Subcellular distribution of calcium-sensitive potassium channels (IK1) in migrating cells

Cell migration is crucial for wound healing, immune defense, or formation of tumor metastases. In addition to the cytoskeleton, Ca2+ sensitive K+ channels (IK1) are also part of the cellular "migration machinery." We showed that Ca2+ sensitive K+ channels support the retraction of the rear part of migrating MDCK-F cells by inducing a localized shrinkage at this cell pole. So far the molecular nature and in particular the subcellular distribution of these channels in MDCK-F cells is unknown. We compared the effect of IK1 channel blockers and activators on the current of a cloned IK1 channel from MDCK-F cells (cIK1) and the migratory behavior of these cells. Using IK1 channels labeled with a HA-tag or the enhanced green fluorescent protein we studied the subcellular distribution of the canine (cIK1) and the human (hIK1) channel protein in different migrating cells. The functional impact of cIK1 channel activity at the front or rear part of MDCK-F cells was assessed with a local superfusion technique and a detailed morphometric analysis. We show that it is cIK1 whose activity is required for migration of MDCK-F cells. IK1 channels are found in the entire plasma membrane, but they are concentrated at the cell front. This is in part due to membrane ruffling at this cell pole. However, there appears to be only little cIK1 channel activity at the front of MDCK-F cells. In our view this apparent discrepancy can be explained by differential regulation of IK1 channels at the front and rear part of migrating cells.     

Identification of differentially methylated sites within unmethylated DNA domains in normal and cancer cells

Altered DNA methylation has been linked to neoplastic cell transformation and is a hallmark of cancer progression. Therefore, the screening for differentially methylated sequences as tumor biomarkers has a significant implication in the clinical setting. To determine the cancer-linked alterations in DNA methylation pattern, we have applied an endonuclease, McrBC, to the existing methylation-sensitive arbitrarily primed polymerase chain reaction (msAP-PCR) method and developed McrBC-msAP-PCR. This modified approach allows detection of differentially methylated sites within unmethylated DNA domains enriched by regulatory sequences and CpG islands. In this method, we used digestion of DNA with the McrBC methylation-sensitive endonuclease to selectively exclude the methylated fraction of DNA, which comprises interspersed and tandem-repeated sequences and exons other than first exons, from analysis. The subsequent digestion of unmethylated DNA fragments with SmaI and HpaII methylation-sensitive restriction endonucleases followed by AP-PCR amplification resulted in the detection of unknown unique sequences associated with cancer-linked methylation changes in genomic DNA. Hypermethylation and hypomethylation are visualized by the increase or decrease in the band intensity of DNA fingerprints. By using this technique, we were able to differentiate clearly, identify, and characterize a number of novel unique DNA sequences with differentially methylated sites in normal and breast cancer cell lines and in normal and rat tumor liver tissues.     

Single plasma membrane K+ channel detection by using dual-color quantum dot labeling

K⁺ channels are widely expressed in eukaryotic and prokaryotic cells, where one of their key functions is to set the membrane potential. Many K⁺ channels are tetramers that share common architectural properties. The crystal structure of bacterial and mammalian K⁺ channels has been resolved and provides the basis for modeling their three-dimensional structure in different functional states. This wealth of information on K⁺ channel structure contrasts with the difficulties to visualize single K⁺ channel proteins in their physiological environment. We describe a method to identify single Ca²⁺-activated K⁺ channel molecules in the plasma membrane of migrating cells. Our method is based on dual-color labeling with quantum dots. We show that >90% of the observed quantum dots correspond to single K⁺ channel proteins. We anticipate that our method can be adopted to label any other ion channel in the plasma membrane on the single molecule level. Ca²⁺-activated K⁺ channel; migration     

Circadian Input Kinases and Their Homologs in Cyanobacteria: Evolutionary Constraints Versus Architectural Diversification

The circadian input kinase A (cikA) gene encodes a protein relaying environmental signal to the central circadian oscillator in cyanobacteria. The CikA protein has a variable architecture and usually consists of four tandemly arrayed domains: GAF, histidine kinase (HisKA), histidine kinase-like ATPase (HATPase_c), and a pseudo-receiver (REC). Among them, HisKA and HATPase_c are the least polymorphic, and REC is not present in heterocystic filamentous cyanobacteria. CikA contains several conserved motifs that are likely important for circadian function. There are at least three types of circadian systems, each of which possesses a different set of circadian genes. The originally described circadian system (kaiABC system) possesses both cikA and kaiA, while the others lack either only cikA (kaiABC ^Δ) or both (kaiBC). The results we obtained allowed us to approximate the time of the cikA origin to be about 2600–2200 MYA and the time of its loss in the species with the kaiABC ^Δ or kaiBC system between 1100 and 600 MYA. Circadian specialization of CikA, as opposed to its non-circadian homologs, is a result of several factors, including the unique conserved domain architecture and high evolutionary constraints of some domains and regions, which were previously identified as critical for the circadian function of the gene.     

Temperature increase results in oxidative stress in goldfish tissues. 1. Indices of oxidative stress

Levels of lipid peroxides (LOOH), thiobarbituric-acid reactive substances (TBARS), protein carbonyls and low- and high-molecular weight thiols were measured in brain, liver, kidney, and white muscle of goldfish, Carassius auratus L., over 1-12 h of high temperature (35 degrees C) exposure followed by 4 or 24 h of lower (21 degrees C) temperature recovery. LOOH and TBARS contents increased during heat shock exposure with a maximal rise of 20-fold for liver TBARS, but both mainly reversed at recovery. Protein carbonyl content was unaffected by heat shock but rose in brain, liver, and kidney during recovery. Low-molecular weight thiol concentrations unexpectedly increased up to approximately 4-fold in brain, kidney and muscle under heat shock and remained high during recovery. Protein thiol contents also rose in liver and muscle during high temperature exposure by 2- and 3-fold, respectively, and decreased to control values or below in all tissues at late recovery. Low- and high-molecular weight thiol levels inversely correlated in liver (R2=0.87) suggesting that the former was used to reduce the latter over the experiment. It is concluded that the redox balance in goldfish tissues is strictly maintained probably contributing to the high tolerance of this species to heat shock.     

