Evaluation of lipid‐binding properties of the N‐terminal helical segments in human apolipoprotein A‐I using fragment peptides

Although the N‐terminal region in human apolipoprotein (apo) A‐I is thought to stabilize the lipid‐free structure of the protein, its role in lipid binding is unknown. Using synthetic fragment peptides, we examined the lipid‐binding properties of the first 43 residues (1–43) of apoA‐I in comparison with residues 44–65 and 220–241, which have strong lipid affinity in the molecule. Circular dichroism measurements demonstrated that peptides corresponding to each segment have potential propensity to form α‐helical structure in trifluoroethanol. Spectroscopic and thermodynamic measurements revealed that apoA‐I (1–43) peptide has the strong ability to bind to lipid vesicles and to form α‐helical structure comparable to apoA‐I (220–241) peptide. Substitution of Tyr‐18 located at the center of the most hydrophobic region in residues 1–43 with a helix‐breaking proline resulted in the impaired lipid binding, indicating that the α‐helical structure in this region is required to trigger the lipid binding. In contrast, apoA‐I (44–65) peptide exhibited a lower propensity to form α‐helical structure upon binding to lipid, and apoA‐I (44–65/S55P) peptide exhibited diminished, but not completely impaired, lipid binding, suggesting that the central region of residues 44–65 is not pivotally involved in the formation of the α‐helical structure and lipid binding. These results indicate that the most N‐terminal region of apoA‐I molecule, residues 1–43, contributes to the lipid interaction of apoA‐I through the hydrophobic helical residues. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Oxytocin hypersensitivity in pregnant P-LAP deficient mice

AimsOxytocin (OT) is the strongest uterotonic substance and has been used widely to induce labor. The physiological importance of OT in modulating the initiation and progression of labor remains unclear. In this study, we showed the roles of OT with onset of labor and also the arginine vasopressin (AVP) effect on urine volume in vivo using both wild type (WT) and placental leucine aminopeptidase (P-LAP)-deficient (KO) mice.Main methodsOT (1, 2, 2.5 U/day) or recombinant P-LAP (0.01 U/day) was continuously infused from gestation day 15.5 in WT and P-LAP KO mice. Duration until onset of labor was observed. Before and after administration of AVP (1 U/day) in WT and P-LAP KO mice, urine volume was measured.Key findingsA significant shortening of pregnancy term was observed in P-LAP KO mice. Continuous infusion of OT (1 U/day) revealed that P-LAP KO mice resulted in premature delivery (OT hypersensitivity). We could observe a significant decrease of urine volume in P-LAP KO mice by administration of AVP. Administration of recombinant P-LAP in WT mice resulted in the delay of the onset of labor about 1.5 days compared with control mice.SignificanceOur present study shows that the regulation of the onset of labor mainly depends on OT and its degradation by P-LAP and also the possible role of P-LAP in the regulation of urine output. P-LAP might be involved in the increased OT sensitivity just prior to onset of labor and also in the onset of labor by degradation of OT.     

Recognition of the Qa-2k tumor antigen by T cell receptor γ/δ of an immunopotentiator-induced tumoricidal T cell of mice

Tumor-specific expression of Qa-2^k antigen coded by the Q5^k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2^– mice. Lymphocytes stimulated in vivo withP. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (L^Q7b/Kb), which expressed the 1 and 2 domains of the Qa-2 antigen (Q7^b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5 half of the Q7^b gene and the 3 half of the H-2K^b gene (pQ7^b/K^b). Using L^Q7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes fromP. acnes-stimulated (C3H/He × B6.K1)F1 mice (H-2^k, Qa-2^–) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the 1/2 region of the Q7^b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the / type (TCR/). The (C3H/He × B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2^k+) derived from AKR mice (Qa-2^–, H-2^k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed L^Q7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the 1/2 region of the Qa-2^k tumor antigen by TCR/.     

The synthesis of evernitrose and 3-epi-evernitrose

Evernitrose (2,3,6-trideoxy-3-C-methyl-4-O-methyl-3-nitro-L-arabino-hexopyranose) was synthesized from methyl 2,6-dideoxy-4-O-methyl-α-L-erythro-hexopyranosid-3-ulose (2) through introduction of an amino group attached to the tertiary branching carbon by the method of Bourgeois, and subsequent oxidation of the amino group by m-chloroperoxybenzoic acid to a nitro group. 3-Cyano-3-O-mesylation of 2 by Bourgeois's method gave exclusively the desired product having the L-ribo configuration; furthermore, the β anomer of 2 gave the L-ribo and L-arabino products in the ratio of 1:2. The latter compound was converted into 3-epi-evernitrose by a similar sequence of reactions.     

