Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering

To monitor the fast compaction process during protein folding, we have used a stopped-flow small-angle X-ray scattering technique combined with a two-dimensional charge-coupled device-based X-ray detector that makes it possible to improve the signal-to-noise ratio of data dramatically, and measured the kinetic refolding reaction of alpha-lactalbumin. The results clearly show that the radius of gyration and the overall shape of the kinetic folding intermediate of alpha-lactalbumin are the same as those of the molten globule state observed at equilibrium. Thus, the identity between the kinetic folding intermediate and the equilibrium molten globule state is firmly established. The present results also suggest that the folding intermediate is more hydrated than the native state and that the hydrated water molecules are dehydrated when specific side-chain packing is formed during the change from the molten globule to the native state.     

Mitochondrial permeability transition mediates apoptosis induced by N-methyl(R)salsolinol,an endogenous neurotoxin,and is inhibited by Bcl-2 and rasagiline,N-propargyl-1(R)-aminoindan

The role of mitochondrial permeability transition (PT) in apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol [NM(R)Sal], was studied by use of dopaminergic neuroblastoma SH-SY5Y cells. NM(R)Sal reduced mitochondrial membrane potential, DeltaPsim, in the early phase of apoptosis, which was not suppressed by a pan-caspase inhibitor, but was antagonized by Bcl-2 and cyclosporin A, suggesting the involvement of the PT in NM(R)Sal-induced loss of DeltaPsim. NM(R)Sal-induced apoptosis was completely inhibited not only by Bcl-2 and a pan-caspase inhibitor, but also by cyclosporin A, suggesting the essential role of the PT in NM(R)Sal-induced apoptosis. In mitochondria isolated from rat liver, NM(R)Sal induced swelling and reduced DeltaPsim, which was inhibited by cyclosporin A and Bcl-2 overexpression. These results indicate that NM(R)Sal induced the PT by direct action on the mitochondria. Rasagiline, N-propargyl-1(R)-aminoindan, which is a now under a clinical trial for Parkinson's disease, suppressed the DeltaPsim reduction, release of cytochrome c, and apoptosis induced by NM(R)Sal in SH-SY5Y cells. Rasagiline also inhibited the NM(R)Sal-induced loss of DeltaPsim and swelling in the isolated mitochondria, proving that rasagiline directly targets the mitochondria also. Altogether, mitochondrial PT plays a key role both in NM(R)Sal-induced cell death and the neuroprotective effect of rasagiline.     

Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display-positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5-trisphosphate (IP(3)) 5-phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large-type Kenyon cells, suggesting that IP(3)-mediated Ca(2+) signaling is enhanced in these neurons.     

Charge conversion enables quantification of the proximity between a normally-neutral mu-conotoxin (GIIIA) site and the Na+ channel pore

mu-Conotoxin (mu-CTX) inhibits Na+ flux by obstructing the Na+ channel pore. Previous studies of mu-CTX have focused only on charged toxin residues, ignoring the neutral sites. Here we investigated the proximity between the C-terminal neutral alanine (A22) of mu-CTX and the Na+ channel pore by replacing it with the negatively charged glutamate. The analog A22E and wild-type (WT) mu-CTX exhibited identical nuclear magnetic resonance spectra except at the site of replacement, verifying that they have identical backbone structures. A22E significantly reduced mu-CTX affinity for WT mu1 Na+ channels (90-fold), as if the inserted glutamate repels the anionic pore receptor. We then looked for the interacting partner(s) of residue 22 by determining the potency of block of Y401K, Y401A, E758Q, D762K, D762A, E765K, E765A and D1241K channels by WT mu-CTX and A22E, followed by mutant cycle analysis to assess their individual couplings. Our results show that A22E interacts strongly with E765K from domain II (DII) (deltadeltaG=2.2 +/- 0.1 vs. <1 kcal/mol for others). We conclude that mu-CTX residue 22 closely associates with the DII pore in the toxin-bound channel complex. The approach taken may be further exploited to study the proximity of other neutral toxin residues with the Na+ channel pore.     

