Kinesin‐1/Hsc70‐dependent mechanism of slow axonal transport and its relation to fast axonal transport

Cytoplasmic protein transport in axons (‘slow axonal transport’) is essential for neuronal homeostasis, and involves Kinesin‐1, the same motor for membranous organelle transport (‘fast axonal transport’). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin‐1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ‐like domain of the kinesin light chain in the Kinesin‐1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin‐1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant‐negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction.     

Serum metabolomics as a novel diagnostic approach for pancreatic cancer

Pancreatic cancer is one of the leading causes of cancer-related death, and there is currently little hope of a cure because there are no effective biomarkers for its early detection. Therefore, the search for novel biomarkers that would allow the early detection of pancreatic cancer is ongoing. In this study, the differences between the metabolomes of pancreatic cancer patients with Stage III, Stage IVa, or Stage IVb disease (n = 20) and healthy volunteers (n = 9) were evaluated by metabolomics, which is the endpoint of the Omics cascade and therefore the last step in the cascade before the phenotype. In our experimental conditions using gas chromatography mass spectrometry (GC/MS), a total of 60 metabolites were detected in serum, and the levels of 18 of the 60 metabolites were significantly changed in pancreatic cancer patients compared with those in healthy volunteers. Then, Principal Component Analysis (PCA), which is a basic form of Multiple Classification Analysis, was performed, and the PCA scores plots based on the 60 metabolites highlighted the metabolomic differences between the pancreatic cancer patients and healthy volunteers. The differences between different stages of pancreatic cancer were also assessed by Partial Least Squares Discriminant Analysis (PLS-DA), which is one of Multiple Classification Analysis, and we found that it was possible to discriminate among the Stage III, Stage IVa, and Stage IVb groups. In addition, values of the 9 metabolites in 1 Stage I pancreatic cancer patient were similar to those obtained from the Stage III, Stage IVa, and Stage IVb pancreatic cancer patients. Our findings will aid the discovery of novel biomarkers that allow the early detection of pancreatic cancer by metabolomic approaches.     

Hyperactivation is the mode conversion from constant-curvature beating to constant-frequency beating under a constant rate of microtubule sliding

Flagellar beating of hyperactivated golden hamster spermatozoa was analyzed in detail using digital image analysis and was compared to that of nonhyperactivated (activated) spermatozoa in order to understand the change in flagellar beating during hyperactivation and the active microtubule sliding that brought about the change in flagellar beating. Hyperactivated flagellar beating, which was characterized by a sharp bend in the proximal midpiece and low beat frequency, was able to alter the waveform with little change in beat frequency (constant-frequency beating), whereas activated flagellar beating, which was characterized by a slight bend in the proximal midpiece and high beat frequency, was able to alter beat frequency with little change in the waveform (constant-curvature beating). These results demonstrate that flagellar beating of hyperactivated and activated spermatozoa were essentially different modes and that hyperactivation was the mode conversion from constant-curvature beating to constant-frequency beating. Detailed analysis of flagellar bends revealed that the increase in curvature in the proximal midpiece during hyperactivation was due to the increase in total length of microtubule sliding in a nearly straight region between bends, while the rate of microtubule sliding remained almost constant.     

Suramin prevents fulminant hepatic failure resulting in reduction of lethality through the suppression of NF-kappaB activity

AIM: Suramin is a symmetrical polysulfonated naphthylamine derivative of urea. There have been few studies on the effect of suramin on cytokines. We examined the effects of suramin on production of inflammatory cytokines. METHODS: We made an acute liver injury model treated with d-galactosamine (GalN) and lipopolysaccharide (LPS). Plasma AST, ALT, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 levels were measured. We compared with survival rate, histological found and NF-kappaB activity between with and without treatment of suramin. In macrophage like cell line, TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity was measured. RESULTS: The lethality of mice administered suramin with GalN/LPS was significantly decreased compared with that in mice without suramin. Changes of hepatic necrosis and apoptosis were slight in suramin-treated mice. Serum AST, ALT, TNF-alpha, IL-6 levels and NF-kappaB activity in the liver were significantly lower in mice administered suramin. In an in vitro model, suramin preincubation inhibited TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity. CONCLUSIONS: Suramin inhibits TNF-alpha and IL-6 production through the suppression of NF-kappaB activity from macrophages and shows therapeutic effects on acute liver damage.     

