Inheritance of alleles for glutelin alpha-2 subunit genes in rice and identification of their corresponding cDNA clone

Rice glutelins consist of acidic (alpha) and basic (beta) subunits which are further separated into three polypeptide components assigned as alpha-1, alpha-2, and alpha-3 subunit components and beta-1, beta-2 and beta-3 subunit components. Nine rice mutant lines with a decreased amount of the glutelin alpha-2 subunit component (alpha-2L) were obtained by screening about 6,800 potential mutant lines derived from the fertilized egg treatment with N-methyl-N-nitrosourea (MNU) using the SDS-PAGE method. The mutants were classified into three types of the increased alpha-1 subunit (alpha-1H/alpha-2L), the decreased beta-2 subunit (beta-2L/alpha-2L) and the increased alpha-3 subunit (alpha-3H/alpha-2L) represented by EM278, CM1707 and EM659, respectively. Iso-electric focus (IEF) analysis revealed that all of the mutants had an extremely low amount of a polypeptide with a 6.71 pI value, whereas a polypeptide with either a 6.50 pI value or with a 6.90 pI value increased significantly in alpha-1H/alpha-2L mutants or in alpha-3H/alpha-2L mutants, respectively. The beta-2L/alpha-2L mutants had a decreased amount of a basic polypeptide with a 8.74 pI value. Genetic analysis revealed that the three types of mutants were controlled by a single incomplete dominant gene respectively, and the three are alleles. The gene was temporarily named glu4, which was found to be located on chromosome 1 linked with the eg and spl6 genes. Two-dimensional electrophoresis analysis revealed that the glu4 encoded polypeptides of pI 6.71/alpha-2 and pI 8.74/beta-2. Amino acid sequence analysis suggested that the mutated acidic polypeptide was the product of a GluA subfamily gene. Northern and RT-PCR analyses revealed that glu4 corresponded to the GluA-1 gene.     

Activation of the lectin complement pathway by H-ficolin (Hakata antigen)

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.     

Generation of amyloid beta protein from a presenilin-1 and betaAPP complex

Presenilin-1 (PS1) is a causative gene in early onset familial Alzheimer's disease (FAD). FAD-linked mutant PS1s significantly increased Abeta40 and Abeta42(43) levels (P < 0.001) and decreased the production of an 11.4 kD (beta-stub) and an 8.7 kD (alpha-stub) carboxyl-terminal fragment of amyloid beta precursor protein (betaAPP-CTFs) (P < 0.01). In the 2% CHAPS extracted lysates, the complex containing the amino-terminal fragment of PS1 (PS1-NTF), the carboxyl-terminal fragments of PS1 (PS1-CTF), and betaAPP-CTFs was identified. Incubation of this isolated complex at pH 6.4 showed the direct generation of Abeta40 and gamma-stub from this complex. This reaction was inhibited by a gamma-secretase inhibitor. The degrading rate of a co-precipitated beta-stub was facilitated under the presence of FAD-linked mutant PS1s. This findings suggest that the direct generation of Abeta from the complex may play an important role in the pathogenesis of Alzheimer's disease.     

Apoptosis induction by wheat-flour sphingoid bases in DLD-1 human colon cancer cells

The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.     

Existence of cerebroside in Saccharomyces kluyveri and its related species

Sphingolipids are ubiquitous compounds derived from ceramide that consist of a sphingoid long-chain base with a 2-amino group amide linked to fatty acid and are present in the membranes of many organisms. As a principal sphingolipid, Saccharomyces cerevisiae contains a free ceramide and its inositol-phosphorylated derivatives (acidic types) but not a neutral glycosylated ceramide, glucosylceramide (cerebroside), which usually appears in eukaryotic cells. When 31 strains accepted in the genera Saccharomyces, Torulaspora, Zygosaccharomyces, and Kluyveromyces were analyzed for sphingolipids, cerebrosides were found in S. kluyveri, Z. cidri, Z. fermentati, K. lactis, K. thermotolerans, and K. waltii. The cerebrosides of S. kluyveri and K. lactis included 9-methyl 4-trans, 8-trans-sphingadienine and its putative metabolic intermediates. A unique characteristic of S. kluyveri was the presence of a trihydroxy sphingoid base, which rarely occurs in fungal cerebrosides. A polymerase chain reaction with primers targeted to the glucosylceramide synthase gene of other microorganisms amplified the fragments of the expected size from S. kluyveri and K. lactis and further extended to the adjacent regions. The presumed protein of S. kluyveri had 54.4% similarity to that of K. lactis, higher than the glucosylceramide synthases from Candida albicans, Pichia pastoris, and other organisms. From these observations, the divergence of S. kluyveri from the lineage of K. lactis in their evolution is discussed.     

Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.     

A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.     

Effects of surgical manipulation of the intestine on peptide YY and its physiology

PYY is a gastrointestinal hormone, mainly released from the distal intestine in response to intraluminal nutrients or via a neurohormonal pathway originating in the proximal intestine. Although there are several molecular forms of circulating PYY with different bioactivity, and further more than six subtypes of Y-receptors, the function is essentially inhibitory to digestive organs located upstream of the digestive tract. These inhibitory mechanisms are named jejunal, ileal and colonic brakes, and play an important supplementary role in adaptation following intestinal resection. When massive resection of the small intestine is performed, the release of PYY from the distal intestine increases, suppressing gastric acid secretion and motility of the gastrointestinal tract, and stimulating pancreatic secretion. After total colectomy, PYY release is reduced first due to reduction of PYY-containing cells, then gradually increases with time, contributing to adaptation of the digestive organs to the new condition.     

The microtubule stabilizing agent laulimalide does not bind in the taxoid site,kills cells resistant to paclitaxel and epothilones,and may not require its epoxide moiety for activity

Laulimalide is a cytotoxic natural product that stabilizes microtubules. The compound enhances tubulin assembly, and laulimalide is quantitatively comparable to paclitaxel in its effects on the reaction. Laulimalide is also active in P-glycoprotein overexpressing cells, while isolaulimalide, a congener without the drug's epoxide moiety, was reported to have negligible cytotoxic and biochemical activity [Mooberry et al. (1999) Cancer Res. 59, 653-660]. We report here that laulimalide binds at a site on tubulin polymer that is distinct from the taxoid site. We found that laulimalide, while as active as paclitaxel, epothilone A, and eleutherobin in promoting the assembly of cold-stable microtubules, was unable to inhibit the binding of radiolabeled paclitaxel or of 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]paclitaxel, a fluorescent paclitaxel derivative, to tubulin. Confirming this observation, we demonstrated that microtubules formed in the presence of both laulimalide and paclitaxel contained near-molar quantities, relative to tubulin, of both drugs. Laulimalide was active against cell lines resistant to paclitaxel or epothilones A and B on the basis of mutations in the M40 human beta-tubulin gene. We also report that a laulimalide analogue lacking the epoxide moiety, while less active than laulimalide in biochemical and cellular systems, is probably more active than isolaulimalide. Further exploration of the role of the epoxide in the interaction of laulimalide with tubulin is therefore justified.