In vitro neurotoxicity of amyloid β-peptide cross-linked by transglutaminase

Transglutaminase catalyzes the intermolecular cross-linking of peptides between Gln and Lys residues, forming an -(-glutamyl) lysine bond. Amyloid -peptide, a major constituent of the deposits in Alzheimer disease, contains Lys16, Lys28, and Gln15 which may act as substrates of transglutaminase. Transglutaminase treatment of amyloid -peptide (1–28) and amyloid -peptide (1–40) yielded cross-linked oligomers. Transglutaminase-treated A retarded neurite extension of PC12 cells, and rat cultured neurons of hippocampus and septum, brain areas severely affected by Alzheimer disease, and subsequently caused cell death, whereas the transglutaminase-untreated counterparts did not show harmful effects. The transglutaminase-catalyzed oligomers of amyloid -peptide and their neurotoxicity may be involved in two characteristics in Alzheimer disease, neuronal degeneration and formation of the insoluble deposits.Abbreviations: AD – Alzheimer disease, A – amyloid -peptide, DMEM – Dulbecco's modified Eagle's medium, DMEM/F–12–1:1 mixture of DMEM and Ham's F–12 medium, FCS – fetal calf serum, HS – horse serum, PAGE – polyacrylamide gel electrophoresis, MTT – 3-(4,5-dimethylthiazol–2-yl)–2,5-diphenyltetrazolium bromide, NGF – nerve growth factor, TGase – transglutaminase.     

Does endothelin-1 participate in the exercise-induced changes of blood flow distribution of muscles in humans?

Maeda, Seiji, Takashi Miyauchi, Michiko Sakane, MakotoSaito, Shinichi Maki, Katsutoshi Goto, and Mitsuo Matsuda. Does endothelin-1 participate in the exercise-induced changes of blood flowdistribution of muscles in humans? J. Appl.Physiol. 82(4): 1107-1111, 1997.Endothelin-1(ET-1) is an endothelium-derived potent vasoconstrictor peptide thatpotentiates contractions to norepinephrine in human vessels. Wepreviously reported that the circulating plasma concentration of ET-1is significantly increased after exercise (S. Maeda, T. Miyauchi, K. Goto, and M. Matsuda. J. Appl.Physiol. 77: 1399-1402, 1994). Tostudy the roles of ET-1 during and after exercise, we investigatedwhether endurance exercise affects the production of ET-1 in thecirculation of working muscles and nonworking muscles. Male athletesperformed one-leg cycle ergometer exercise of 30-min duration atintensity of 110% of their individual ventilatory threshold. Plasmaconcentrations of ET-1 in both sides of femoral veins (veins in theworking leg and nonworking leg) and in the femoral artery (artery inthe nonworking leg) were measured before and afterexercise. The plasma ET-1 concentration in the femoralvein in the nonworking leg was significantly increased after exercise,whereas that in femoral vein in the working leg was not changed. Thearteriovenous difference in ET-1 concentration was significantlyincreased after exercise in the circulation of the nonworking leg butnot of the working leg, which suggests that the production of ET-1 wasincreased in the circulation of the nonworking leg by exercise. Thepresent study also demonstrated that the plasma norepinephrineconcentrations were elevated by exercise in the femoral veins of boththe working and nonworking legs, suggesting that the sympathetic nerveactivity was augmented in both legs during exercise. Therefore, thepresent study demonstrates the possibility that the increase inproduction of ET-1 in nonworking muscles may cause vasoconstriction andhence decrease blood flow in nonworking muscles through its directvasoconstrictive action or through an indirect effect of ET-1 toenhance vasoconstrictions to norepinephrine and that these responsesmay be helpful in increasing blood flow in workingmuscles. We propose that endogenous ET-1 contributes tothe exercise-induced redistribution of blood flow in muscles.

     

Identification of Influenza C Virus Phosphoproteins

The HMV-II cells infected with influenza C virus were labeled with inorganic [³²P]phosphate to identify phosphorylated proteins. Analysis by radioimmunoprecipitation with antiviral serum or monoclonal antibodies revealed that three major structural proteins of the virus, hemagglutinin-esterase (HE), nucleoprotein (NP), and matrix protein (M1) are all phosphorylated in both infected cells and virions. It was also observed that, in the presence of trypsin (10 μg/ml), the unphosphorylated form of the HE glycoprotein was cleaved efficiently whereas the phosphorylated form was not, raising the possibility that phosphorylation of HE may influence its susceptibility to degradation by proteolytic enzymes.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Sulphides and furanones from steam volatile oils of Allium fistulosum and Allium chinense

The constituents of the steam volatile oils from two kinds of Allium fistulosum, A. fistulosum var. caespitosum and A. chinense, have been investigated by GC and spectral techniques (IR, UV, GC/MS, ¹H NMR and ¹³C NMR). The compounds identified from the neutral fraction of each volatile oil included sulphides, thiolanes, alcohols, aldehydes, ketones, furanones and others. Among the sulphur compounds, dipropyl disulphide comprised ca 28% of A. fistulosum oil, ca 23% of A. fistulosum var. caespitosum oil and ca 30% of A. chinense oil. A. fistulosum oil was characterized by a large quantity of tridecan-2-one (ca 52%) and 2,3-dihydro-2-octyl-5-methylfuran-3-one (ca 16%). Also, a large amount of 2,3-dihydro-2-hexyl-5-methylfuran-3-one (ca 20%) was isolated from A. chinense oil.     

43Ca NMR studies of Ca2+-Tetrahymena calmodulin complexes

⁴³Ca NMR spectra of Ca²⁺-Tetrahymena calmodulin(Tet. CaM.) complexes have been observed under various conditions. Off-rate of Ca²⁺ from Tet. CaM. is estimated to be approx. 2.7 × 10³ s^?1 under a certain assumption. Relaxation rates of ⁴³Ca NMR of Ca²⁺-Tet. CaM. are remarkably increased(by one order in magnitude) by adding trifluoperazine(TFP), a potent calmodulin antagonist. Relaxation parameters estimated suggest that Ca²⁺ mobility is reduced by the TFP binding. A stoichiometry of TFP is two moles per Tet. CaM. molecule. The relaxation rates of ⁴³Ca NMR signals are increased by adding excessive Mg²⁺ to the Ca²⁺-Tet. CaM. solutions. The addition of Mg²⁺ to the Ca²⁺-Tet. CaM. complex decreases apparent pKa value of the complex as well.