Functional differentiation in the leucine-rich repeat domains of closely related plant virus-resistance proteins that recognize common avr proteins

The N' gene of Nicotiana sylvestris and L genes of Capsicum plants confer the resistance response accompanying the hypersensitive response (HR) elicited by tobamovirus coat proteins (CP) but with different viral specificities. Here, we report the identification of the N' gene. We amplified and cloned an N' candidate using polymerase chain reaction primers designed from L gene sequences. The N' candidate gene was a single 4143 base pairs fragment encoding a coiled-coil nucleotide-binding leucine-rich repeat (LRR)-type resistance protein of 1,380 amino acids. The candidate gene induced the HR in response to the coexpression of tobamovirus CP with the identical specificity as reported for N'. Analysis of N'-containing and tobamovirus-susceptible N. tabacum accessions supported the hypothesis that the candidate is the N' gene itself. Chimera analysis between N' and L(3) revealed that their LRR domains determine the spectrum of their tobamovirus CP recognition. Deletion and mutation analyses of N' and L(3) revealed that the conserved sequences in their C-terminal regions have important roles but contribute differentially to the recognition of common avirulence proteins. The results collectively suggest that Nicotiana N' and Capsicum L genes, which most likely evolved from a common ancestor, differentiated in their recognition specificity through changes in the structural requirements for LRR function.     

An Improved Assay Method for Antifouling Substances Using the Blue Mussel,Mytilus edulis

The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37°C and pH 8.5 in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10 mm Tris-HCl, 7 mm 2-mercaptoethanol, 7 mm MnCl₂ and 50 mm NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5′-GACGTC-3′, cuts between T and C and produces a 3′-tetranucleotide extension in the presence of MnCl₂, as it does in the presence of MgCl₂.     

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.     

Cardio-Metabolic Disease Risks and Their Associations with Circulating 25-Hydroxyvitamin D and Omega-3 Levels in South Asian and White Canadians

Objectives

This study compared cardio-metabolic disease risk factors and their associations with serum vitamin D and omega-3 status in South Asian (SAC) and White Canadians (WC) living in Canada’s capital region.

Methods

Fasting blood samples were taken from 235 SAC and 279 WC aged 20 to 79 years in Ottawa, and 22 risk factors were measured.

Results

SAC men and women had significantly higher fasting glucose, insulin, homeostasis model assessment for insulin resistance (HOMA-IR), apolipoprotein B (ApoB), ratios of total (TC) to HDL cholesterol (HDLC) and ApoB to ApoA1, leptin, E-selectin, P-selectin, ICAM-1 and omega-3 (p < 0.05), but lower HDLC, ApoA1, vitamin D levels than WC (p < 0.05). SAC women had higher CRP and VEGF than WC women. Adequate (50–74.9 nmol/L) or optimal (≥ 75 nmol/L) levels of 25(OH)D were associated with lower BMI, glucose, insulin, HOMA-IR, TG, TC, low density lipoprotein cholesterol (LDLC), ApoB/ApoA1 ratio, CRP, leptin, and higher HDLC, ApoA1, omega-3 index, L-selectin levels in WC, but not in SAC. Intermediate (>4%-<8%) or high (≥ 8%) levels of omega-3 indices were related to lower E-selectin, P-selectin, ICAM-1 and higher HDLC, 25(OH)D levels in WC, but not in SAC. The BMIs of ≤ 25 kg/m² were related to lower LDLC, ApoB, VEGF, creatinine and higher 25(OH)D in WC, but not in SAC.

Conclusions

The associations of vitamin D, omega-3 status, BMI and risk factors were more profound in the WC than SAC. Compared to WC, vitamin D status and omega-3 index may not be good predictive risk factors for the prevalence of CVD and diabetes in SAC.     

Antisense oligonucleotide to cofilin enhances respiratory burst and phagocytosis in opsonized zymosan-stimulated mouse macrophage J774.1 cells

Phagocytes play a central role in the host defense system, and the relationship between the mechanism of their activation and cytoskeletal reorganization has been studied. We have previously reported a possible involvement of cofilin, an actin-binding protein, in phagocyte functions through its phosphorylation/dephosphorylation and translocation to the plasma membrane regions. In this work, we have obtained a new line of evidence showing an important role of cofilin in phagocyte functions using the mouse macrophage cell line J774.1 and an antisense oligonucleotide to cofilin. Upon stimulation with opsonized zymosan (OZ), cofilin was phosphorylated, and it accumulated around phagocytic vesicles. As the antisense oligonucleotide to cofilin, a 20-mer S-oligo corresponding to the sequence including the AUG translational initiation site was found to be effective. In the cells treated with the antisense oligonucleotide, the amount of cofilin was less than 30% of that in the control cells, and the level of F-actin was two or three times higher than that in the control cells before and throughout the cell activation. In the antisense oligonucleotide-treated cells, OZ-triggered superoxide production was three times faster than that in the control cells. Furthermore, phagocytosis of OZ was enhanced by the antisense. These results show that cofilin plays an essential role in the control of phagocyte function through regulation of actin filament dynamics.     

Class VI myosin moves processively along actin filaments backward with large steps.

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot take this scenario. We found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes "hot spots" on actin filaments that attract myosin heads. Myosin VI may step on these "hot spots" on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps.     

Mycosphaerella buna sp. nov. with aPseudocercospora anamorph isolated from the leaves of Japanese beech

A species ofMycosphaerella with aPseudocercospora anamorph was collected on overwintered fallen leaves of Japanese beech,Fagus crenata. Based on comparison of morphology withMycosphaerella species on Fagaceae, the fungus was newly described asMycosphaerella buna. ThePseudocercospora anamorph derived from a single ascospore of the fungus was morphologically identical to an endophytic anamorph isolated from asymptomatic living leaves of Japanese beech. Contribution No. 150, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.     

Regulation of gonadotropin secretion and puberty onset by neuromedin U

Neuromedin U (NMU), an anorexigenic peptide, was originally isolated from porcine spinal cord in 1985. As NMU is abundant in the anterior pituitary gland, we investigated the effects of NMU on gonadotropin secretion. Both NMU and its receptors, NMUR1 and NMUR2, were expressed in the pituitary gland. NMU suppressed LH and FSH releases from rat anterior pituitary cells. Moreover, NMU-deficient mice exhibit an early onset of vaginal opening. The LHbeta/FSHbeta ratio, which is an index of puberty onset, is high in young NMU-deficient mice. These results indicate that NMU suppresses gonadotropin secretion and regulates the onset of puberty.