A compact microelectrode acceleration device which aids penetration of small cells

An improved piezoelectric electrode acceleration device is described, which requires no specially designed electronic circuitry. The device is simple in design, compact and readily adaptable to most standard neurophysiological equipment. It will produce either sinusoidal motion or pulses, singly or continuously. A relatively pure axial motion of the electrode tip with little lateral whip is produced by the device.     

Mechanosensory calcium-selective cation channels in epidermal cells

This paper explores the properties and likely functions of an epidermal Ca(2+)-selective cation channel complex activated by tension. As many as eight or nine linked or linkable equivalent conductance units or co-channels can open together. Open time for co-channel quadruplets and quintuplets tends to be relatively long with millimolar Mg2+ (but not millimolar Ca2+) at the cytosolic face of excised plasma membrane. Sensitivity to tension is regulated by transmembrane voltage and temperature. Under some circumstances channel activity is sychronized in rhythmic pulses. Certain lanthanides and a cytoskeleton-disturbing herbicide that inhibit gravitropic reception act on the channel system at low concentrations. Specifically, ethyl-N-phenylcarbamate promotes tension-dependent activity at micromolar levels. With moderate suction, Gd3+ provided at about 0.5 micromole at the extracellular face of the membrane promotes for several seconds but may then become inhibitory. Provision at 1-2 micromoles promotes and subsequently inhibits more vigorously (often abruptly and totally), and at high levels inhibits immediately. La3+, a poor gravitropic inhibitor, acts similarly but much more gradually and only at much higher concentrations. These properties, particularly these susceptibilities to modulation, indicate that in vivo the mechanosensitive channel must be mechanosensory and mechanoregulatory. It could serve to transduce the shear forces generated in the integrated wall-membrane-cytoskeleton system during turgor changes and cell expansion as well as transducing the stresses induced by gravity, touch and flexure. In so far as such transduction is modulated by voltage and temperature, the channels would also be sensors for these modalities as long as the wall-membrane-cytoskeleton system experiences mechanical stress.     

A novel dual mode microelectrode array for neuroelectrical and neurochemical recording in vitro

This paper presents an electrical method for measurement of Hematocrit (HCT) using a novel HCT estimation parameter. Particularly in the case of electrical HCT measurements, the measurement error generally increases with changes in the electrical conditions of the plasma such as conductivity and osmolality. This is because the electrical properties of blood are a function not only of HCT, but also of the electrical conditions in the plasma. In an attempt to reduce the measurement errors, we herein propose a novel HCT estimation parameter reflecting the characteristics of both the changes in volume of red blood cells (RBCs) and electrical conditions of plasma, simultaneously. In order to characterize the proposed methods under various electrical conditions of plasma, we prepared twelve blood samples such as four kinds of plasma conditions (hypotonic, isotonic, two kinds of hypertonic conditions) at three different HCT levels. Using linear regression analysis, we confirmed that the proposed parameter was highly correlated with reference HCT (HCT(ref.)) values measured by microcentrifugation. Thus, the HCT measurement error was less than 4%, despite considerable variations in the conductivity and osmolality of the plasma at conditions of the HCT(ref.) of 20%. Multiple linear regression analysis showed that the proposed HCT estimation parameter also yielded a lower measurement error (1%) than the other parameter previously used for the same purpose. Thus, these preliminary results suggest that proposed method could be used for accurate, fast, easy, and reproducible HCT measurements in medical procedures.     

Intracellular microelectrode measurements in small cells evaluated with the patch clamp technique.

Microelectrode penetration of small cells leads to a sustained depolarization of the resting membrane potential due to a transmembrane shunt resistance (Rs) introduced by the microelectrode. This has led to underestimation of the resting membrane potential of various cell types. However, measurement of the fast potential transient occurring within the first few milliseconds after microelectrode penetration can provide information about pre-impalement membrane electrophysiological properties. We have analyzed an equivalent circuit of a microelectrode measurement to establish the conditions under which the peak of the impalement transients (Ep) approaches the pre-impalement resting membrane potential (Em) of small cells most closely. The simulation studies showed that this is the case when the capacitance of the microelectrode is low and the membrane capacitance of the cell high. In experiments performed to assess the reliability of Ep as a measure of Em, whole-cell patch clamp measurements were performed in the current clamp mode to monitor, free from the effects of Rs, Em in cultured human monocytes. Microelectrode impalement of such patch clamped cells and measurement of Ep made it possible to detect correlation between Ep and Em and showed that for small cells such as human monocytes Ep is on average 6 mV less negative than the resting membrane potential.     

