N-linked oligosaccharide chains of Sendai virus fusion protein determine the interaction with endoplasmic reticulum molecular chaperones

The selectivity and individual roles of the N-linked oligosaccharide chains of Sendai virus fusion protein (F protein) in the interaction with endoplasmic reticulum molecular chaperones were investigated by analyses of transient expression of single N-glycosylation mutants and sequential immunoprecipitation. We demonstrated differential interactions depending on the location of the N-linked oligosaccharide chain, and showed that these interactions were correlated with the folding and transport of F proteins. Moreover, mutant F proteins that lacked the specific N-linked oligosaccharide chains required for disulfide bond formation showed increased association with ERp57.     

Tim50 is a subunit of the TIM23 complex that links protein translocation across the outer and inner mitochondrial membranes

Based on the results of site-specific photocrosslinking of translocation intermediates, we have identified Tim50, a component of the yeast TIM23 import machinery, which mediates translocation of presequence-containing proteins across the mitochondrial inner membrane. Tim50 is anchored to the inner mitochondrial membrane, exposing the C-terminal domain to the intermembrane space. Tim50 interacts with the N-terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti-Tim50 antibodies block the protein translocation across the inner membrane. A translocation intermediate accumulated at the TOM complex is crosslinked to Tim50. We suggest that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the TIM23 complex     

Chemical modification of amine groups on PS II protein(s) retards photoassembly of the photosynthetic water-oxidizing complex

Four Mn atoms function as catalysts in the water-oxidizing complex located on the oxidizing side of PS II. We have studied the involvement of amine groups of the PS II proteins in photoligation of Mn2+ to the apo water-oxidizing complex, using the combined techniques of photoactivation and chemical modification with the modifiers methyl acetimidate (MAI), acetic acid N-hydroxysuccinimide ester (NHS), and 2,4,6-trinitrobenzenesulfonic acid (TNBS). Chemical modification of hydroxylamine-treated PS II core complexes decreased their capacity for restoration of oxygen evolution and photoligation of Mn2+ to the apo water-oxidizing complex (WOC), but did not affect their electron transfer activity in the vicinity of PS II. The number of functional high-affinity Mn-binding sites, but not of low-affinity sites, was significantly modulated by chemical modification. Kinetic analysis of photoactivation with the repetitive flashes revealed that the intermediate generated during a photoactivation process was destabilized by the chemical modification. To identify which proteins possess the amine groups involved in ligation of functional Mn, we examined the difference in NHS biotinylation between PS II core complexes with and without the Mn cluster. NHS biotinylation resulting in altered ligation of functional Mn apparently occurred on three proteins: an antenna chlorophyll binding protein (CP47), a light-harvesting chlorophyll protein (CP29), and another chlorophyll binding protein (PS II-S). Of these proteins, only the Mn-dependent biotinylation of CP47 was found to occur independently of the application of an NHS-masking concentration before removal of the functional Mn. These results suggest that lysyl residues of CP47, and perhaps also CP29 and PS II-S, function in direct photoligation of Mn2+ to the apo WOC.     

Identification of thermoacidophilic bacteria and a new Alicyclobacillus genomic species isolated from acidic environments in Japan

Sixty strains of thermoacidophilic bacteria have been isolated from soil and water samples obtained from various acidic environments in Japan. An initial comparative sequence analysis of the hypervariable regions of the 16S rDNA revealed that all strains could be assigned to the Alicyclobacillus acidocaldarius- Alicyclobacillus genomic species 1 group, which could be further subdivided into three clusters (Clusters I-III). On the basis of phenotypic characteristics, chemotaxonomic profiles, and phylogenetic data of six selected strains, five strains were identified as either A. acidocaldarius or Alicyclobacillus genomic species 1; however, one strain (MIH 332) could not be determined to belong to either of these species. 16S rDNA sequence homology values between strain MIH 332 and the reference strains of A. acidocaldarius (ATCC 27009(T)) and Alicyclobacillus genomic species 1 (DSM 11984) were 98.8% and 99.1%, respectively, which were higher than the corresponding similarity between the reference strains (98.4%). On the other hand, DNA-DNA hybridization levels between strain MIH 332 and the reference strains were 39% and 44%, respectively, which were lower than the value between the reference strains (59% or 65%). However, the phenotype of strain MIH 332 was also similar to those of the reference strains, and a typical phenotype could not be found for the strain, thus indicating that the strain may be a new genomic species of A. acidocaldarius, for which the name Alicyclobacillus genomic species 2 is tentatively proposed. The results of this study suggest that A. acidocaldarius and its related species are widely distributed in acidic environments in Japan, with slight regional variations in morphological and genotypic characteristics.     

PEX12, the Pathogenic Gene of Group III Zellweger Syndrome: cDNA Cloning by Functional Complementation on a CHO Cell Mutant, Patient Analysis, and Characterization of Pex12p

Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11–20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for ²⁹¹Asn and ²⁹²Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.     

Protective effects of nicaraven,a new hydroxyl radical scavenger,on the endothelial dysfunction after exposure of pig coronary artery to hydroxyl radicals

Recently, we have reported that a new synthetic compound, 1,2bis(nicotinamido)-propane (nicaraven), improved cardiac function following preservation and reperfusion. In this study, we investigated the efficacy of nicaraven as a radical scavenger by using an in vitro model of oxidative stress, to clarify mechanisms of the protective effect of this new compound on reperfusion injury in rat heart. Ring segments of epicardial right coronary arteries (RCA) of pig were suspended in organ chambers and exposed to hydroxyl radicals (·OH), generated (by two different systems ) by 0.28 mM FeSO₄/0.28 mM H₂O₂ and DHF/Fe³⁺-ADP (2.4 mM, 43 nM, and 1.56 uM, respectively) to the bathing solution for 60 min. Prior exposure of the coronary arteries to ·OH significantly produced right-ward shift of the dose-response curves of the bradykinin-induced endothelium-dependent relaxations (an increase in the ED₅₀ value for bradykinin by 4.37 and 1.98 times than control in two different ·OH generating systems, respectively), but did not affect the maximum relaxation responses. The presence of nicaraven (10^-4 and 10^-5 M) in the ·OH generating system, shifted the dose-response curves to bradykinin to the control level, suggesting a significant hydroxyl radical scavenging effect of the drug. These results indicate that nicaraven, a new hydroxyl radical scavenger, exhibits a protective effect on hydroxyl radicalinduced endothelial dysfunctions of pig coronary artery.     

Chromosomes of Phagocata kawakatsui and Bdellocephala annandalei from Lake Biwa-ko in Honshú, central Japan

Two lake-dwelling species of paludicolen triclads from Lake Biwa-ko (Honshû, Japan) were studied taxonomically and karyologically. (1) Phagocata kawakatsui Okugawa, 1956, is an epigean species usually inhabiting shallow springs and spring-fed streams in Central Japan. In Lake Biwa-ko, animals were obtained from several bottom stations of the littoral area in the southern part of the northern basin (3–70 m in depth). Chromosome numbers and karyotype: 2x=24 (2m+2sm+2sm+2m+2sm+2m+2sm+2m+2m+2sm+2m+2m). The first pair of metacentric chromosomes is very large in size. (2) Bdellocephala annandalei Ijima et Kaburaki, 1916, an endemic species, is distributed widely in the deep areas of the northern basin (30 to over 100 m in depth). Chromosome numbers and karyotype: 2x=28 (2m+2sm+2sm+2sm+2sm+2m+2m+2m+2m+2m+2m+2m+2m+2m) with the first pair of metacentric chromosomes very long.