Hypoxia and recovery perturb free radical processes and antioxidant potential in common carp (Cyprinus carpio) tissues

The effects of hypoxia exposure and subsequent normoxic recovery on the levels of lipid peroxides (LOOH), thiobarbituric acid reactive substances (TBARS), carbonylproteins, total glutathione levels, and the activities of six antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of the common carp Cyprinus carpio. Hypoxia exposure (25% of normal oxygen level) for 5h generally decreased the levels of oxidative damage products, but in liver TBARS content were elevated. Hypoxia stimulated increases in the activities of catalase (by 1.7-fold) and glutathione peroxidase (GPx) (by 1.3-fold) in brain supporting the idea that anticipatory preparation takes place in order to deal with the oxidative stress that will occur during reoxygenation. In liver, only GPx activity was reduced under hypoxia and reoxygenation while other enzymes were unaffected. Kidney showed decreased activity of GPx under aerobic recovery but superoxide dismutase (SOD) and catalase responded with sharp increases in activities. Skeletal muscle showed minor changes with a reduction in GPx activity under hypoxia exposure and an increase in SOD activity under recovery. Responses by antioxidant defenses in carp organs appear to include preparatory increases during hypoxia by some antioxidant enzymes in brain but a more direct response to oxidative insult during recovery appears to trigger enzyme responses in kidney and skeletal muscle.     

Analysis of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> M145 genes <Emphasis Type="Italic">SCO4164</Emphasis> and <Emphasis Type="Italic">SCO5854</Emphasis> encoding putative rhodaneses

Streptomyces coelicolor genome carries two apparently paralogous genes, SCO4164 and SCO5854, that encode putative thiosulfate sulfurtransferases (rhodaneses). These genes (and their presumed translation products) are highly conserved and widely distributed across actinobacterial genomes. The SCO4164 knockout strain was unable to grow on minimal media with either sulfate or sulfite as the sole sulfur source. The SCO5854 mutant had no growth defects in the presence of various sulfur sources; however, it produced significantly less amounts of actinorhodin. Furthermore, we discuss possible links between basic interconversions of inorganic sulfur species and secondary metabolism in S. coelicolor.     

Evolutionary History of the Photolyase/Cryptochrome Superfamily in Eukaryotes

Background

Photolyases and cryptochromes are evolutionarily related flavoproteins, which however perform distinct physiological functions. Photolyases (PHR) are evolutionarily ancient enzymes. They are activated by light and repair DNA damage caused by UV radiation. Although cryptochromes share structural similarity with DNA photolyases, they lack DNA repair activity. Cryptochrome (CRY) is one of the key elements of the circadian system in animals. In plants, CRY acts as a blue light receptor to entrain circadian rhythms, and mediates a variety of light responses, such as the regulation of flowering and seedling growth.

Results

We performed a comprehensive evolutionary analysis of the CRY/PHR superfamily. The superfamily consists of 7 major subfamilies: CPD class I and CPD class II photolyases, (6–4) photolyases, CRY-DASH, plant PHR2, plant CRY and animal CRY. Although the whole superfamily evolved primarily under strong purifying selection (average ω = 0.0168), some subfamilies did experience strong episodic positive selection during their evolution. Photolyases were lost in higher animals that suggests natural selection apparently became weaker in the late stage of evolutionary history. The evolutionary time estimates suggested that plant and animal CRYs evolved in the Neoproterozoic Era (~1000–541 Mya), which might be a result of adaptation to the major climate and global light regime changes occurred in that period of the Earth’s geological history.     

Changes in Diffusion Kurtosis Imaging and Magnetic Resonance Spectroscopy in a Direct Cranial Blast Traumatic Brain Injury (dc-bTBI) Model

Explosive blast-related injuries are one of the hallmark injuries of veterans returning from recent wars, but the effects of a blast overpressure on the brain are poorly understood. In this study, we used in vivo diffusion kurtosis imaging (DKI) and proton magnetic resonance spectroscopy (MRS) to investigate tissue microstructure and metabolic changes in a novel, direct cranial blast traumatic brain injury (dc-bTBI) rat model. Imaging was performed on rats before injury and 1, 7, 14 and 28 days after blast exposure (~517 kPa peak overpressure to the dorsum of the head). No brain parenchyma abnormalities were visible on conventional T2-weighted MRI, but microstructural and metabolic changes were observed with DKI and proton MRS, respectively. Increased mean kurtosis, which peaked at 21 days post injury, was observed in the hippocampus and the internal capsule. Concomitant increases in myo-Inositol (Ins) and Taurine (Tau) were also observed in the hippocampus, while early changes at 1 day in the Glutamine (Gln) were observed in the internal capsule, all indicating glial abnormality in these regions. Neurofunctional testing on a separate but similarly treated group of rats showed early disturbances in vestibulomotor functions (days 1–14), which were associated with imaging changes in the internal capsule. Delayed impairments in spatial memory and in rapid learning, as assessed by Morris Water Maze paradigms (days 14–19), were associated with delayed changes in the hippocampus. Significant microglial activation and neurodegeneration were observed at 28 days in the hippocampus. Overall, our findings indicate delayed neurofunctional and pathological abnormalities following dc-bTBI that are silent on conventional T2-weighted imaging, but are detectable using DKI and proton MRS.