Intracellular delivery of proteins in complexes with oligoarginine-modified liposomes and the effect of oligoarginine length

The intracellular delivery of proteins using cell-penetrating peptides (CPPs) including oligoarginine (oligo-Arg) carriers raises the possibility of establishing novel therapeutic methods. We compared the effect of the length of oligo-Arg in modified liposomes ((Arg)(n)-L; n = 4, 6, 8, 10) on the delivery of proteins by flow cytometry, fluorescence microscopy, and spectrofluorimetry. As a free liposome, Arg4-modified liposome Arg4-L was most efficiently internalized in cells. The efficiency decreased depending on the length of oligo-Arg. For the intracellular delivery of proteins, (Arg)(n)-L was physically associated with proteins. Concerning the effect of oligo-Arg length, liposome/protein complexes showed a different behavior. Arg4-L carried bovine serum albumin (BSA, 66 kDa) and beta-galactosidase (beta-Gal, 120 kDa) 6-fold higher than free BSA and free beta-Gal. Arg10-L showed similar performance for these two proteins to Arg4-L. The enzymatic activity of beta-Gal in the cells showed that proteins were transported as a biologically active form. Arg10-L carried 100-fold more immunoglobulin G (IgG, 150 kDa) than free IgG, and 3-fold more than Arg4-L into cells. Shorter oligo-Arg chain on liposomes may be enough for liposomes alone to be taken up in cells, but more Arg residues may be needed to form a complex with high molecular weight proteins and deliver them into cells. This information will aid in the design of (Arg)(n)-L as a carrier for delivering proteins into cells.     

The role of a HSV thymidine kinase stimulating substance, scopadulciol, in improving the efficacy of cancer gene therapy

BACKGROUND: The most extensively investigated strategy of suicide gene therapy for treatment of cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as acyclovir (ACV) and ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid, scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system. METHODS: The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+) cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK- cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values. RESULTS: SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels, while no effect of SDC was observed on the phosphorylation of ACV-MP to ACV-TP by cellular kinases. The SDC/prodrug combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of ACV/GCV and SDC. CONCLUSIONS: SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of ACV and GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic ACV. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy.     

Lymphatic development

The lymphatic system is essential for fluid homeostasis, immune responses, and fat absorption, and is involved in many pathological processes, including tumor metastasis and lymphedema. Despite its importance, progress in understanding the origins and early development of this system has been hampered by lack of defining molecular markers and difficulties in observing lymphatic cells in vivo and performing genetic and experimental manipulation of the lymphatic system. Recent identification of new molecular markers, new genes with important functional roles in lymphatic development, and new experimental models for studying lymphangiogenesis has begun to yield important insights into the emergence and assembly of this important tissue. This review focuses on the mechanisms regulating development of the lymphatic vasculature during embryogenesis. Birth Defects Research (Part C) 87:222–231, 2009. © 2009 Wiley‐Liss, Inc.     

The evolution of mammalian chemokine genes

Chemokines play an important role in orchestrating cell recruitment and localization in both physiological and pathological conditions. More than 44 ligands have been identified in the human genome. A significantly different set of chemokines, however, is found in the mouse genome, suggesting a rapid evolution of the chemokine system in mammalian genomes. Thus, there are lineage and even individual-specific differences in chemokine genes in mammals. Differences in the expression and function between even recently duplicated genes are also evident. In this review, we discuss how evolutionary events such as gene duplication and gene conversion have shaped the diverse arrays of chemokines in mammalian genomes.     

Chemokine PARC gene (SCYA18) generated by fusion of two MIP-1alpha/LD78alpha-like genes

Two loci in the human genome, chromosomes 4q12-q21 and 17q11.2, contain clusters of CXC and CC chemokine subfamily genes, respectively. Since mice appear to contain fewer chemokine genes than humans, numerous gene duplications might have occurred in each locus of the human genome. Here we describe the genomic organization of the human pulmonary and activation-regulated CC chemokine (PARC), also known as DC-CK1 and AMAC-1. Despite high sequence similarity to a CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha)/LD78alpha, PARC is chemotactic for lymphocytes and not for monocytes and does not share its receptor with MIP-1alpha. Analyses of the BAC clones containing the human PARC gene indicated that the gene is located most closely to MIP-1alpha (HGMW-approved symbol SCYA3) and MIP-1beta (HGMW-approved symbol SCYA4) on chromosome 17q11.2. Dot-plot comparison suggested that the PARC gene had been generated by fusion of two MIP-1alpha-like genes with deletion and selective usage of exons. Base changes accumulated before and after the fusion might have adapted the gene to a new function. Since there are variably duplicated copies of the MIP-1alpha gene called LD78beta (HGMW-approved symbol SCYA3L) in the vicinity of the MIP-1alpha gene, the locus surrounding the MIP-1alpha gene seems to be a "hot spring" that continuously produces new family genes. This evidence provides a new model, duplication and fusion, of the molecular basis for diversity within a gene family.