Influence of surface anesthesia on the pressure pain threshold measured with different-sized probes

Transcutaneous pressure with pressure probes of arbitrary diameters have been commonly used for measuring the threshold and magnitude of muscle pain, yet this procedure lacks scientific validation. To examine the valid probe dimensions, we conducted physiological experiments using 34 human subjects. Pin-prick pain, pressure pain threshold (PPT) to pressure probes of various diameters, heat pain threshold, and electrical pain threshold of deep tissues were measured before and after application of surface lidocaine anesthesia to the skin surface over the brachioradial muscle in a double-blinded manner. The anesthesia neither affected PPT with larger probes (diameters: 1.6 and 15?mm) nor increased electric pain threshold of deep structures, whereas it diminished pain count in pin-prick test and PPT with a 1.0?mm diameter probe, suggesting that mechanical pain thresholds measured with 1.6 and 15?mm probes reflect the pain threshold of deep tissues, possibly muscle. Pain thresholds to heat did not change after application of the anesthesia. These results suggest that larger pressure probes can give a better estimation of muscular pain threshold.     

Isolation of a cDNA clone encoding mouse 3-hydroxyacyl CoA dehydrogenase

Rae-38, a cDNA clone isolated from mouse embryonal carcinoma F9 cells, was sequenced, and the deduced RAE-38 protein showed about 86% homology to pig 3-hydroxyacyl CoA dehydrogenase (HCDH; EC 1.1.1.35). This clone can be used to elucidate the regulatory mechanism of HCDH gene expression in mammals.     

Experimental Approach Reveals the Role of alx1 in the Evolution of the Echinoderm Larval Skeleton

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.     

Vascular Ehlers-Danlos Syndrome Mutations in Type III Collagen Differently Stall the Triple Helical Folding

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)₇ with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)₇-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.     

A Novel Acylaminoimidazole Derivative,WN1316, Alleviates Disease Progression via Suppression of Glial Inflammation in ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron degenerative disease. Given that oxidative stress and resulting chronic neuronal inflammation are thought to be central pathogenic, anti-oxidative agents and modulators of neuronal inflammation could be potential therapies for ALS. We report here that the novel small molecular compound, 2-[mesityl(methyl)amino]-N-[4-(pyridin-2-yl)-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316) selectively suppresses oxidative stress-induced cell death and neuronal inflammation in the late-stage ALS mice. WN1316 has high blood-brain-barrier permeability and water solubility, and boosts both neuronal apoptosis inhibitory protein (NAIP) and NF-E2-related factor 2 (Nrf2) which governed glutathione (GSH)-related anti-oxidation pathway protecting motor neurons against oxidative injuries. Post-onset oral administration of low dose (1–100 µg/kg/day) WN1316 in ALS(SOD1^H46R) and ALS(SOD1^G93A) mice resulted in sustained improved motor function and post onset survival rate. Immunohistochemical analysis revealed less DNA oxidative damage and motor neuronal inflammation as well as repression of both microgliosis and astrocytosis, concomitant down regulation of interleukin-1β and inducible nitric oxide synthase, and preservation of the motoneurons in anterior horn of lumbar spinal cord and skeletal muscle (quadriceps femoris). Thus, WN1316 would be a novel therapeutic agent for ALS.     

Direct molecular interaction of caveolin-3 with KCa1.1 channel in living HEK293 cell expression system

Caveolin family is supposed to be essential molecules for the formation of not only caveola structure on cell membrane but also functional molecular complexes in them with direct and/or indirect interaction with other membrane and/or submembrane associated proteins. The direct coupling of caveolin-1 (cav1) with large conductance Ca²⁺-activated K⁺ channel, KCa1.1 has been established in several types of cells and in expression system as well. The possible interaction of caveolin-3 (cav3), which shows expression in some differential tissues from cav1, with KCa1.1 remains to be determined. In the present study, the density of KCa1.1 current expressed in HEK293 cells was significantly reduced by the co-expression of cav3, as well as cav1. The co-localization and direct interaction of GFP- or CFP-labeled cav3 (GFP/CFP-cav3) with YFP- or mCherry-labeled KCa1.1 (KCa1.1-YFP/mCherry) were clearly demonstrated by single molecular image analyses using total internal reflection fluorescence (TIRF) microscopy and fluorescence resonance energy transfer (FRET) analyses with acceptor photobleaching method. The deletion of suggested cav1-binding motif in C terminus region of KCa1.1 (KCa1.1ΔCB-YFP) resulted in the marked decrease in cell surface expression, co-localization and FRET efficiency with CFP-cav3 and CFP-cav1. The FLAG-KCa1.1 co-immunoprecipitation with GFP-cav3 or GFP-cav1 also supported their direct molecular interaction. These results strongly suggest that cav3 possesses direct interaction with KCa1.1, presumably at the same domain for cav1 binding. This interaction regulates KCa1.1 expression to cell surface and the formation of functional molecular complex in caveolae in living cells.     

Origin and transport of the A-I and arginine-rich apolipoproteins in mesenteric lymph of rats.

Transport of apolipoprotein A-I and argininerich apolipoprotein in mesenteric lymph was examined in rats given constant intraduodenal infusions of saline, glucose in saline, or emulsified fat. Lymph flow in all groups was constant from 5 to 50 hr after beginning the infusions. Lymphatic transport of triglycerides was about 20-fold greater and transport of apoprotein A-I was about twofold greater in fat-infused rats than in the other two groups. In each group transport of apoprotein A-I bore a significant positive relationship to transport of triglycerides. Lymphatic transport of the arginine-rich apoprotein was only 6-12% of that of apoprotein A-I and was more closely related to lymphatic transport of total protein than to that of triglycerides. In fat-infused rats given [(3)H]lysine intraduodenally, about two-thirds of the (3)H in the chylomicron proteins was in apoprotein A-I and only about 1% was in the arginine-rich apoprotein. Estimated specific activity of chylomicron proteins was highest for apoprotein A-I and apoprotein A-IV, and lowest for the arginine-rich apoprotein and proteins of low molecular weight (mainly C apoproteins). In fat-infused rats given constant intravenous infusions of radioiodinated high density lipoproteins from blood plasma, the specific activity of apoprotein A-I in lymph chylomicrons was only about 5% of that of apoprotein A-I in blood high density lipoproteins, indicating that more than 90% of the apoprotein A-I in chylomicrons was synthesized in the intestine. From these and other data it is concluded that both the intestine and liver are significant sources of apoprotein A-I whereas only the liver synthesizes significant amounts of the arginine-rich apoprotein.     

On the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. The wavelength dependence of observed oxygen equilibrium parameter values

Spectral changes of oxyhemoglobin induced by such anions as 2,3-diphosphoglycerate, inositol hexaphosphate, and Cl- may affect the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. Therefore, the anion-induced difference spectra were extensively measured under a variety of conditions and accurate oxygen equilibrium curves were determined under representative conditions with detection at different wavelengths selected from peaks, troughs, and zero difference points of the difference spectra in the visible and Soret regions. Oxygen equilibrium parameters including the four Adair constants (i.e., equilibrium constants for four steps of oxygenation) estimated from the equilibrium curves did not show any dependence on wavelength within the limits of experimental error. These results indicate that anion-induced spectral changes do not invalidate the spectrophotometric determination of oxygen saturation and confirm the validity of the previous conclusions drawn in our series of studies on the effects of anions, pH and temperature on oxygen equilibrium parameters.     

Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes)

Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.