Probing Functional Properties of Nociceptive Axons Using a Microfluidic Culture System

Pathological changes in axonal function are integral features of many neurological disorders, yet our knowledge of the molecular basis of axonal dysfunction remains limited. Microfluidic chambers (MFCs) can provide unique insight into the axonal compartment independent of the soma. Here we demonstrate how an MFC based cell culture system can be readily adapted for the study of axonal function in vitro. We illustrate the ease and versatility to assay electrogenesis and conduction of action potentials (APs) in naïve, damaged or sensitized DRG axons using calcium imaging at the soma for pharmacological screening or patch-clamp electrophysiology for detailed biophysical characterisation. To demonstrate the adaptability of the system, we report by way of example functional changes in nociceptor axons following sensitization by neurotrophins and axotomy in vitro. We show that NGF can locally sensitize axonal responses to capsaicin, independent of the soma. Axotomizing neurons in MFC results in a significant increase in the proportion of neurons that respond to axonal stimulation, and interestingly leads to accumulation of Nav1.8 channels in regenerating axons. Axotomy also augmented AP amplitude following axotomy and altered activation thresholds in a subpopulation of regenerating axons. We further show how the system can readily be used to study modulation of axonal function by non-neuronal cells such as keratinocytes. Hence we describe a novel in vitro platform for the study of axonal function and a surrogate model for nerve injury and sensitization.     

Stimulating effects of low-power He-Ne laser on growth of Saccharomyces cerevisiae under high pressure

Summary A low-power He-Ne laser decreased the growth inhibitory effect of hydrostatic pressure (50 MPa) on Saccharomyces cerevisiae.     

Bioluminescence‐based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells

We have developed a bioluminescence‐based non‐destructive cytotoxicity assay in which cell viability and membrane damage are simultaneously evaluated using Emerald luciferase (ELuc) and endoplasmic reticulum (ER)‐targeted copepod luciferase (GLuc‐KDEL), respectively, by using multi‐integrase mouse artificial chromosome (MI‐MAC) vector. We have demonstrated that the time‐dependent concentration response curves of ELuc luminescence intensity and WST‐1 assay, and GLuc‐KDEL luminescence intensity and lactate dehydrogenase (LDH) activity in the culture medium accompanied by cytotoxicity show good agreement in toxicant‐treated ELuc‐ and GLuc‐KDEL‐expressing HepG2 stable cell lines. We have clarified that the increase of GLuc‐KDEL luminescence intensity in the culture medium reflects the type of cell death, including necrosis and late apoptosis, but not early apoptosis. We have also uncovered a strong correlation between GLuc‐KDEL luminescence intensity in the culture medium and the extracellular release of high mobility group box 1 (HMGB1), a representative damage‐associated molecular pattern (DAMP) molecule. The bioluminescence measurement assay using ELuc and GLuc‐KDEL developed in this study can simultaneously monitor cell viability and membrane damage, respectively, and the increase of GLuc‐KDEL luminescence intensity in the culture medium accompanied by the increase of cytotoxicity is an index of necrosis and late apoptosis associated with the extracellular release of DAMP molecules.     

Thermostable DNA helicase improves the sensitivity of digital PCR

DNA/RNA helicases, which catalyze the unwinding of duplex nucleic acids using the energy of ATP hydrolysis, contribute to various biological functions involving DNA or RNA. Euryarchaeota-specific helicase Tk-EshA (superfamily 2) from the hyperthermophilic archaeon Thermococcus kodakarensis has been used to decrease generation of mis-amplified products (noise DNAs) during PCR. In this study, we focused on another type (superfamily 1B) of helicase, Tk-Upf1 (TK0178) from T. kodakarensis, and compared its effectiveness in PCR and digital PCR with that of Tk-EshA. For this purpose, we obtained Tk-Upf1 as a recombinant protein and assessed its enzymatic characteristics. Among various double-stranded DNA (dsDNA) substrates (forked, 5′ overhung, 3′ overhung, and blunt-ended duplex), Tk-Upf1 had the highest unwinding activity toward 5′ overhung DNAs. Noise DNAs were also eliminated in the presence of Tk-Upf1 at concentrations 10-fold lower than those required to yield a comparable reduction with Tk-EshA. When a 5′ or 3′ overhung mis-annealed primer was included as a competitive primer along with specific primers, noise DNAs derived from the mis-annealed primer were eliminated in the presence of Tk-Upf1. In digital PCR, addition of Tk-EshA or Tk-Upf1 increased fluorescent intensities and improved separation between common and risk allele clusters, indicating that both helicases functioned as signal enhancers. In comparison with Tk-EshA, a smaller amount of Tk-Upf1 was required to improve the performance of digital PCR.     

Genome instability syndromes caused by impaired DNA repair and aberrant DNA damage responses

Maintenance of genome integrity is essential for all organisms because genome information regulates cell proliferation, growth arrest, and vital metabolic processes in cells, tissues, organs, and organisms. Because genomes are constantly exposed to intrinsic and extrinsic genotoxic stress, cellular DNA repair machinery and proper DNA damage responses (DDR) have evolved to quickly eliminate genotoxic DNA lesions, thus maintaining the genome integrity suitably. In human, germline mutations in genes involved not only in cellular DNA repair pathways but also in cellular DDR machinery frequently predispose hereditary diseases associated with chromosome aberrations. These genetic syndromes typically displaying mutations in DNA repair/DDR-related genes are often called “genome instability syndromes.” Common features of these hereditary syndromes include a high incidence of cancers and developmental abnormalities including short stature, microcephaly, and/or neurological deficiencies. However, precisely how impaired DNA repair and/or dysfunctional DDR pathologically promote(s) these syndromes are poorly understood. In this review article, we summarize the clinical symptoms of several representatives “genome instability syndromes” and propose the plausible pathogenesis thereof.     