CMOS microelectrode array for the monitoring of electrogenic cells

Signal degradation and an array size dictated by the number of available interconnects are the two main limitations inherent to standalone microelectrode arrays (MEAs). A new biochip consisting of an array of microelectrodes with fully-integrated analog and digital circuitry realized in an industrial CMOS process addresses these issues. The device is capable of on-chip signal filtering for improved signal-to-noise ratio (SNR), on-chip analog and digital conversion, and multiplexing, thereby facilitating simultaneous stimulation and recording of electrogenic cell activity. The designed electrode pitch of 250 microm significantly limits the space available for circuitry: a repeated unit of circuitry associated with each electrode comprises a stimulation buffer and a bandpass filter for readout. The bandpass filter has corner frequencies of 100 Hz and 50 kHz, and a gain of 1000. Stimulation voltages are generated from an 8-bit digital signal and converted to an analog signal at a frequency of 120 kHz. Functionality of the read-out circuitry is demonstrated by the measurement of cardiomyocyte activity. The microelectrode is realized in a shifted design for flexibility and biocompatibility. Several microelectrode materials (platinum, platinum black and titanium nitride) have been electrically characterized. An equivalent circuit model, where each parameter represents a macroscopic physical quantity contributing to the interface impedance, has been successfully fitted to experimental results.     

An extracellular microelectrode array for monitoring electrogenic cells in culture

This paper describes a planar array of microelectrodes developed for monitoring the electrical activity of cells in culture. The device allows the incorporation of surface topographical features in an insulating layer above the electrodes. Semiconductor technology is employed for the fabrication of the gold electrodes and for the deposition and patterning of an insulating layer of silicon nitride. The electrodes have been tested using a cardiac cell culture of chick embryo myocytes, and the physical beating of the cultured cells correlated with the simultaneous extracellular voltage measurements obtained. It was found that extracellular stimulation of the cells was possible via the same electrodes used for recording.     

A voltage-gated calcium-selective channel encoded by a sodium channel-like gene

BSC1, which was originally identified by its sequence similarity to voltage-gated Na(+) channels, encodes a functional voltage-gated cation channel whose properties differ significantly from Na(+) channels. BSC1 has slower kinetics of activation and inactivation than Na(+) channels, it is more selective for Ba(2+) than for Na(+), it is blocked by Cd(2+), and Na(+) currents through BSC1 are blocked by low concentrations of Ca(2+). All of these properties are more similar to voltage-gated Ca(2+) channels than to voltage-gated Na(+) channels. The selectivity for Ba(2+) is partially due to the presence of a glutamate in the pore-forming region of domain III, since replacing that residue with lysine (normally present in voltage-gated Na(+) channels) makes the channel more selective for Na(+). BSC1 appears to be the prototype of a novel family of invertebrate voltage-dependent cation channels with a close structural and evolutionary relationship to voltage-gated Na(+) and Ca(2+) channels.     

A novel method for constructing pathogen-regulated small RNA cDNA library

Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp. carotovora (Ecc), often leads to soft rot disease in the plant Brassica campestris L. ssp. pekinensis (Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem-loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 3′ end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing.     

A novel procedure for separating small peptides on polyacrylamide gels

Summary A simple and fast procedure that allows the separation of small (1–3 kDa) peptides on glycine-SDS gels is described. Peptides were separated by glycine-SDS/PAGE as a result ofin situ complexation of peptide/SDS during electrophoretic migration and visualized by Coomassie blue staining. The data presented here shows the separation of small peptides of different isoelectric points, sizes, and hydrophobicity on polyacrylamide mini gels. Ten different peptides have been tested with this method. The data suggest the dependence of SDS/peptide complex formation and migration due to the number of basic amino acid residues, length of peptide and the hydrophobicity/hydrophilicity ratio.     

A novel method for activity monitoring in small non-human primates

Patterns of spontaneous activity are valuable reflections of well-being in animals and humans and, because of this, investigations have frequently incorporated some form of activity monitoring into their studies. It is widely believed that activity monitoring, alongside assessments of general behaviour, should be included in initial CNS safety pharmacology screening. As the number of marmoset studies having actimetry as their focus, or as an adjunct, is increasing, we wished to evaluate an alternative approach to those commonly used. The method is based on miniaturized accelerometer technologies, currently used for human activity monitoring.Actiwatch-Minis were used to monitor the activity of two groups of differently housed marmosets for 14 consecutive days. Group A consisted of four mixed-sex pairs of animals and group B comprised eight group-housed males. Activity profiles were generated for weekday and weekend periods. The devices captured quantifiable data which showed differences in total activity between the two differently housed groups and revealed intragroup variations in the temporal spread of activity between weekdays and weekends. The Actiwatch-Mini has been shown to generate retrospective, data-logged activity counts recorded from multiple animals in a single arena by means of non-invasive monitoring.     

A novel procedure for separating small peptides on polyacrylamide gels

A simple and fast procedure that allows the separation of small(1–3 kDa) peptides on glycine-SDS gels is described. Peptideswere separated by glycine-SDS/PAGE as a result of in situ complexation of peptide/SDS during electrophoretic migration and visualized by Coomassie blue staining. The data presented here shows the separation of small peptides of different isoelectric points, sizes, and hydrophobicity on polyacrylamidemini gels. Ten different peptides have been tested with this method. The data suggest the dependence of SDS/peptide complex formation and migration due to the number of basic amino acid residues, length of peptide and the hydrophobicity/hydrophilicity ratio.     

