Insights into the mode of action of 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89) against adult Schistosoma mansoni worms

Control of morbidity associated with schistosomiasis via chemotherapy largely relies on the drug praziquantel. Repeated therapy with praziquantel has created concerns about the possible selection of resistant worms and necessitated the search for novel drugs to treat schistosomiasis. Here, a murine model was infected with Schistosoma mansoni and treated with oral 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89), which caused a significant reduction in fecundity and egg burden and reduced morbidity when administered at 5-weeks post-infection.The analysis showed that the mode of action occurred through the ingestion of activated N-89 by the worms, and that there was no direct external effect on the S. mansoni worms. Ultrastructural analysis of the treated worms showed disruptions in the gut lumen and the presence of large volumes of material, suggestive of undigested blood meals or red blood cells. In addition, there were reduced vitelline cells in female worms and damage to sub-tegmental musculature in male worms. Eggs recovered from the treated mice showed both damage to the eggs and the production of immature eggs. Expression of mRNA responsible for gut and digestive function and egg production was also significantly affected by N-89 treatment, whereas control genes for musculature showed no significant changes.Thus, N-89 drastically affected the total digestive function and egg production of S. mansoni worms. Physiological processes requiring heme uptake such as egg production and eggshell formation were subsequently affected, suggesting that the compound could be a possible therapeutic drug candidate for schistosomiasis control.     

Expression,Purification, and Characterization of Recombinant Human α<Subscript>1</Subscript>-Antitrypsin Produced Using Silkworm–Baculovirus Expression System

Human α₁-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α₁-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~?15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.     

Identification of a functionally important population in phenol-digesting activated sludge with antisera raised against isolated bacterial strains.

Antisera were raised against nine strains which had been isolated from phenol-acclimated oil refinery activated sludge. Although several antisera reacted significantly with the activated sludge during a period of adaptation to phenol, only an antiserum against one of the isolates, Alcaligenes sp. E2, reacted with the activated sludge after the adaptation period. A kinetic pattern of phenol-oxygenating activity of the activated sludge after the adaptation period was similar to that of strain E2. These results suggest that a functionally important population in the phenol-digesting activated sludge was serologically identified.     

cDNA structure, alternative splicing and exon-intron organization of the predisposing tuberous sclerosis (Tsc2) gene of the Eker rat model.

The Eker rat hereditary renal carcinoma (RC) is an excellent example of a Mendelian dominant predisposition to a specific cancer in an experimental animal. We recently reported that a germline insertion in the rat homologue of the human tuberous sclerosis gene (TSC2) gives rise to the dominantly inherited cancer in the Eker rat model. We now describe the entire cDNA (5375 bp without exons 25 and 31) and genomic structure of the rat Tsc2 gene. The deduced amino acid sequence (1743 amino acids) shows 92% identity to the human counterpart. Surprisingly, there are a great many (> or = 41) coding exons with small sized introns spanning only approximately 35 kb of genomic DNA. Two alternative splicing events [involving exons 25 (129 bp) and 31 (69 bp)] make for a complex diversity of the Tsc2 product. The present determination of the Tsc2 gene and establishment of strong conservation between the rat and man provide clues for assessing unknown gene functions apart from that already predicted from the GTPase activating proteins (GAP3) homologous domain and for future analysis of intragenic mutations in tumors using methods such as PCR-SSCP and for insights into diverse phenotypes between species.     

Activity of H(+)-ATPase in ruminal bacteria with special reference to acid tolerance.

Batch culture experiments showed that permeabilized cells and membranes of Ruminococcus albus and Fibrobacter succinogenes, acid-intolerant celluloytic bacteria, have only one-fourth to one-fifth as much H(+)-ATPase as Megasphaera elsdenii and Streptococcus bovis, which are relatively acid tolerant. Even in the cells grown in continuous culture at pH 7.0, the acid-intolerant bacteria contained less than half as much H(+)-ATPase as the acid-tolerant bacteria. The amounts of H(+)-ATPase in the acid-tolerant bacteria were increased by more than twofold when the cells were grown at the lowest pH permitting growth, whereas little increase was observed in the case of the acid-intolerant bacteria. These results indicate that the acid-intolerant bacteria not only contain smaller amounts of H(+)-ATPase at neutral pH but also have a lower capacity to enhance the level of H(+)-ATPase in response to low pH than the acid-tolerant bacteria. In addition, the H(+)-ATPases of the acid-intolerant bacteria were more sensitive to low pH than those of the acid-tolerant bacteria, although the optimal pHs were similar.     

