The quantitative assay of the clustering activity of the lymphocytosis-promoting factor (pertussis toxin) of Bordetella pertussis on Chinese hamster ovary (CHO) cells

An experimental design and a statistical method for the estimation of the clustering-response activity of lymphocytosis-promoting factor (LPF) in Chinese hamster ovary cells growing in wells on a microplate were investigated. The scoring method introduced by Ipsen was adopted to express the grade of the clustering response rather than the end-point method generally used. The scoring method was validated by statistical analyses. The grade of response varied with the location of the wells on a microplate, and thus the expression of the clustering activity of a test sample in terms of the end-point may be inadequate in terms of accuracy and reproducibility. It was shown that the allocation of test samples to individual wells according to a Latin square design minimized the effect of the location of wells on the clustering response. Under such experimental conditions, a fairly precise and reproducible method for the quantification of the clustering activity was developed.     

The complete mitogenome of the hydrothermal vent crab Gandalfus yunohana (Crustacea: Decapoda: Brachyura): a link between the Bythograeoidea and Xanthoidea

Yang, J.‐S., Nagasawa, H., Fujiwara, Y., Tsuchida, S. & Yang, W.‐J. The complete mitogenome of the hydrothermal vent crab Gandalfus yunohana (Crustacea: Decapoda: Brachyura): a link between the Bythograeoidea and Xanthoidea. —Zoologica Scripta, 39, 621–630. Metazoan mitochondrial genomes (mitogenomes) are often used for all‐level phylogenetic analyses and evolution modelling. Although mitochondrial fragments facilitate studying the occurrence and dispersal of hydrothermal‐vent species, few complete mitogenomes have been determined for comprehensive analyses. We determined the complete nucleotide sequence of the bythograeid crab Gandalfus yunohana. The G. yunohana mitogenome is 15 567 bp in length and with an AT content of 69.9%. A putative control region of 625 bp was identified due to its position (between rrnS and trnI) and AT richness (72.8%), which exhibits high similarity with that of the Australian giant crab Pseudocarcinus gigas. The mitochondrial gene order is identical to the typical brachyuran mode. Codon usage, nucleotide composition and bias are well conserved as the Brachyura. Phylogenetic analyses from protein‐coding genes indicated its closest relationship with P. gigas. All the results support the close evolution distance between the Bythograeoidea and Xanthoidea, which might imply the possible origin that the only superfamily of vent crabs underwent. The G. yunohana mitogenome exhibits highly conserved characteristics with those of other decapods, especially its close relative brachyurans. A recent origin rather than the relic fauna was suggested. The present study will supply considerable data of use for both genomics and evolutionary research on hydrothermal vent ecosystems.     

Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain

Previous studies indicated that single-headed smooth muscle myosin and S1 (a single head fragment) are not regulated through phosphorylation of the regulatory light chain (RLC). To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of Delta MD lacked the motor domain, that of Delta(MD+ELC) lacked the motor and essential light chain binding domains, and single-headed HMM had one intact head alone. The basal ATPase activities of the three mutants decreased as the KCl concentration became less than 0.1 M. Such a decrease was not observed for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of Delta MD and Delta(MD+ELC), but that of single-headed HMM, were phosphorylated. When their RLCs were unphosphorylated, the three mutants exhibited similar actin-activated ATPase levels. However, when they were phosphorylated, the actin-activated ATPase activities of Delta MD and Delta(MD+ELC) increased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We propose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs. We also propose that a cooperative interaction between two motor domains is required for a higher level of actin activation.     

Functional roles of ionic and hydrophobic surface loops in smooth muscle myosin: their interactions with actin

This investigation ascertains whether, in (smooth muscle) myosin, certain residues engage in functional interactions with their actin conjugates in an actomyosin complex. Such interactions have been postulated from putting together crystallographic models of the two proteins [Rayment, I., Rypniewski, W. R., Schmidt-B?se, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G., and Holden, H. M. (1993) Science 261, 50-58]. Here, in several instances, we ask whether mutation of a particular residue significantly impairs a function, and find that the answers are largely rationalized by the original postulation. Additionally, a novel element emerges from our investigation. To assess function, we test the wild type and mutant systems as they perform in the steady state of ATP degradation. In doing so, we assume, as usual, that degradation proceeds from an early stage in which the complex forms (and is described by parameter K(app)) to a later stage during which the product leaves the complex (and is described by parameter V(max)). Interestingly, certain defects induced by the mutations are associated with changes in K(app), and other defects are associated with changes in V(max), suggesting that our procedure at least roughly distinguishes between events according to the time in the degradation at which they occur. In this framework, we suggest that (1) in the actin-myosin association phase, cationic residues Lys-576 and Lys-578 interact with anionic residues of the so-called second actin, and (2) in the product leaving phase, hydrophobic residues Trp-546, Phe-547, and Pro-548, as well as the Thr-532/Asn-533/Pro-534/Pro-535 sequence, sever connections with the so-called first actin. The role of Glu-473 is also examined.     