2-aminopropionitrile polymer. I. The hydrolyzate of the basic fraction

The basic fraction of a 2-aminopropionitrile polymer was subjected to acid hydrolysis and was analyzed by means of GC-MS, after trimethylsilylation. The fundamental polymer structural units were alanine, 2,2-iminodipropionic acid, N-(1-cyanoethyl)alanine, N-ethylalanine, glycine, cyanoglycine, and 5-amino-4-carboxyimidazole residues. The last three units may be derived from hydrogen cyanide. Oligomeric combinations of these units were also detected in the hydrolyzate, due to partial hydrolysis of the polymer.     

Furosemide: a Specific Inhibitor of Pi Transport across the Plasma Membrane of Plant Cells

A search was made for inhibitors of P_i uptake that act directlyon the P_i transporter in the plasma membranes of Catharanthusroseus cells to inhibit P_i uptake without inhibition of protonpumping. Using standard electrodes, we monitored changes inpH and in the concentration of K⁺ ions, as well as the rateof P_i uptake, when an inhibitor to be tested was applied tothe cells in unbuffered medium. A9C (28 µM), a blockerof anion channels, inhibited P_i uptake but it also inhibitedthe proton pump. However, a structurally similar inhibitor,furosemide, inhibited P_i uptake without inhibiting proton pumping. It is suggested that the carboxylic group of these inhibitorsinteracts with the P_i-binding site (probably an amino group)of the P_i transporter in the plasma membrane and that the hydrophobicstructure of these inhibitors facilitates their accumulationin the plasma membrane. ³Present address: Department of Biology, Hitotsubashi University,2-1 Naka, Kunitachi, Tokyo, 186 Japan     

Characterization of Nontoxic-Nonhemagglutinin Component of the Two Types of Progenitor Toxin (M and L) Produced by Clostridium botulinum Type D CB-16

A 9.8-kbp DNA fragment which contained a neurotoxin gene and its upstream region was cloned from Clostridium botulinum type D strain CB-16. Nucleotide sequencing of the fragment revealed that genes encoding for hemagglutinin (HA) subcomponents and one for a nontoxic-nonhemagglutinin (NTNH) component were located upstream of the neurotoxin gene. This strain produced two toxins of different molecular size (approximately 300 kDa and 500 kDa) which were designated as progenitor toxins (M and L toxins). The molecular size of the NTNH component of L toxin was approximately 130 kDa on SDS-PAGE and its N-terminal amino acid sequence was M-D-I-N-D-D-L-N-I-N-S-P-V-D-N-K-N-V-V-I which agreed with that deduced from the nucleotide sequence. In contrast, the M toxin had a 115-kDa NTNH component whose N-terminal sequence was S-T-I-P-F-P-F-G-G-Y-R-E-T-N-Y-I-E, corresponding to the sequence from Ser141 of the deduced sequence. A 15-kDa fragment, which was found to be associated with an M toxin preparation, possessed the same N-terminal amino acid sequence as that of the 130-kDa NTNH component. Furthermore, five major fragments generated by limited proteolysis with V8 protease were shown to have N-terminal amino acid sequences identical to those deduced from the nucleotide sequence of 130-kDa NTNH. These results indicate that the 130-kDa NTNH of the L toxin is cleaved at a unique site, between Thr and Ser, leading to the 115-kDa NTNH of the M toxin.     

Acidification and Alkalinization of Culture Medium by Catharanthus roseus cellsIs Anoxic Production of Lactate a Cause of Cytoplasmic Acidification?

Catharanthus roseus(L.) G. Don cells acidified Mura-shige-Skoogmedium rapidly. Upon transfer to fresh medium, the medium pH(initially5.3) dropped below 4 within 2 d. This acidificationwas reversed under hypoxic conditions. The cells induced a similaracidification in a simple medium consisting of CaCl₂, KCl, andglucose: medium pH dropped below 4 within 6 h. The acidificationwas accompanied by an influx of K⁺ at a H⁺(efflux)/K⁺ ratioof ca 0.6 as well as by an expansion of endogenous organic acidpool, in which malic and citric acids were the major components.Anoxia reversed all these processes: the direction of both K⁺and H⁺ fluxes reversed with a H⁺/K⁺ ratio of 1.70. Anoxia induceda cytoplasmic acidification from pH 7.6 (aerobic) to 7.4 asmeasured by 31P-NMR, accompanied by a rapid, long-lasting lactateaccumulation at expense of malic and citric acids. Evidencesuggested that accumulation of lactic acid was not a cause ofcytoplasmic acidification under anoxia, but a result of pH regulationby the biochemical pH-stat [Davies (1973) Symp. Soc. Exp. Biol.27: 513]. The anoxic acidification of the cytoplasm was ascribedto the influx of H⁺ from the medium. (Received April 18, 1997; Accepted July 8, 1997)