Yoshihiko Uratani

INTRACORTICAL REGIONALITY REPRESENTED BY SPECIFIC TRANSCRIPTION FOR A NOVEL PROTEIN, LATEXIN

The monoclonal antibody (mAb) PC3.1 recognizes a subset of neurons distributed in the infragranular layers of the lateral neocortex of the rat. Immunoaffinity chromatography with mAb PC3.1 showed that this antibody specifically binds a peptide epitope on a 29 kDa protein named latexin. To study the molecular details of the protein, we isolated four independent cDNA clones for latexin from cDNA libraries of the rat cerebral cortex and whole brain using the amino acid sequences of latexin fragments. Analysis of these cDNA clones showed that the predicted primary structure of latexin consists of 223 amino acids, and has no strict homology to any sequences so far known. Western and Northern blots demonstrated that the latexin and its mRNA were expressed predominantly in neural tissues with some expression in non‐neural tissues. The gene that encodes latexin in the rat appeared to have homologues in other mammalian species and in the chick. In situ hybridization showed that latexin mRNA is synthesized in a subset of neurons in the lateral but not the dorsal neocortex, and that the distribution profile of these neurons is quite similar to that of neurons expressing latexin. These results indicate that latexin is a novel class of neuronal protein which represents intracortical regionality, and suggest that the regional specification of the neocortex involves selective parcellation of neurons which express a particular gene.

Latexin, a carboxypeptidase A inhibitor, is expressed in rat peritoneal mast cells and is associated with granular structures distinct from secretory granules and lysosomes.

Latexin, a protein possessing inhibitory activity against rat carboxypeptidase A1 (CPA1) and CPA2, is expressed in a neuronal subset in the cerebral cortex and cells in other neural and non-neural tissues of rat. Although latexin also inhibits mast-cell CPA (MCCPA), the expression of latexin in rat mast cells has not previously been confirmed. In the present study we examined the expression and subcellular localization of latexin in rat peritoneal mast cells. Western blot and reverse-transcriptase-mediated PCR analyses showed that latexin was contained and expressed in the rat peritoneal mast cells. Immunocytochemically, latexin immunofluorescence was localized on granular structures distinct from MCCPA-, histamine- or cathepsin D-immunopositive granules. Immunoelectron microscopy revealed that latexin was associated with a minority population of granules. The latexin-associated granules were separated from MCCPA- or histamine-containing granules on a self-generating density gradient of polyvinylpyrrolidone-coated silica-gel particles (Percoll). Treatments with high ionic strength and heparinase released latexin from the granules, suggesting that latexin is non-covalently associated with a heparin-like component of the granules. MCCPA and histamine were released from the mast cells after non-immunological and immunological stimulation with compound 48/80, A23187 and anti-IgE antibody, whereas latexin was not released. These results show that latexin is synthesized in rat peritoneal mast cells and suggest that it is associated with a unique type of intracellular granules distinct from MCCPA- and histamine-containing secretory granules and lysosomes.

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

SummaryThe gene braB, encoding the Na–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated Mr of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.

Latexin expression in smaller diameter primary sensory neurons in the rat.

Most of the smaller diameter neurons of dorsal root and trigeminal ganglia in adult rats expressed latexin, which has the inhibitor activity of carboxypeptidase A. Most of the dorsal root ganglion (DRG) neurons containing either calcitonin gene-related peptide (CGRP), substance P (SP) or somatostatin (SST) coexpressed latexin. Latexin was widely distributed in the cytoplasm of the cell body and in axonal fibers of cultured DRG neurons which were sensitive to capsaicin. In addition, latexin-immunoreactivity was observed throughout lamina II of the spinal cord in normal animals, but was lost following sciatic nerve-axotomy, suggesting the presence of latexin-immunoreactive axonal fibers and/or terminals from DRG neurons. Immunoelectron microscopy indeed revealed latexin-immunoreactive axonal terminals and thinly myelinated and unmyelinated axonal fibers within the dorsal horn. These observations suggest that latexin may be involved in nociceptive information transmission or its modulation.

Na+(Li+)/Branched-chain amino acid cotransport inPseudomonas aeruginosa

SummaryA transport system for branched-chain amino acids (designated as LIV-II system) inPseudomonas aeruginosa requires Na+ for its operation. Coupling cation for this system was identified by measuring cation movement during substrate entry using cation-selective electrodes. Uptakes of Na+ and Li− were induced by the imposition of an inwardly-directed concentration gradient of leucine, isoleucine, or valine. No uptake of H− was found, however, under the same conditions. In addition, effects of Na+ and Li+ on the kinetic property of the system were examined. At chloride salt concentration of 2.5mm, values of apparentKm andVmax for leucine uptake were larger in the presence of Na+ than Li+. These results indicate that the LIV-II transport system is a Na+(Li+)/substrate cotransport system, although effects of Na+ and Li+ on kinetics of the system are different.