Expression and differential intracellular localization of two major forms of human 8-oxoguanine DNA glycosylase encoded by alternatively spliced OGG1 mRNAs

We identified seven alternatively spliced forms of human 8-oxoguanine DNA glycosylase (OGG1) mRNAs, classified into two types based on their last exons (type 1 with exon 7: 1a and 1b; type 2 with exon 8: 2a to 2e). Types 1a and 2a mRNAs are major in human tissues. Seven mRNAs are expected to encode different polypeptides (OGG1-1a to 2e) that share their N terminus with the common mitochondrial targeting signal, and each possesses a unique C terminus. A 36-kDa polypeptide, corresponding to OGG1-1a recognized only by antibodies against the region containing helix-hairpin-helix-PVD motif, was copurified from the nuclear extract with an activity introducing a nick into DNA containing 8-oxoguanine. A 40-kDa polypeptide corresponding to a processed form of OGG1-2a was detected in their mitochondria using antibodies against its C terminus. Electron microscopic immunocytochemistry and subfractionation of the mitochondria revealed that OGG1-2a locates on the inner membrane of mitochondria. Deletion mutant analyses revealed that the unique C terminus of OGG1-2a and its mitochondrial targeting signal are essential for mitochondrial localization and that nuclear localization of OGG1-1a depends on the NLS at its C terminus.     

Effects of total parenteral nutrition on rat bile canaliculi: a stereologic analysis

OBJECTIVE: To quantitate, in a stereologic manner, changes in bile canalicular morphology before and after choleretic infusion of total parenteral nutrition (TPN) and to determine whether TPN produces changes in localized regions within the hepatic lobule. STUDY DESIGN: Livers were obtained from sham-operated on normal adult male rats (control) and from rats that received intravenous TPN solution containing 20% glucose and 3.5% Molipron F. The tissues, obtained by a rigorous sampling procedure, were systematically subjected to stereologic analysis. Measurements were made on electron micrographs at two levels of magnification by point, intersection and profile counts, and then volume, surface area and length were estimated per unit parenchymal volume. RESULTS: The surface area of the canalicular wall per parenchymal volume increased significantly (from 5.33 x 10(-2) to 6.73 x 10(-2) microns 2/micron 3) after TPN treatment, as did the length of microvilli (from 0.241 to 0.267 microns/micron 3). However, the volume of bile canalicular lumina per parenchymal volume (0.306% and 0.320%), total length of bile canaliculi (1.05 x 10(-2) and 1.06 x 10(-2) microns/micron 3) and diameter of microvilli (8.73 x 10(-2) and 8.94 x 10(-2) microns) remained constant. CONCLUSION: These results indicate that changes in canalicular shape and microvillus hypertrophy may cause lowering efficiency of the bile flow rate.     

Purification and characterization of a novel protein produced by Bifidobacterium longum SBT2928 that inhibits the binding of enterotoxigenic Escherichia coli Pb176 (CFA/II) to gangliotetraosylceramide

A novel protein (BIF) which shows inhibitory activity on the binding of enterotoxigenic Escherichia coli Pb176 (ETEC with colonization factor antigen (CFA) II, which consists of coli surface-associated antigens CS1 and CS3) to gangliotetraosylceramide (asialo GM1 or GA1) was isolated from the culture supernatant fluid of Bifidobacterium longum SBT2928 (BL2928) at its stationary phase. The homogeneity of the final preparation of BIF was demonstrated by SDS-PAGE, polyacrylamide gel electrofocusing and N-terminal amino acid sequencing. The BIF was characterized as (i) a protein with an M(r) of approximately 104 kDa when chromatographed on a gel filtration column, and 52 kDa when separated on SDS-PAGE, and (ii) having an isoelectric point of 5.9. No change in size was produced by thiol reduction. These results suggest that BIF is a homodimer consisting of identical 52 kDa monomers. The purified BIF at the concentration of 25 micrograms protein ml-1 caused a 50% reduction in binding of the ETEC strain to GA1.     

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(α- -rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(α- -rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), β-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6′-O-oleoyl-β- -glucopyranosyl)-β-sitosterol (7), and β-sitosterol-3-O-β- -glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.