Ionic, structural, and temperature effects on DNA nanoparticles formed by natural and synthetic polyamines

We synthesized analogues of spermine and studied the effects of chemical structure, ionic strength, and temperature on lambda-DNA nanoparticle formation. Effective concentration of polyamines for DNA condensation (EC50) was lowest for hexamines (0.2 microM) and highest for spermine (tetramine, 4.2 microM). The EC50 value increased with [Na+]. Dynamic light scattering showed nanoparticles with hydrodynamic radii (R(h)) of 40-50 nm. Effect of temperature on R(h) was measured between 20 and 70 degrees C. For spermine, R(h) remained relatively stable until 50 degrees C and increased significantly at >60 degrees C. In contrast, the hexa- and penta-valent analogues exhibited a gradual increase in R(h) between 20 and 70 degrees C. The nanoparticles were mainly toroidal, as revealed by electron microscopy (EM). EM studies showed changes in morphology and size of condensed structures with an increase in temperature. A possible mechanism for the differential effects of temperature on DNA nanoparticles might involve different modes of DNA-polyamine interactions.     

Characterization of structural and catalytic differences in rat intestinal alkaline phosphatase isozymes

To understand the differences between the rat intestinal alkaline phosphatase isozymes rIAP-I and rIAP-II, we constructed structural models based on the previously determined crystal structure for human placental alkaline phosphatase (hPLAP). Our models of rIAP-I and rIAP-II displayed a typical alpha/beta topology, but the crown domain of rIAP-I contained an additional beta-sheet, while the embracing arm region of rIAP-II lacked the alpha-helix, when each model was compared to hPLAP. The representations of surface potential in the rIAPs were predominantly positive at the base of the active site. The coordinated metal at the active site was predicted to be a zinc triad in rIAP-I, whereas the typical combination of two zinc atoms and one magnesium atom was proposed for rIAP-II. Using metal-depleted extracts from rat duodenum or jejunum and hPLAP, we performed enzyme assays under restricted metal conditions. With the duodenal and jejunal extract, but not with hPLAP, enzyme activity was restored by the addition of zinc, whereas in nonchelated extracts, the addition of zinc inhibited duodenal IAP and hPLAP, but not jejunal IAP. Western blotting revealed that nearly all of the rIAP in the jejunum extracts was rIAP-I, whereas in duodenum the percentage of rIAP-I (55%) correlated with the degree of AP activation (60% relative to that seen with jejunal extracts). These data are consistent with the presence of a triad of zinc atoms at the active site of rIAP-I, but not rIAP-II or hPLAP. Although no differences in amino acid alignment in the vicinity of metal-binding site 3 were predicted between the rIAPs and hPLAP, the His153 residue of both rIAPs was closer to the metal position than that in hPLAP. Between the rIAPs, a difference was observed at amino acid position 317 that is indirectly related to the coordination of the metal at metal-binding site 3 and water molecules. These findings suggest that the side-chain position of His153, and the alignment of Q317, might be the major determinants for activation of the zinc triad in rIAP-I.     

Collagenous Alzheimer amyloid plaque component assembles amyloid fibrils into protease resistant aggregates

Recently, a novel plaque-associated protein, collagenous Alzheimer amyloid plaque component (CLAC), was identified in brains from patients with Alzheimer's disease. CLAC is derived from a type II transmembrane collagen precursor protein, termed CLAC-P (collagen XXV). The biological function and the contribution of CLAC to the pathogenesis of Alzheimer's disease and plaque formation are unknown. In vitro studies indicate that CLAC binds to fibrillar, but not to monomeric, amyloid beta-peptide (Abeta). Here, we examined the effects of CLAC on Abeta fibrils using assays based on turbidity, thioflavin T binding, sedimentation analysis, and electron microscopy. The incubation of CLAC with preformed Abeta fibrils led to increased turbidity, indicating that larger aggregates were formed. In support of this contention, more Abeta was sedimented in the presence of CLAC, as determined by gel electrophoresis. Moreover, electron microscopy revealed an increased amount of Abeta fibril bundles in samples incubated with CLAC. Importantly, the frequently used thioflavin T-binding assay failed to reveal these effects of CLAC. Digestion with proteinase K or trypsin showed that Abeta fibrils, incubated together with CLAC, were more resistant to proteolytic degradation. Therefore, CLAC assembles Abeta fibrils into fibril bundles that have an increased resistance to proteases. We suggest that CLAC may act in a similar way in vivo.     

Echinococcus shiquicus n. sp., a taeniid cestode from Tibetan fox and plateau pika in China

The taeniid cestode Echinococcus shiquicus n. sp. was found from the Tibetan fox Vulpes ferrilata and the plateau pika Ochotona curzoniae in the Qinghai-Tibet plateau region of China. In the adult stage, E. shiquicus from the foxes is morphologically similar to Echinococcus multilocularis. However, the new species is differentiated by its smaller rostellar hooks, fewer segments, distinct position of genital pore in the mature segment and fewer eggs in the gravid segment. Hydatid cysts of E. shiquicus found in the livers from the pikas were essentially unilocular but an oligovesicular cyst was also found. The data of mitochondrial and nuclear DNA sequences proved E. shiquicus to be a valid taxon.     

