Simultaneous determination of catechols in thalamic slices with liquid chromatography/electrochemistry

A method was developed for the simultaneous determination of dopamine (DA), epinephrine (E), norepinephrine (NE), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylglycol (MHPG), as well as L-3,4-dihydroxyphenylalanine (L-DOPA) with liquid chromatography (LC) using electrochemical (EC) detection. With a ODS column and a mobile phase consisting of a sodium acetate-citrate with heptasulfonic acid, this method was applied on simultaneous determination of catechols released from thalamic slices of ddY mouse. The pretreatment of the bathing medium required only centrifugation, and the supernatant was injected directly into the LCEC system. The high potassium stimulation of catecholaminergically innervated thalamic slices led to increase in the levels of DA, NE, DOPAC and MHPG, especially of NE, but not that of L-DOPA itself. In the present study, we designed to make simultaneous determination of catechols released from thalamic slices for estimation of the physiological status of catecholaminergic neuronal activity.     

Analysis of insertions and extensions in the functional evolution of the ribonucleotide reductase family

Ribonucleotide reductases (RNRs) are used by all free‐living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical‐based mechanism for nucleotide reduction. In this work, we expand on our recent phylogenetic inference of the entire RNR family and describe the evolutionarily relatedness of insertions and extensions around the structurally homologous catalytic barrel. Using evo‐velocity and sequence similarity network (SSN) analyses, we show that the N‐terminal regulatory motif known as the ATP‐cone domain was likely inherited from an ancestral RNR. By combining SSN analysis with AlphaFold2 predictions, we also show that the C‐terminal extensions of class II RNRs can contain folded domains that share homology with an Fe‐S cluster assembly protein. Finally, using sequence analysis and AlphaFold2, we show that the sequence motif of a catalytically essential insertion known as the finger loop is tightly coupled to the catalytic mechanism. Based on these results, we propose an evolutionary model for the diversification of the RNR family.     

Characterization of proteins expressed abundantly in the fruit-body ofFlammulina velutipes

Chronological changes of protein expression in the vegetative mycelium ofFlammulina velutipes and expression of these proteins in the fruit-body were investigated by two-dimensional polyacrylamide gel electrophoresis. Four proteins (FBA 1-4) expressed abundantly in the fruit-body were found to have different expression patterns in the vegetative mycelium after the fruiting treatment. FBA 1-4 had similar amino acid sequences and displayed a high similarity with the deduced amino acid sequence of theC1 cDNA, which has an Arg-Gly-Asp (RGD) cell-attachment sequence. This suggests that FBA 1-4 may have cell-to-cell attachment activity.     

Fossil Flowers and Associated Plant Fossils from the Kamikitaba Locality (Ashizawa Formation, Futaba Group, Lower Coniacian, Upper Cretaceous) of Northeast Japan

in situ spores, and megaspores also document the presence of Selaginellaceae and Schizaeaceae. Received 8 February 1999/ Accepted in revised form 7 April 1999     

Isolation and characterization of novel neutral glycolipids from Thermoplasma acidophilum.

Several novel neutral glycolipids (GL-1a, GL-1b, GL-2a, GL-2b and GL-2c) were isolated from Thermoplasma acidophilum by high-performance liquid chromatography using phenylboronic acid-silica and preparative thin-layer chromatography. The tentative structures of these lipids were characterized by the combination of gas-liquid chromatography, the methylation procedure, and (1)H-NMR and FAB-mass spectrometries. The lipophilic portion of the neutral glycolipids was composed of a simple molecular species named caldarchaeol (dibiphytanyl-diglycerol tetraether). The sugar moieties of these glycolipids were composed of gulose and glucose which formed monosaccharide residues on one side or both sides of the core lipids. Gulose was attached to the terminal glycerol OH group of the core lipid with a beta-configuration and glucose being attached with an alpha-configuration. The proposed structure of GL-1a was gulosylcaldarchaeol and that of GL-1b was glucosylcaldarchaeol. The structures of GL-2a, GL-2b, and GL-2c were the analogs of the caldarchaeol derivatives attached by a variety of gulosyl residues or glucosyl residues on both sides of the terminal OH groups.     

SHC-alpha5beta1 integrin interactions regulate breast cancer cell adhesion and motility.