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43^−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43^−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43^−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.     

Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28–58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.     

Cytotoxic T-lymphocyte tolerance to minor H-43a alloantigen is induced exclusively in the context of the self major histocompatibility complex class I H-2Kb molecules

We elucidated previously that cytotoxic T lymphocyte precursors (CTLp) against H-43a allo-antigen, which we had discovered as a new mouse minor H antigen, were primed in H-43b mice only in the context of self H-2Kb restriction element, and that anti-H-43a CTLp tolerance was induced in H-43b mice by injection with H-43a spleen cells (SC) from H-43 congenic mice, i.e., under the condition of disparity at only the H-43 locus. The present study attempted to determine whether the H-2Kb restriction element for anti-H-43a CTLp priming is also implicated in the induction of anti-H-43a CTLp tolerance. For this purpose, we used a newly established H-43b C3W (H-2k) strain which is H-43 congenic to H-43a C3H/HeN. When (C3W X B10.MBR)F1 (H-43b, H-2Kk/b, Ik/k, Dk/q) mice were injected with H-43a-bearing (C3H/HeN X B10.AKM)F1 (H-43a/b;H-2Kk/k,Ik/k,Dk/q)SC, their selfH-2Kb-restricted anti-H-43a CTLp were were primed (cross-priming). By contrast, injection of H-43a-bearing (C3H/HeN X B10.MBR)F1 (H-43a/b; H-2Kk/b,Ik/k, Dk/q)SC, which differ from (C3H/HeN x B10.AKM) F1 SC solely at H-2K and possess H-2Kb molecules, did not prime but specifically inactivated the anti-H-43a CTLp of (C3W x B10.MBR)F1 mice. These results indicate clearly that anti-H-43a CTLp tolerance is induced exclusively in the context of the H-2Kb element expressed on the antigenic H-43a SC.     

Cytotoxic T lymphocyte response to minor H-42a alloantigen in H-42b mice: clonal inactivation of the precursor cytotoxic T lymphocytes by veto-like spleen cells that express the H-42a antigen

When (B10.BR X CWB)F1 (BWF1; H-2k/b) mice carrying the H-42b allele at the minor H-42 locus were injected with H-42a C3H.SW (CSW; H-2b) or C3H (H-2k) spleen cells (SC), self-H-2Kb restricted anti-H-42a pCTL in the BWF1 recipients were primed and differentiated to anti-H-42a CTL after in vitro stimulation with (B10.BR X CSW)F1 (BSF1; H-2k/b, H-42b/a) SC. In contrast, anti-H-42a pCTL in H-42b mice were inactivated by injection with H-42-congenic H-42a SC, and stable anti-H-42a CTL tolerance was induced. Preference of H-2Kb restriction of anti-H-42a CTL was strict, and self-H-2Kb-restricted anti-H-42a CTL did not lyse target cells carrying H-42a antigen in the context of H-2Kbm1. Involvement of suppressor cells in the anti-H-42a CTL tolerance was ruled out by the present cell transfer study and the previous cell-mixing in vitro study. Notably, treatment with anti-Thy-1.2 antibody (Ab) plus complement (C) wiped out the ability of CSW SC in the priming of anti-H-42a pCTL of BWF1 mice but left that of C3H SC unaffected, and injection of the anti-Thy-1.2 Ab plus C-treated CSW SC induced anti-H-42a CTL tolerance in the BWF1 recipients. Furthermore, H-42a/b, I-Ab/bm12 [CSW X B6.CH-2bm12 (bm12)]F1 SC could not prime anti-H-42a pCTL in H-42b, I-Ab (CWB X B6)F1 recipients, whereas H-42a/b, I-Ab (CSW X B6)F1 SC primed anti-H-42a pCTL in H-42b, I-Ab/bm12 (CWB X bm12)F1 recipients. The unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC was sometimes corrected by immunization of H-42b female mice with H-42-congenic H-42a male SC. Taking all of the results together, we propose the following. Unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC is caused by "veto cells" contained in the antigenic H-42a SC. Anti-H-42a pCTL in the H-42b recipients directly interacting with H-42-congenic H-42a SC, which bear H-42a antigen and H-2Kb restriction element, are inactivated or vetoed.(ABSTRACT TRUNCATED AT 400 WORDS)