Accumulation and aggregation of amyloid beta-protein in late endosomes of Niemann-pick type C cells

There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid beta-protein (Abeta) metabolism. However, the precise cellular linkage sites between cholesterol and Abeta have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable Abeta accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This Abeta accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of Abeta42 and insolubility in SDS, suggesting the presence of aggregated Abeta in late endosomes. Most importantly, Abeta accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated Abeta42 as well as cholesterol, suggesting a direct interaction between aggregated Abeta and cholesterol.     

Relationship between calnexin and BiP in suppressing aggregation and promoting refolding of protein and glycoprotein substrates

Calnexin (CNX) is a membrane protein of the endoplasmic reticulum that has been defined primarily as a lectin, yet is capable of functioning as a molecular chaperone with non-glycosylated proteins in vitro. Here, we assess the relative contributions of the oligosaccharide- and polypeptide-binding sites of CNX to its in vitro chaperone functions by comparing it with the Hsp70 chaperone of the endoplasmic reticulum, BiP. Both proteins were equally effective in preventing the aggregation of non-glycosylated citrate synthase, indicating that the polypeptide-binding site of CNX is capable of functioning at a level similar to that of Hsp70. However, when confronted with glycoprotein substrates, the lectin site of CNX provided a significant advantage over BiP in suppressing aggregation. CNX also cooperated with BiP and the J domain of Sec63p in the ATP-dependent refolding of glycoprotein and non-glycosylated substrates. The lectin site of CNX was essential for refolding of the glycoprotein. These findings reinforce the function of CNX as a bona fide chaperone and illustrate how its lectin site confers advantages relative to other chaperones when confronted with glycoprotein substrates.     

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.     

Super agonist actions of clothianidin and related compounds on the SAD beta 2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes

To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SAD beta 2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist action.     

Dietary polyunsaturated fatty acids suppress acute hepatitis, alter gene expression and prolong survival of female Long-Evans Cinnamon rats, a model of Wilson disease

In the Long-Evans Cinnamon rat, copper accumulates in the liver because of a mutation in the copper-transporting ATPase gene, and peroxidative stresses are supposed to be augmented. We examined the effects of dietary fatty acids on hepatitis, hepatic gene expression, and survival. Rats were fed a conventional, low-fat diet (CE2), a CE2 diet supplemented with 10 wt% of lard (Lar), high-linoleic soybean oil (Soy), or a mixture of docosahexaenoic acid (DHA)-rich fish oil and soybean oil (DHA/Soy). Among female rats, the mean survival times of the DHA/Soy and the Soy groups were longer by 17 approximately 20% than in the Lar and the CE2 groups. Among male rats, the survival times were much longer than in the females, but no significant difference in survival was observed among the dietary groups. Serum ceruloplasmin levels in female and male rats of all of the dietary groups were similar. Serum transaminase levels of the DHA/Soy group tended to be lower than in the CE2 group. Histological examinations revealed a marked degeneration in hepatic tissue integrity in the Lar and CE2 groups but not in the DHA/Soy group. Hepatic levels of metal-related genes, transferrin and ceruloplasmin, as well as those related to bile acid synthesis were up-regulated, and an inflammation-related gene (cyclooxygenase [COX]-2) was down-regulated in the DHA/Soy group. Some proliferation-related genes were also affected by the dietary fatty acids. These results indicate that polyunsaturated fatty acids suppress the development of acute hepatitis and prolong survival in females, regardless of whether they are of the n-6 or n-3 type, which are associated with altered gene expressions.     

Structure and direct electrochemistry of cytochrome P450 from the thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7

Cytochrome P450 from thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 (P450st) has been expressed in Escherichia coli and purified at high homogeneity. P450st was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=53.6 A, b=55.1 A, and c=130.9 A, and the structure was determined at a 3.0 A resolution. The final R-factor was 0.194 (Rfree=0.235). Structural comparison with cytochrome P450 from S. solfataricus (CYP119) suggests that the region composed of the F to G helices and the Cl- binding site is responsible for the affinity for a ligand coordinating heme iron. Direct electrochemistry of P450st in a didodecyldimethylammonium bromide (DDAB) film on a plastic formed carbon (PFC) electrode has also been demonstrated. A quasi-reversible redox response has been observed even at elevated temperatures of up to 80 degrees C.