The oncogenic SHC proteins are signaling substrates for most receptor and cytoplasmic tyrosine kinases (TKs) and have been implicated in cellular growth, transformation, and differentiation. In tumor cells overexpressing TKs, the levels of tyrosine phosphorylated SHC are chronically elevated. The significance of amplified SHC signaling in breast tumorigenesis and metastasis remains unknown. Here we demonstrate that seven- to ninefold overexpression of SHC significantly altered interactions of cells with fibronectin (FN). Specifically, in human breast cancer cells overexpressing SHC (MCF-7/SHC) the association of SHC with alpha5beta1 integrin (FN receptor) was increased, spreading on FN was accelerated, and basal growth on FN was reduced. These effects coincided with an early decline of adhesion-dependent MAP kinase activity. Basal motility of MCF-7/SHC cells on FN was inhibited relative to that in several cell lines with normal SHC levels. However, when EGF or IGF-I was used as the chemoattractant, the locomotion of MCF-7/SHC cells was greatly (approx fivefold) stimulated, while it was only minimally altered in the control cells. These data suggest that SHC is a mediator of the dynamic regulation of cell adhesion and motility on FN in breast cancer cells.     

Immunological identification of an inward rectifier K+ channel (Kir4.1) in the intermediate cell (melanocyte) of the cochlear stria vascularis of gerbils and rats

The cochlear stria vascularis produces the positive endocochlear potential (EP) and the endolymph. Both the EP and the endolymph are essential for the physiological function of hair cells. The intermediate cell is one of several cell types constituting the stria vascularis. It is known that inward rectifier K+ channels can play a constitutive role in the determination of the resting membrane potential. Localization of a member of the inward rectifier K+ channel family, Kir4.1, in the stria vascularis of gerbils and rats was investigated by immunological methods. A polyclonal antibody specific to the C-terminus of the rat Kir4.1 channel was raised in rabbits. Immunostaining of dissociated cells revealed that the Kir4.1 channel was localized to the intermediate cell, but not to the epithelial marginal cell. Subcellular localization of the Kir4.1 channel to the plasma membrane of the intermediate cell was confirmed by immunoelectron microscopy. Immunostaining of whole-tissue preparations revealed a network-like structure composed of intermediate cells. It seems likely that the Kir4.1 channel mediates the inwardly rectifying K+ current in the intermediate cell as shown previously by electrophysiological methods, and that this channel plays key roles in the production of the EP and K+ transport in the stria vascularis.     

A relationship between diffusional transport in lipid membranes and a lipophilic-eutectic parameter

A theoretical model has been developed to relate passive diffusional transport with a parameter termed the lipophilic-eutectic coefficient, Le. Based on this parameter it is proposed that the lipophilicity of a substance can be estimated. The method is based on the melting point lowering of a pure substance in the presence of an impurity of similar structure. Cholesterol was chosen as a model biological lipid because it is a neutral lipid that is found in relatively high concentrations in a variety of membranes. Transdermal absorption in humans and intestinal absorption in the rat both show a high degree of correlation with respect to Le. For our studies, Le appears to be as satisfactory as the partition coefficient for estimating lipophilicity and its determination is less demanding analytically.     

Immunohistochemical study of TAFII250 in the rat laryngeal nervous system

The cause of spasmodic dysphonia, a dystonic disorder of the larynx, remains unclear. Recently, TAFII250, TATA-box binding protein associated factor, was suggested to be involved in dystonia parkinsonism. There is a possibility that TAFII250 is involved in spasmodic dysphonia, but little information is available about the expression of TAFII250 in the laryngeal nervous system. In this study, we investigated the localization of TAFII250 protein in the rat laryngeal nervous system by immunohistochemistry. TAFII250-immunoreactivity was detected in the nodose ganglion and superior cervical ganglion. In these nuclei, TAFII250 was localized in the nucleus of NeuroTrace-positive neurons but not in GFAP-positive glial cells. No positive cells were detected in the motor and parasympathetic nervous system. TAFII250-immunoreactivity was sustained between 3 and 7 days after vagotomy, but at 14 days expression was down-regulated in the distal part of the nodose ganglion. These findings suggest that TAFII250 plays an important role in the laryngeal innervation of the sensory and sympathetic nervous systems.     

Detailed analysis of Japanese population substructure with a focus on the southwest islands of Japan

Uncovering population structure is important for properly conducting association studies and for examining the demographic history of a population. Here, we examined the Japanese population substructure using data from the Japan Multi-Institutional Collaborative Cohort (J-MICC), which covers all but the northern region of Japan. Using 222 autosomal loci from 4502 subjects, we investigated population substructure by estimating F(ST) among populations, testing population differentiation, and performing principal component analysis (PCA) and correspondence analysis (CA). All analyses revealed a low but significant differentiation between the Amami Islanders and the mainland Japanese population. Furthermore, we examined the genetic differentiation between the mainland population, Amami Islanders and Okinawa Islanders using six loci included in both the Pan-Asian SNP (PASNP) consortium data and the J-MICC data. This analysis revealed that the Amami and Okinawa Islanders were differentiated from the mainland population. In conclusion, we revealed a low but significant level of genetic differentiation between the mainland population and populations in or to the south of the Amami Islands, although genetic variation between both populations might be clinal. Therefore, the possibility of population stratification must be considered when enrolling the islander population of this area, such as in the J-MICC study.