Phosphorylation of Fanconi anemia protein, FANCA, is regulated by Akt kinase

Phosphorylation of the Fanconi anemia complementation group A (FANCA) protein is thought to be important for the function of the FA pathway. However, the kinase for FANCA (so-called FANCA-PK) remains to be identified. FANCA has a consensus sequence for Akt kinase near serine 1149 (Ser1149), suggesting that Akt can phosphorylate FANCA. We performed in vitro kinase assays using as substrate either a GST-fusion wild-type (WT) FANCA fragment or a GST-fusion FANCA fragment containing a mutation from serine to alanine at 1149 (FANCA-S1149A). These experiments confirmed that FANCA is phosphorylated at Ser 1149, in vitro. However, (32)P-orthophosphate labeling experiments revealed that FANCA-S1149A was more efficiently phosphorylated than WT-FANCA. Furthermore, phosphorylation of wild-type FANCA was blocked by coexpression of a constitutively active (CA)-Akt and enhanced by a dominant-negative (DN) Akt. Our results suggest that Akt is a negative regulator of FANCA phosphorylation.     

Chemokine-independent preference for T-helper-1 cells in transendothelial migration

We analyzed differences in the transendothelial migration (TEM) ability of T-helper (Th)-1 and Th2 cells across a murine endothelial cell line (F-2) under static conditions. The TEM abilities of Th1 cells from mice bearing autoimmune diseases and antigen-specific Th1 cell lines were severalfold higher than those of Th2 cells and lines of the same origin. These preferences were observed without exogenous chemoattractant and were insensitive to pertussis toxin, which completely blocks TEM induced by exogenous chemoattractants. Antibodies against LFA-1 and ICAM-1 as well as CD44 markedly blocked the TEM of Th1 cells. TEM ability was also blocked by pharmacological inhibitors of Src family protein-tyrosine kinases (PP2 and herbimycin A), phosphatidylinositol 3-kinase (wortmannin), and phosphatidylinositol-specific phospholipase C (). Cross-linking of CD44 strongly induced highly elongated morphology in Th1 lines, but weakly in Th2 lines. The pharmacological inhibitors that blocked TEM also inhibited this morphological change, whereas pertussis toxin did not. These data indicate that there are signaling pathways for TEM independent of chemokine attraction, but through adhesion molecules including CD44, and that the preferential TEM ability of Th1 over Th2 cells is formed, at least in part, by intrinsic differences in these pathways.     

Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine-serine-proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.     

Negative correlations between resistance to three organophosphate insecticides and productivity within a natural population of Drosophila melanogaster (Diptera: Drosophilidae)

To investigate the relationship between resistance to organophosphate insecticides and fitness components, we first measured resistance to three organophosphates, malathion, prothiophos, and fenitrothion, and productivity, a measure of fitness components, for each of the isofemale lines from the same natural population of Drosophila melanogaster (Meigen). Pearson correlation coefficients indicated that positive correlations among resistance to the organophosphates and negative correlations between resistance to each of the organophosphates and the productivity existed within the natural population. We further investigated the genetic basis of the correlations among resistance to the organophosphates and the productivity, by using chromosome-substituted lines between a resistant and a susceptible inbred line established from the same natural population. Chromosomal analyses indicated that the third chromosome from the resistant line exhibited not only significant, positive effects on resistance to all of the organophosphates tested but also a significant negative effect on the productivity, suggesting positive genetic correlations between resistance to each organophosphate and negative genetic correlations between resistance to each organophosphate and the productivity. In addition, a significant negative effect on the productivity was also detected from the second chromosome, which did not exhibit significant major effects on resistance to the organophosphates. This suggests that fitness components of resistant lines could be also affected by factors independent of insecticide resistance. The dynamics of genetic variation in resistance to the organophosphates within the natural population of D. melanogaster are discussed from the standpoint of negative genetic correlations between resistance to the organophosphates and the productivity.     

Factors influencing eating ability of old in-patients in a rehabilitation hospital in Japan

Objectives: This study was designed to determine the factors influencing eating ability of old in‐patients in a rehabilitation hospital. Design: Cross‐sectional investigation. Setting: Forty‐six in‐patients in the rehabilitation ward of Hashimoto Hospital in Kagawa Prefecture in Japan were investigated using a multi‐disciplinary approach. Main outcome measures: Age, gender, state of dentition, muscle activity of lip, cheek and tongue, biting force, salivary flow rate per a minute (SFR), masticatory ability for gummy jelly, swallowing ability, texture of meal, independency of walking (Functional Independence Measure=FIM) and ability to communicate. Results: Bivariate analysis for the relationship between surveyed items and masticatory ability (chi‐square test) identified that better masticatory ability for gummy jelly was associated with age (<85 years), gender (male), state of dentition (dentate), SFR (high), activity of lip (good), biting force (high), swallowing ability (good) and activity of communication (high). Among these items, SFR (p=0.001), gender (p=0.004), ability to communicate (p=0.005) and age (p=0.012) were found having an influence on the masticatory ability (logistic regression analysis). On the other hand, age (<85years), gender (male), SFR (high), activity of lip (good), activity of cheek (good), biting force (high), masticatory ability (good) and swallowing ability (good) had a relationship with normal texture of meal. In regression analysis, only two items, activity of lip (p=0.003) and swallowing ability (p=0.024) emerged as factors on texture of meal. Conclusions: Masticatory ability for gummy jelly was influenced by cognitive function and was excluded from the factors on the state of meal. These results suggested the limitation of evaluation using test food, so dentists should observe eating behaviour of in‐patients. In addition, dentists should pay attention to the activity of the lip and swallowing ability as well as dentition and prostheses in the rehabilitation of eating ability. As SFR was the most significant factor on masticatory ability, this emphasizes the necessity of care for dry mouth caused by side effects of multi‐medication     

Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity

Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218-222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPbeta, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). PMA induced expression of RPTPbeta mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D(3) and interferon-gamma, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPbeta mRNA, whereas 1. 2% Me(2)SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPbeta antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPbeta. Double immunostaining studies also indicated that newly expressed RPTPbeta colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPbeta cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPbeta, a protein that functions as the VacA receptor.     

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins.     

Semisynthesis of C-ring modified triptolide analogues and their cytotoxic activities

Several C-ring modified analogues of a potent antileukemic diterpene, triptolide (1), were synthesized and their structure-activity relationships were studied.     

Homostachydrine is a Xenobiotic Substrate of OCTN1/SLC22A4 and Potentially Sensitizes Pentylenetetrazole-Induced Seizures in Mice

Neurochemical Research - Understanding of the underlying mechanism of epilepsy is desired since some patients fail to control their seizures. The carnitine/organic cation transporter OCTN1/SLC22A4...