Association of plasma viral RNA load with prognosis in cats naturally infected with feline immunodeficiency virus

We measured the quantity of plasma feline immunodeficiency virus (FIV) RNA using a real-time sequence detecting system. Plasma viral RNA load was shown to correlate with the clinical stage, survival time, and disease progression in naturally FIV-infected cats. The present study indicates that the plasma viral RNA load can be used as a clinical marker representing the impairment of the immune system and predicting the clinical outcome in FIV-infected cats.     

Suppressive effect of polysaccharides from the edible and medicinal mushrooms,Lentinus edodes and Agaricus blazei,on the expression of cytochrome P450s in mice

To investigate the effects of lentinan from Lentinas edodes and polysaccharides from Agaricus blazei (ABPS) on the expression of cytochrome P450s (CYPs), lentinan (10 mg/kg/day) or ABPS (200 mg/kg/day) was administered to female BALB/c mice four times every other day by intraperitoneal injection. Lentinan and ABPS suppressed both the constitutive and 3-methylcholanthrene-induced CYP1A expression and ethoxyresorufin-O-deethylation activity in the liver.     

Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots

Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.     

Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule

Leukocyte common antigen-related molecule (LAR) is a receptor-like protein tyrosine phosphatase (PTPase) with two PTPase domains. In the present study, we detected the expression of LAR in the brain, kidney, and thymus of mice using anti-LAR PTPase domain subunit monoclonal antibody (mAb) YU1. In the thymus, LAR was expressed on CD4(-)CD8(-) and CD4(-)CD8(low) thymocytes. The development of thymocytes in CD45 knockout mice is blocked partially in the maturation of CD4(-)CD8(-) to CD4(+)CD8(+). We postulated that LAR regulates Lck and Fyn in the immature thymocytes. Transfection of wild-type LAR activated extracellular signal-regulated kinase signal transduction pathway in CD45-deficient Jurkat cells stimulated with anti-CD3 mAb. LAR mutants, with Cys to Ser mutation in the catalytic center of PTPase D1, bound to tyrosine-phosphorylated Lck and Fyn, and LAR PTPase domain 2 was tyrosine phosphorylated by Fyn tyrosine kinase. The phosphorylated LAR was associated with Fyn Src homology 2 domain. Moreover, LAR dephosphorylated phosphorylated tyrosine residues in both the COOH terminus and kinase domain of Fyn in vitro. Our results indicate that Lck and Fyn would be substrates of LAR in immature thymocytes and that each LAR PTPase domain plays distinct functional roles in phosphorylation and dephosphorylation.     

Proteome approaches to characterize seed storage proteins related to ditelocentric chromosomes in common wheat (Triticum aestivum L.)

Changes in protein composition of wheat endosperm proteome were investigated in 39 ditelocentric chromosome lines of common wheat (Triticum aestivum L.) cv. Chinese Spring. Two-dimensional gel electrophoresis followed by Coomassie Brilliant Blue staining has resolved a total of 105 protein spots in a gel. Quantitative image analysis of protein spots was performed by PDQuest. Variations in protein spots between the euploid and the 39 ditelocentric lines were evaluated by spot number, appearance, disappearance and intensity. A specific spot present in all gels was taken as an internal standard, and the intensity of all other spots was calculated as the ratio of the internal standard. Out of the 1755 major spots detected in 39 ditelocentric lines, 1372 (78%) spots were found variable in different spot parameters: 147 (11%) disappeared, 978 (71%) up-regulated and 247 (18%) down-regulated. Correlation studies in changes in protein intensities among 24 protein spots across the ditelocentric lines were performed. High correlations in changes of protein intensities were observed among the proteins encoded by genes located in the homoeologous arms. Locations of structural genes controlling 26 spots were identified in 10 chromosomal arms. Multiple regulators of the same protein located at various chromosomal arms were also noticed. Identification of structural genes for most of the proteins was found difficult due to multiple regulators encoding the same protein. Two novel subunits (1B(Z,) 1BDz), the structure of which are very similar to the high molecular weight glutenin subunit 12, were identified, and the chromosome arm locations of these subunits were assigned.