Amino acids in positions 48, 52, and 73 differentiate the substrate specificities of the highly homologous chlorocatechol 1,2-dioxygenases CbnA and TcbC

Chlorocatechol 1,2-dioxygenase (CCD) is the first-step enzyme of the chlorocatechol ortho-cleavage pathway, which plays a central role in the degradation of various chloroaromatic compounds. Two CCDs, CbnA from the 3-chlorobenzoate-degrader Ralstonia eutropha NH9 and TcbC from the 1,2,4-trichlorobenzene-degrader Pseudomonas sp. strain P51, are highly homologous, having only 12 different amino acid residues out of identical lengths of 251 amino acids. But CbnA and TcbC are different in substrate specificities against dichlorocatechols, favoring 3,5-dichlorocatechol (3,5-DC) and 3,4-dichlorocatechol (3,4-DC), respectively. A study of chimeric mutants constructed from the two CCDs indicated that the N-terminal parts of the enzymes were responsible for the difference in the substrate specificities. Site-directed mutagenesis studies further identified the amino acid in position 48 (Leu in CbnA and Val in TcbC) as critical in differentiating the substrate specificities of the enzymes, which agreed well with molecular modeling of the two enzymes. Mutagenesis studies also demonstrated that Ile-73 of CbnA and Ala-52 of TcbC were important for their high levels of activity towards 3,5-DC and 3,4-DC, respectively. The importance of Ile-73 for 3,5-DC specificity determination was also shown with other CCDs such as TfdC from Burkholderia sp. NK8 and TfdC from Alcaligenes sp. CSV90 (identical to TfdC from R. eutropha JMP134), which convert 3,5-DC preferentially. Together with amino acid sequence comparisons indicating high conservation of Leu-48 and Ile-73 among CCDs, these results suggested that TcbC of strain P51 had diverged from other CCDs to be adapted to conversion of 3,4-DC.     

Structural and functional characterization of rabbit and human L-gulonate 3-dehydrogenase

L-Gulonate 3-dehydrogenase (GDH) catalyzes the NAD(+)-linked dehydrogenation of L-gulonate into dehydro-L-gulonate in the uronate cycle. In this study, we isolated the enzyme and its cDNA from rabbit liver, and found that the cDNA is identical to that for rabbit lens lambda-crystallin except for lacking a codon for Glu(309). The same cDNA species, but not the lambda-crystallin cDNA with the codon for Glu(309), was detected in the lens, which showed the highest GDH activity among rabbit tissues. In addition, recombinant human lambda-crystallin that lacks Glu(309) displays enzymatic properties similar to rabbit GDH. These data indicate that GDH is recruited as lambda-crystallin without gene duplication. An outstanding feature of GDH is modulation of its activity by low concentrations of P(i), which decreases the catalytic efficiency in a dose dependent manner. P(i) also protects the enzyme against both thermal and urea denaturation. Kinetic analysis suggests that P(i) binds to both the free enzyme and its NAD(H)-complex in the sequential ordered mechanism. Furthermore, we examined the roles of Asp(36), Ser(124), His(145), Glu(157 )and Asn(196) in the catalytic function of rabbit GDH by site-directed mutagenesis. The D36R mutation leads to a switch in favor of NADP(H) specificity, suggesting an important role of Asp(36) in the coenzyme specificity. The S124A mutation decreases the catalytic efficiency 500-fold, and the H145Q, N196Q and N195D mutations result in inactive enzyme forms, although the E157Q mutation produces no large kinetic alteration. Thus, Ser(124), His(145) and Asn(196) may be critical for the catalytic function of GDH.     

Galectin-4 binds to sulfated glycosphingolipids and carcinoembryonic antigen in patches on the cell surface of human colon adenocarcinoma cells

Galectin-4, a member of the galectin family, is expressed in the epithelium of the alimentary tract. It has two tandemly repeated carbohydrate recognition domains and specifically binds to an SO3- -->3Galbeta1-->3GalNAc pyranoside with high affinity (Ideo, H., Seko, A., Ohkura, T., Matta, K. L., and Yamashita, K. (2002) Glycobiology 12, 199-208). In this study, we found that galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, such as SB1a, SM3, SM4s, SB2, SM2a, and GM1, but not to glycosphingolipids with 3-O-sialylated Gal, such as sLc4Cer, snLc4Cer, GM3, GM2, and GM4, using both an enzyme-linked immunosorbent assay and a surface plasmon resonance assay. A confocal immunocytochemical assay showed that galectin-4 was colocalized with SB1a, GM1, and carcinoembryonic antigen (CEA) in the patches on the cell surface of human colon adenocarcinoma CCK-81 and LS174T cells. This localization was distinct from caveolin/VIP21 localization. Furthermore, immobilized galectin-4 promoted adhesion of CCK-81 cells through the sulfated glycosphingolipid, SB1a. CEA also bound to galectin-4 with KD value of 2 x 10(-8) m by surface plasmon resonance and coimmunoprecipitated with galectin-4 in LS174T cell lysates. These findings suggest that SB1a and CEA in the patches on the cell surface of human colon adenocarcinoma cells could be biologically important ligands for galectin-4.     

Ceramide 1-phosphate, a mediator of phagocytosis

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the FcgammaRIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.     

A mode of assembly of P0, P1, and P2 proteins at the GTPase-associated center in animal ribosome: in vitro analyses with P0 truncation mutants

Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CDelta65 and CDelta81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0.P1-P2 binding site. The mutant CDelta107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NDelta21-NDelta92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants NDelta6, NDelta14, and CDelta18-CDelta81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10.L7/L12 complex and L11. Analysis of incorporation of (32)P-labeled P1/P2 into the 50 S subunits with WT-P0 and CDelta81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CDelta81 bound only one copy each. The hybrid ribosome with CDelta81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.