in both reductive steps. Compared to the wild-type Pdx, the D38A, W106A, and delta106 mutants exhibited considerably higher Kd values for ferric P450cam and retained ca. 20% of the first electron transferring ability. In contrast, the binding affinity of the mutants for oxy-P450cam was not substantially altered while the second ET rates were <1%. On the basis of the kinetic and modeling data we conclude that (i) P450cam-Pdx interaction is highly specific in part because it is guided/controlled by the redox state of both partners; (ii) there are alternative ET routes from Pdx(r) to ferric P450cam and a unique pathway to oxy-P450cam involving Asp38; (iii) Pdx Trp106 is a key structural element that couples the second ET event to product formation possibly via its "push" effect on the heme-binding loop.     

A biophysical approach to the optimisation of dendritic-tumour cell electrofusion

Electrofusion of tumour and dendritic cells (DCs) is a promising approach for production of DC-based anti-tumour vaccines. Although human DCs are well characterised immunologically, little is known about their biophysical properties, including dielectric and osmotic parameters, both of which are essential for the development of efficient electrofusion protocols. In the present study, human DCs from the peripheral blood along with a tumour cell line used as a model fusion partner were examined by means of time-resolved cell volumetry and electrorotation. Based on the biophysical cell data, the electrofusion protocol could be rapidly optimised with respect to the sugar composition of the fusion medium, duration of hypotonic treatment, frequency range for stable cell alignment, and field strengths of breakdown pulses triggering membrane fusion. The hypotonic electrofusion consistently gave a tumour-DC hybrid rate of up to 19%, as determined by counting dually labelled fluorescent hybrids in a microscope. This fusion rate is nearly twice as high as that usually reported in the literature for isotonic media. The experimental findings and biophysical approach presented here are generally useful for the development of efficient electrofusion protocols, especially for rare and valuable human cells.     

Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner

The microtubule cytoskeleton and the mitotic spindle are highly dynamic structures, yet their sizes are remarkably constant, thus indicating that the growth and shrinkage of their constituent microtubules are finely balanced. This balance is achieved, in part, through kinesin-8 proteins (such as Kip3p in budding yeast and KLP67A in Drosophila) that destabilize microtubules. Here, we directly demonstrate that Kip3p destabilizes microtubules by depolymerizing them--accounting for the effects of kinesin-8 perturbations on microtubule and spindle length observed in fungi and metazoan cells. Furthermore, using single-molecule microscopy assays, we show that Kip3p has several properties that distinguish it from other depolymerizing kinesins, such as the kinesin-13 MCAK. First, Kip3p disassembles microtubules exclusively at the plus end and second, remarkably, Kip3p depolymerizes longer microtubules faster than shorter ones. These properties are consequences of Kip3p being a highly processive, plus-end-directed motor, both in vitro and in vivo. Length-dependent depolymerization provides a new mechanism for controlling the lengths of subcellular structures.     

Anti-inflammatory properties of intestinal Bifidobacterium strains isolated from healthy infants

Certain Bifidobacterium strains have been shown to inhibit inflammatory responses in intestinal epithelial cells. However, the precise mechanisms of these effects, including the chemical nature of the active compounds, remain to be elucidated. Here partial characterization of the anti-inflammatory properties of Bifidobacterium strains isolated from feces of healthy infants is reported. It was found that conditioned media (CM) of all strains studied are capable of attenuating tumor necrosis factor-α (TNF-α) and lipopolysaccharide- (LPS) induced inflammatory responses in the HT-29 cell line. In contrast, neither killed bifidobacterial cells, nor cell-free extracts showed such activities. Further investigations resulted in attribution of this activity to heat-stable, non-lipophilic compound(s) resistant to protease and nuclease treatments and of molecular weight less than 3 kDa. The anti-inflammatory effects were dose- and time-dependent and associated with inhibition of IκB phosphorylation and nuclear factor-κ light chain enhancer of activated B cells (NF-κB)-dependent promoter activation. The combined treatments of cells with CMs and either LPS or TNF-α, but not with CMs alone, resulted in upregulation of transforming growth factor-β1, IκBζ, and p21(CIP) mRNAs. Our data suggest certain species-specificities of the anti-inflammatory properties of bifidobacteria. This observation should prompt additional validation studies using larger set of strains and employing the tools of comparative genomics.     

Micro CT-based multiscale elasticity of double-porous (pre-cracked) hydroxyapatite granules for regenerative medicine

Hundred micrometers-sized porous hydroxyapatite globules have proved as a successful tissue engineering strategy for bone defects in vivo, as was shown in studies on human mandibles. These granules need to provide enough porous space for bone ingrowth, while maintaining sufficient mechanical competence (stiffness and strength) in this highly load-bearing organ. This double challenge motivates us to scrutinize more deeply the micro- and nanomechanical characteristics of such globules, as to identify possible optimization routes. Therefore, we imaged such a (pre-cracked) granule in a microCT scanner, transformed the attenuation coefficients into voxel-specific nanoporosities, from which we determined, via polycrystal micromechanics, voxel-specific (heterogeneous) elastic properties. The importance of the latter and of the presence of one to several hundred micrometers-sized cracks for realistically estimating the load-carrying behavior of the globule under a typical two-point compressive loading (as in a "splitting" test) is shown through results of large-scale Finite Element analyses, in comparison to analytical results for a sphere loaded at its poles: Use of homogeneous instead of heterogeneous elastic properties would overestimate the structure's stiffness by 5% (when employing a micromechanics-based process as to attain homogeneous properties)-the cracks, in comparison, weaken the structure by one to two orders of magnitudes.