N-glycan structures of a recombinant mouse soluble Fcγ receptor II

N-glycans of a recombinant mouse soluble Fc receptor II (sFcRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226: 139–46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was -(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and -(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry.     

Nucleic Acid Metabolism Involved in Auxin-induced Elongation of Yeast Cells

The following results were obtained using a variant yeast strain, N55, which can respond to the cell-elongating action of auxin. Base analogs of nucleic acids (2-thiouracil, 8-azaguanine, and 5-fluorouracil) inhibited the auxin-induced elongation of yeast cells only when they were added to the preculture prior to auxin treatment. The inhibitory effect of 2-thiouracil and 5-fluorouracil was reversed by uracil and that of 8-azaguanine by guanine. Actino-mycin D inhibited the auxin-induced elongation when given to the culture containing auxin, but not when given to the preculture. The similarity in these respects between yeast and tissues of higher plants is discussed.     

Fatty acid incorporation by Rhodnius prolixus midgut

[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.     

In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5' splice sites

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (R_i) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity = 97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions −3, −2 and −1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site.     

Growth and cell wall changes in azuki bean epicotyls I. Changes in wall polysaccharides during intact growth

Measurement of endogenous growth rates and the mechanical propertyof the cell wall in various regions of light-grown azuki beanepicotyls revealed diat the minimum stress-relaxation time (T_o)was the shortest in the upper region (0–30 mm below theapex) of the epicotyl, where vigorous endogenous growth tookplace, and became longer toward the basal region, which wasmature and not growing. In the upper region of the epicotyl, a lower percentage of a-celluloseand a higher percentage of pectic substances than in the lowerregion were found. The percentage of hemicellulose content wasalmost constant over the whole epicotyl. Major components ofnoncellulosic neutral sugars in the cell wall were galactoseand xylose. The percentage of the galactose content to the noncellulosicpolysaccharide was highest in the upper region and lowest inthe basal region of the epicotyl, and a clearly negative correlationbetween the galactose composition and the T_o value was obtained.On the contrary, the percentage of die xylose content was highestin the basal region and lowest in die upper region, and a clearlypositive correlation between die xylose composition and theT_o value was obtained. During die endogenous growth of die intactepicotyl, all die neutral sugars, particularly galactose, increasedin die upper region, whereas in die middle and basal regions,only xylose increased. Similar changes in sugar compositionswere observed during IAA-induced elongation of die segment excisedfrom various regions of die epicotyl. (Received July 27, 1978; )     

Tumor suppressor in lung cancer (TSLC)1 suppresses epithelial cell scattering and tubulogenesis

The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases.     

Change in enantioselectivity in bufuralol 1''-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6

The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1'-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1'-hydroxylase activities at a substrate concentration of 100 microM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF > S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF < or = S-BF), whereas the product diastereoselectivity of (1'R-OH-BF < 1'-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K(m) values were similar among the recombinant enzymes, and V(max) values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1'-hydroxylation by CYP2D6.     

Reinstatement of Grateloupia subpectinata (Rhodophyta, Halymeniaceae) based on morphology and rbcL sequences

Morphological observations and molecular analyses of the north‐western Pacific species of the red algal genus Grateloupia (Halymeniaceae) indicate the presence of an entity, which is somewhat similar in gross morphology to G. asiatica Kawaguchi et Wang but is distinguished from the latter species by some morphological features. These include: (i) a somewhat fleshy texture; (ii) wider and much thicker (4.5–10 mm wide and up to 1300 μm thick) axes, of which an inner cortex consists of more (6–9) cells; (iii) generally longer (up to 17 cm), marginal and surface proliferations that are clearly constricted (terete) at bases; and (iv) much elongated, oblong auxiliary cells. Phylogenetic analysis using the ribulose‐l,5‐bisphosphate carboxylase/ oxygenase (rbcL) gene of G. asiatica and the alga in question shows them to be distantly related and strongly supports the differentiation of these two entities at the species level. Judging from the literature, this entity is actually Grateloupia subpectinata Holmes, which has been placed into synonymy under G. asiatica [as G. filicina (Lamouroux) C. Agardh] or G. prolongata J. Agardh in previous reports, and therefore the Holmes name is reinstated.     

Effects of various growth conditions in a chemostat on expression of virulence factors in Porphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.     

Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica

We cloned two forms of the secreted and thermostable luciferase genes, MpLuc1 and MpLuc2, from the marine copepod, Metridia pacifica. The 840-bp MpLuc1 cDNA comprised a 630-bp open reading frame encoding a 210-amino acid polypeptide (22.7 kDa). MpLuc1 had the closest homology with Metridia longa luciferase. The 753-bp MpLuc2 cDNA consisted of a 567-bp open reading frame (20.3 kDa), and it had the closest homology with Gaussia princeps luciferase. Single-specimen genomic PCR confirmed the presence of two luciferase genes in M. pacifica, and single-specimen RT-PCR revealed that both luciferase mRNAs were expressed. Both MpLuc1 and MpLuc2 (MpLucs) specifically reacted with the substrate coelenterazine producing identical bioluminescent spectra (lambdamax, 485 nm), but with different kinetics. Adding salt such as MgCl2 and CaCl2 to the reaction mixture significantly enhanced MpLuc1 and MpLuc2 activities. Wild-type MpLucs were remarkably thermostable; MpLuc1 retained about 60% of the original activity even after incubation at 90 degrees C for 30 min. MpLucs expressed in NIH-3T3 and HeLa cells were largely secreted into the culture medium. Continuous monitoring of secreted MpLuc1 driven by the c-fos promoter demonstrated the potential usefulness of MpLuc1 in nondisruptive reporter assays.