Molecular cloning of a novel transmembrane protein MOLT expressed by mature oligodendrocytes

A novel oligodendrocyte (OL)-specific cDNA was isolated from brain capillary endothelial cells and characterized. The cDNA encodes a protein of 1099 amino acids that contains a signal peptide and a transmembrane domain. The protein was expressed in mature OLs in vivo and in vitro cell cultures and was thus designated as mature OL transmembrane protein (MOLT). RT-PCR analysis showed that MOLT mRNA was expressed in brain, lung, pancreas, and testis. A polyclonal antibody raised against a part of the mouse MOLT reacted specifically with multipolar OLs possessing radially oriented processes that penetrated into the gray matter. More cells were detected in the white matter, and these had longitudinally oriented processes. In a rat OL lineage culture system, oligodendrocyte precursor cells did not initially produce MOLT mRNA and protein, but when they begun to differentiate into mature OLs, they started expressing MOLT. Consequently, MOLT may function as OLs become mature and may serve as a cell-surface marker for OL differentiation.     

Purification and characterization of the recombinant Thermus sp. strain T2 alpha-galactosidase expressed in Escherichia coli

The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable alpha-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M(r), 53,514). The observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from Thermus brockianus was over 70%. Thermus sp. strain T2 alpha-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75 degrees C for p-nitrophenyl-alpha-D-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40 degrees C for 1 h. The enzyme acted on the terminal alpha-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea alpha-galactosidase I. The enzyme has only one Cys residue in the molecule. para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.     

Behavioral compensations in a positional learning and memory task by aged monkeys

The present experiment assessed learning and memory of a positional task by evaluating behavioral strategies as well as accuracy of a task in four young and four aged monkeys. They were tested in a delayed response (DR) task that has been widely used to study animal models of aging. The task consisted of two phases; an acquisition of the task and a positional memory test with five delay times (1-30 s). There was no clear difference between age groups in the number of trials needed for acquisition of the task. However, an analysis of behavior revealed differences in behavioral characteristics displayed during testing. The young monkeys showed various irrelevant behaviors during the execution of the task. In contrast, the aged monkeys consistently concentrated on the task exhibiting no behaviors irrelevant to the task. These results showed than the aged monkeys' performance was supported by a different behavioral strategy from the young monkeys. The results of the memory test were similar to those of the acquisition on the accuracy and the behavior. The aged monkeys depended on behavioral cues to preserve their positional memory, especially during the task. The present study suggests that cognitive impairments in aged monkeys can be compensated for by employing behavioral strategies.     

Epidemiological survey of Orientia tsutsugamushi distribution in field rodents in Saitama Prefecture, Japan, and discovery of a new type

There are various antigenic variants of Orientia tsutsugamushi which are distinguished by immunological and molecular genetic methods targeted at the antigenic diversity of 56-kDa type-specific antigen proteins. The present study was performed to analyze 15 strains successfully isolated from rodents in Saitama Prefecture, Japan, by 56-kDa gene sequence homologies, reactivities with type-specific monoclonal antibodies and polymerase chain reaction (PCR) using type-specific primer-pairs. We demonstrated the presence of a new type of O. tsutsugamushi among the isolates. This new type, designated as the Saitama type, was located in the branch of Karp type in the phylogenetic tree based on 56-kDa gene sequences, but distant from the known Karp types, such as Karp, JP-1 and JP-2, showing less than 90% homology. Strains of this type could not be distinguished by immunological methods from Karp type strains, but a new primer-pair for PCR which specifically amplifies the DNA of this new type strain was designed. This primer-pair may serve to find this strain type in future studies.     

Mechanistic study of the oxidation of caffeic acid by digital simulation of cyclic voltammograms

The oxidation mechanism of caffeic acid (CAF) has been studied by means of cyclic voltammetry with the plastic formed carbon or glassy carbon electrode. CAF gives a well-developed two-electron reversible wave in acidic media, whereas it shows an irreversible behavior, i.e., a decrease of the rereduction peak, in less acidic media, suggesting that the oxidation of CAF follows an irreversible chemical reaction(s). Digital simulation analyses based on different oxidation mechanisms have been performed for the voltammograms obtained with the GC electrode in 1:1 (v/v) water:ethanol solutions. The results clearly show that the seeming two-electron oxidation of CAF occurs stepwise via one-electron processes, each of which follows an irreversible chemical reaction. It has also been suggested that the semiquinone radical as an intermediate of the one-electron oxidation should play an important role in the oxidation reaction. Evaluations of the rate constants for the chemical reactions have further suggested that the chemical reactions are dimerization reactions.     

T cells of atomic bomb survivors respond poorly to stimulation by Staphylococcus aureus toxins in vitro: does this stem from their peripheral lymphocyte populations having a diminished naïve CD4 T-cell content?

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer na?ve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (na?ve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (na?ve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their na?ve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.     

Pupal commitment and its hormonal control in wing imaginal discs

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.     

On the role of periodism in the origin of proteins

Two different views have been proposed for origins of genes (or proteins). One is that primordial genes evolved from random sequences. This view underlies the concept of modern in vitro evolution experiments that functional molecules (even proteins) evolved from random sequence-libraries. On the contrary, the second view reminds that "random sequences" would be an unusual state in which to find RNA or DNA, because it is their inherent nature to yield periodic structures during the course of semi-conservative replication. In this second view, the periodicity of DNA (or RNA) is responsible for emergence of primordial genes. Although recent reports on the variety of periodicities present in proteins, genes and genomes are consistent with the second view, it has yet to be experimentally tested. We assessed the significance of periodicities of DNA in the origin of genes by constructing such periodic DNAs. The results showed that periodic DNA produced ordered proteins at very high rates, which is in contrast to the fact that proteins with random sequences lack secondary structures. We concluded that periodicity played a pivotal role in the origin of many genes. The observation should pave the way for new experimental evolution systems for proteins.     

The levels of mature glycosylated nicastrin are regulated and correlate with gamma-secretase processing of amyloid beta-precursor protein

Nicastrin, a type-I transmembrane glycoprotein, is a necessary component of the high molecular weight presenilin (PS) complexes that mediate intramembranous cleavage of beta-amyloid precursor protein (betaAPP) and Notch. Nicastrin undergoes trafficking-dependent glycosylation maturation, and PS1 interacts preferentially with these maturely glycosylated forms of nicastrin. We investigated the effects of differing levels of the immature and mature endoglycosidase-H-resistant forms of nicastrin on Abeta40- and Abeta42-peptide secretion in several cell lines stably expressing a mutant nicastrin (D336A/Y337A) that increases Abeta secretion. There was no correlation between Abeta secretion and the level of over-expression of the immature forms of nicastrin. The total level of mature nicastrin remained constant, but mutant nicastrin replaced endogenous mature nicastrin in varying degrees. Differences in the levels of mature mutant nicastrin positively correlated with Abeta secretion, but did not influence either betaAPP trafficking or processing by alpha- and beta-secretases. Proper trafficking and terminal maturation of nicastrin is therefore a necessary event for the regulated intramembranous proteolysis of betaAPP.