Conformation of antithrombin iii with defective biological functions derived from a thrombophilic patient

Antithrombin III (AT III) with defective biological functions and altered antigenicity has been found in a thrombophilic patient. Circular dichroism (CD) spectra of antithrombin III purified from the patients plasma were measured in near and far-ultraviolet (UV) wavelength regions. The far-UV CD spectrum of the patients AT III was similar at room temperature to that of normal AT III derived from healthy adult males. Mean residue ellipticity at 221 nm of the patients AT III, however, decreased its magnitude by 6% gradually as temperature increased up to 74 degrees C, whereas that of the normal AT III reduced its magnitude sharply at around 63-64 degrees C and by 18%. The near-UV CD spectrum of patients AT III was different from that of normal AT III. These results indicate that the secondary structure of patients AT III is similar to that of normal AT III at room temperature, while local conformation of aromatic amino acid residues in the abnormal protein is different from that in the normal one and that the secondary structure of the patients AT III is heat-stable, while that of the normal one is heat-labile.

A circular dichroism study of sheath contraction in pyocin R1

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, is a protein particle shaped like a bacteriophage tail composed of a contractile sheath, core, baseplate and tail fibers. Alkaline treatment with sodium carbonate caused sheath contraction without considerable disassembly of other components. Circular dichroism (CD) spectra of pyocin R1 before and after the treatment, and of isolated sheath, were measured in wavelength regions around 220 and 290 nm at neutral pH. The alkaline treatment caused a red shift of the minimum from 208 nm to 212 nm. A marked difference in the CD spectrum was found in the near-ultraviolet region. THe difference is considered to be mainly due to a CD spectra change of tryptophan residues in the sheath subunits.

Phospholipids stabilize the secondary structure of the sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa

For functional reconstitution of bacterial cotransporters (carriers or permeases) including the sodium-coupled branched-chain amino acid carrier (LIV-II carrier) of Pseudomonas aeruginosa, the presence of phospholipid is required through the process of solubilization and purification of the transporters from the bacterial membranes, suggesting the possibility that phospholipid may stabilize the structure of the cotransporter proteins to be in a functional form. In this study, this possibility was examined by studying the effect of denaturant on the secondary structure of the LIV-II carrier purified in the absence and presence of phospholipid using circular dichroism (CD) spectroscopy. CD spectra of the purified LIV-II carrier solubilized in n-octyl-beta-D-glucopyranoside (OG), OG/dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) mixture, and dispersed into DOPE/DOPG small unilamellar vesicles were measured in the absence of denaturant. The three spectra were very similar and had a trough at 222 nm with mean residue molar ellipticity of -23000 deg.cm(2)/dmol and a shoulder at 208 nm. CD spectral analyses with three different methods (S.W. Provencher, J. Glöckner, Estimation of globular protein secondary structure from circular dichroism, Biochemistry 20 (1981) 33-37; J.Y. Yang, C.-S.C. Wu, H.Z. Martinez, Calculation of protein conformation from circular dichroism, Methods Enzymol. 130 (1986) 208-269; N. Sreerama, R.W. Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal. Biochem. 209 (1993) 32-44) revealed that the LIV-II carrier solubilized in OG/DOPE/DOPG mixture contained 69-75% alpha-helix and 0-9% beta-sheet. Addition of 6 M guanidine hydrochloride decreased 48% of the amplitude at 222 nm of the CD spectrum of the carrier solubilized in OG alone and 9-14% of the CD amplitude of the carrier solubilized in OG/DOPE/DOPG or OG/dioleoylphosphatidylcholine mixture and dispersed in liposomes composed of DOPE/DOPG. These results show that the ordered secondary structure of the LIV-II carrier is partially unfolded in OG without phospholipid by denaturant but is greatly stabilized with phospholipids with oleoyl chains independently of their polar head group composition and suggest that the alpha-helical structure of the carrier is mainly embedded in the lipid environment.

Colocalization of latexin with carboxypeptidase A in the infragranular neurons of cerebral cortex

Latexin is a carboxypeptidase A (CPA) inhibitor expressed in certain cortico-cortical projection neurons located in the infragranular layers of lateral cortical areas of adult rats and mice. To understand a physiological function of latexin in the cerebral cortex, we previously analyzed colocalization of latexin with protein phosphatase inhibitor-l and with Al-opioid receptor (K. Takiguchi-Hayashi & Y. Arimatsu, Neurosci. Res. Suppl. 21. S44, 1997). In the present study, we examined colocalization of latexin with CPA by immunohistochemistry. Restricted and similar distribution profiles were observed within the cerebral cortex for latexinand CPA-immunopositive neurons. In double immunofluorescence experiments in some cortical areas, it was shown that all of latexin-immunopositive neurons were also CPAimmunopositive. These observations suggest that latexin play a role together with CPA in the cortico-cortical circuit.