A novel small molecule,LAS-0811, inhibits alcohol-induced apoptosis in VL-17A cells

One of the pathways by which alcohol induces hepatocyte apoptosis is via oxidative stress. We screened several chemically-synthesized small molecules and found LAS-0811, which inhibits oxidative stress. In this study, we elucidated its role in inhibiting alcohol-induced apoptosis in hepatocyte-like VL-17A cells. VL-17A cells were pre-incubated with LAS-0811, followed by ethanol incubation. Ethanol-induced reactive oxygen species and apoptosis were significantly inhibited in LAS-0811 pre-treated cells. VL-17A cells were transfected with a reporter (ARE/TK-GFP) plasmid containing green fluorescent protein (GFP) as a reporter gene and the anti-oxidant response element as the promoter. LAS-0811 pre-treatment significantly induced the GFP expression compared to the cells treated with ethanol alone. LAS-0811 induced the activation of nrf2 and enhanced the expression and activity of glutathione peroxidase, one of the downstream targets of nrf2. The results indicate that LAS-0811 protects VL-17A cells against ethanol-induced oxidative stress and apoptosis at least in part via nrf2 activation.     

A glass-membrane pH microelectrode.

By miniaturizing the original MacInnes and Dole glass-membrane pH electrode a new pH microelectrode has been developed. The technique developed utilizes the tip of a high electrical resistance glass pipet that can be sealed with a thin membrane of H⁺-sensitive glass. Single-barreled electrodes have been made with tip diameters ranging from 1.5 to 100 μm and double-barreled electrodes with tip diameters from 2 to 28 μm. The glass-membrane pH microelectrode provided a means for sensing the pH of biological solutions with an electrode having theoretical slope and tip configurational control. The most unique characteristics of the electrode were: the pH sensing surface was quite small, the tip diameter could be controlled, and the problem of electrode insulation was eliminated.     

A versatile strategy for the synthesis of crown ether-bearing heterocycles: discovery of calcium-selective fluoroionophore

Heterocycles have been modified in various ways in the search for new functions, but few examples are known of crown ethers incorporating heterocycles in macro-ring systems. Here we report a simple and versatile synthesis of crown ether-bearing heterocycles. An acylurea moiety in the heterocycles is efficiently transformed to 'crown ether' of various ring sizes. The products included a Ca(2+)-selective fluoroionophore. Our simple methodology is expected to provide many novel functional heterocyclic compounds, including fluoroionophores and candidate pharmaceuticals.     

Substituted benzimidazoles: A novel chemotype for small molecule hKSP inhibitors

Substituted benzimidazoles were profiled as inhibitors of kinesin spindle protein (KSP), an increasingly important target for the development of anticancer drugs. This series demonstrated the monoastral phenotypic response and was found to be active in both enzymatic and cellular-based assays.     

A novel simulation model for stem cells differentiation

A novel mathematical model to simulate mesenchymal stem cells differentiation into specialized cells is proposed. The model is based upon material balances for extracellular matrix compounds, growth factors and nutrients coupled with a mass-structured population balance describing cell growth, proliferation and differentiation. The proposed model is written in a general form and it may be used to simulate a generic cell differentiation pathway occurring in vivo or during in vitro cultivation when specific growth factors are used. Literature experimental data concerning the differentiation of mesenchymal stem cells into chondrocytes in terms of total DNA and glycosaminoglycan content are successfully compared with model results, thus demonstrating the validity of the proposed model as well as its predictive capability. A further test of the model capability is performed for the case of in vivo fracture healing during which mesenchymal stem cells differentiate into chondrocytes and osteoblasts. Considerations about the extension of the proposed model to different pathologies beside fracture healing are reported. Finally, sensitivity analysis of model parameters is also performed in order to clarify what mechanisms most strongly influence differentiation and the distribution of cell types.     

A novel method for culturing neural stem cells

The standard culture method for neural stem cells cannot prevent the attachment of neurospheres, which eventually result in differentiation. This study developed a new method for long-term neural stem cell cultivation. In the antiattachment group, neural stem cells were cultured in flasks coated with 1.5% agarose gel. As a control, cells were cultured in plastic flasks. The 5-bromine-deoxyuridine incorporation assay was used to determine the S-phase labeling index of both groups. The methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to determine the total cell vitality. After a 3-mo culture, the spontaneous differentiation of stem cells was studied using immunocytochemistry for neuroepithelial stem cell protein. We found that neural stem cells grew rapidly in the antiattachment flasks. There was no statistically significant difference between the two groups in terms of the S-phase labeling index or MTT assay. When cultured for 3 mo in vitro, many more cells differentiated in the control than in the antiattachment group (32.05 vs. 0.64%, P < 0.01). Moreover, the neural stem cells in the antiattachment group remained multipotent. Therefore, flasks coated with agarose gel are suitable for long-term neural stem cell culture.