The protein kinase C cascade regulates recruitment of matrix metalloprotease 9 to podosomes and its release and activation

Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.     

Studies on solvatochromic properties of aminophenylstyryl-quinolinum dye, LDS 798, and its application in studying submicron lipid based structure

The styryl group of dyes has been used in cellular studies for over 20 years because of their solvatochromic and/or electrochromic properties. Here we report characterization of solubility and solvatochromic properties of a near infra-red styryl dye, styryl 11 or LDS 798. We have extended our studies to small unilamellar vesicles and lipid based nanoparticles and found that solvatochromic properties of this dye used in tandem with fluorescence correlation spectroscopy can be used to efficiently determine the diffusion coefficient and hence the size of the submicron lipid based particles. This technique has the potential to provide essential information about liposomal and vesicular structures and their movement in vitro and in situ.     

A rapid non-destructive DNA extraction method for insects and other arthropods

Preparation of arthropods for morphological identification often damages or destroys DNA within the specimen. Conversely, DNA extraction methods often destroy the external physical characteristics essential for morphological identification. We have developed a rapid, simple and non-destructive DNA extraction technique for arthropod specimens. This technique was tested on four arthropod orders, using specimens that were fresh, preserved by air drying, stored in ethanol, or collected with sticky or propylene glycol traps. The technique could be completed in 20 min for Coleoptera, Diptera and Hemiptera, and 2 min for the subclass Acarina, without significant distortion, discolouration, or other damage to the specimens.     

Fast- or Slow-inactivated State Preference of Na+ Channel Inhibitors: A Simulation and Experimental Study

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the “steady-state slow inactivation curve”), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated.     

High-resolution multiple-unit EEG in cat auditory cortex reveals large spatio-temporal stochastic interactions

It has been argued that information processing in the cortex is optimised with regard to certain information theoretic principles. We have, for instance, recently shown that spike-timing dependent plasticity can improve an information-theoretic measure called spatio-temporal stochastic interaction which captures how strongly a set of neurons cooperates in space and time. Systems with high stochastic interaction reveal Poisson spike trains but nonetheless occupy only a strongly reduced area in their global phase space, they reveal repetiting but complex global activation patterns, and they can be interpreted as computational systems operating on selected sets of collective patterns or "global states" in a rule-like manner. In the present work we investigate stochastic interaction in high-resolution EEG-data from cat auditory cortex. Using Kohonen maps to reduce the high-dimensional dynamics of the system, we are able to detect repetiting system states and estimate the stochastic interaction in the data, which turns out to be fairly high. This suggests an organised cooperation in the underlying neural networks which cause the data and may reflect generic intrinsic computational capabilities of the cortex.     

Cortical activity pattern computation

Neural computations are modelled in various ways, but still there is no clear understanding of how the brain performs its computational tasks. This paper presents new results about analysis of neural processes in terms of activity pattern computations. It is shown that it is possible to extract from high-resolution EEG data a first order Markov approximation of a neural communication system employing pattern computations, which is significantly different from similar purely random systems. In our view this result shows that it is likely that neural activity patterns measurable at the macro-level by EEG are correlated with underlying neural computations.     

Biotechnological approach to the synthesis of 9alpha-hydroxylated steroids

The steroid 9alpha-hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9alpha-hydroxyprogesterone. In 7 days, 43.6 mg 9alpha-hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4-androstene-3,17-dione in vivo. An extensive analytical method development, including LLE, HPLC-DAD, MS, and NMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion rate were unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.     

Nitric oxide, mitochondrial hyperpolarization, and T cell activation

T lymphocyte activation is associated with nitric oxide (NO) production, which plays an essential role in multiple T cell functions. NO acts as a messenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulates mitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many cell types, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both activation and apoptosis of Tlymphocytes, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cell signaling pathways. Overproduction of NO in systemic lupus erythematosus induces mitochondrial biogenesis and alters Ca(2+) signaling. Thus, whereas NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to the pathogenesis of autoimmunity.     

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

Background  

The mitochondrial DNA (mtDNA) of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT) was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts.