Prototypical type I E-cadherin and type II cadherin-7 mediate very distinct adhesiveness through their extracellular domains

Using a dual pipette assay that measures the force required to separate adherent cell doublets, we have quantitatively compared intercellular adhesiveness mediated by Type I (E- or N-cadherin) or Type II (cadherin-7 or -11) cadherins. At similar cadherin expression levels, cells expressing Type I cadherins adhered much more rapidly and strongly than cells expressing Type II cadherins. Using chimeric cadherins, we found that the extracellular domain exerts by far the dominant effect on cell adhesivity, that of E-cadherin conferring high adhesivity, and that of cadherin-7 conferring low adhesivity. Type I cadherins were incorporated to a greater extent into detergent-insoluble cytoskeletal complexes, and their cytoplasmic tails were much more effective in disrupting strong adherent junctions, suggesting that Type II cadherins form less stable complexes with beta-catenin. The present study demonstrates compellingly, for the first time, that cadherins are dramatically different in their ability to promote intercellular adhesiveness, a finding that has profound implications for the regulation of tissue morphogenesis.     

Distinct roles of cadherin-6 and E-cadherin in tubulogenesis and lumen formation

Classic cadherins are important regulators of tissue morphogenesis. The predominant cadherin in epithelial cells, E-cadherin, has been extensively studied because of its critical role in normal epithelial development and carcinogenesis. Epithelial cells may also coexpress other cadherins, but their roles are less clear. The Madin Darby canine kidney (MDCK) cell line has been a popular mammalian model to investigate the role of E-cadherin in epithelial polarization and tubulogenesis. However, MDCK cells also express relatively high levels of cadherin-6, and it is unclear whether the functions of this cadherin are redundant to those of E-cadherin. We investigate the specific roles of both cadherins using a knockdown approach. Although we find that both cadherins are able to form adherens junctions at the basolateral surface, we show that they have specific and mutually exclusive roles in epithelial morphogenesis. Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation. Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity. In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.     

Biochemical and functional characterization of inositol 1,3,4,5, 6-pentakisphosphate 2-kinases

Synthesis of inositol 1,2,3,4,5,6-hexakisphosphate (IP(6)), also known as phytate, is integral to cellular function in all eukaryotes. Production of IP(6) predominately occurs through phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (IP(5)) by a 2-kinase. Recent cloning of the gene encoding this kinase from Saccharomyces cerevisiae, designated scIpk1, has identified a cellular role for IP(6) production in the regulation of mRNA export from the nucleus. In this report, we characterize the biochemical and functional parameters of recombinant scIpk1. Purified recombinant scIpk1 kinase activity is highly selective for IP(5) substrate and exhibits apparent K(m) values of 644 nm and 62.8 microm for IP(5) and ATP, respectively. The observed apparent catalytic efficiency (k(cat)/K(m)) of scIpk1 is 31,610 s(-)(1) m(-)(1). A sequence similarity search was used to identify an IP(5) 2-kinase from the fission yeast Schizosaccharomyces pombe. Recombinant spIpk1 has similar substrate selectivity and catalytic efficiency to its budding yeast counterpart, despite sharing only 24% sequence identity. Cells lacking sc-IPK1 are deficient in IP(6) production and exhibit lethality in combination with a gle1 mutant allele. Both of these phenotypes are complemented by expression of the spIPK1 gene in the sc-ipk1 cells. Analysis of several inactive mutants and multiple sequence alignment of scIpk1, spIpk1, and a putative Candida albicans Ipk1 have identified residues involved in catalysis. This includes two conserved motifs: E(i/l/m)KPKWL(t/y) and LXMTLRDV(t/g)(l/c)(f/y)I. Our data suggest that the mechanism for IP(6) production is conserved across species.     

Simultaneous isolation of two platelet membrane fractions: Biochemical,immunological and functional characterization

Simultaneous isolation of two platelet membrane subfractions was achieved by centrifugation on 40 % sucrose from a 100.000g crude membrane fraction. Characterization of both types of membranes was carried out by different biochemical and immunological markers. Using a surface label, ³H Concanavaline A (³HCon A), a marker enzyme, phosphodiesterase, and lipid analysis, one of the fraction has been identified as external or plasma membranes, the other consists of intracellular membranes. Further two specific antibodies directed against external membrane antigens (LeK^a and LgG L) react almost exclusively with the external membranes. Finally both kinds of membranes were able to uptake calcium but the affinity for this cation was higher for the internal than for the external membranes. This suggests that both membranes are implicated in the regulation of the cytoplasmic calcium concentration and that the internal membranes (dense tubular system) play the major part in this regulation.     

Characterization of cadherin-24, a novel alternatively spliced type II cadherin

Cadherins comprise a superfamily of calcium-dependent cell-cell adhesion molecules. Within the superfamily are six subfamilies including type I and type II cadherins. Both type I and type II cadherins are composed of five extracellular repeat domains with conserved calcium-binding motifs, a single pass transmembrane domain, and a highly conserved cytoplasmic domain that interacts with beta-catenin and p120 catenin. In this study, we describe a novel cadherin, cadherin-24. It is a type II cadherin with a 781-codon open reading frame, which encodes a type II cadherin protein complete with five extracellular repeats containing calcium-binding motifs, a transmembrane domain, and a conserved cytoplasmic domain. Cadherin-24 has the unusual feature of being alternatively spliced in extracellular repeat 4. This alternative exon encodes 38 in-frame amino acids, resulting in an 819-amino-acid protein. Sequence analysis suggests the presence of beta-catenin and p120 catenin-binding sequences, and immunoprecipitation experiments confirm the ability of both forms of the novel cadherin to associate with alpha-catenin, beta-catenin, and p120 catenin. In addition, aggregation assays show that both forms of cadherin-24 mediate strong cell-cell adhesion.     

The crystal structure of human E-cadherin domains 1 and 2, and comparison with other cadherins in the context of adhesion mechanism

Cell adhesion mediated by type I cadherins involves homophilic "trans" interactions that are thought to be brought about by a strand exchange mechanism involving the N-terminal extracellular domain. Here, we present the high-resolution crystal structure of the N-terminal two domains of human E-cadherin. Comparison of this structure with other type I cadherin structures reveals features that are likely to be critical to facilitate dimerization by strand exchange as well as dimer flexibility. We integrate this structural knowledge to provide a model for type I cadherin adhesive interactions. Intra-molecular docking of the conserved N-terminal "adhesion arm" into the acceptor pocket in monomeric E-cadherin appears largely identical to inter-molecular docking of the adhesion arm in adhesive trans dimers. A strained conformation of the adhesion arm in the monomer, however, may create an equilibrium between "open" and "closed" forms that primes the cadherin for formation of adhesive interactions, which are then stabilized by additional dimer-specific contacts. By contrast, in type II cadherins, strain in the adhesion arm appears absent and a much larger surface area is involved in trans adhesion, which may compensate the activation energy required to peel off the intra-molecularly docked arm. It seems that evolution has selected slightly different adhesion mechanisms for type I and type II cadherins.     

Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria

BLUF (a sensor of Blue-Light Using FAD) is a novel putative photoreceptor domain that is found in many bacteria and some eukaryotic algae. As found on genome analysis, certain cyanobacteria have BLUF proteins with a short C-terminal extension. As typical examples, Tll0078 from thermophilic Thermosynechococcus elongatus BP-1 and Slr1694 from mesophilic Synechocystis sp. PCC 6803 were comparatively studied. FAD of both proteins was hardly reduced by exogenous reductants or mediators except methylviologen but showed a typical spectral shift to a longer wavelength upon excitation with blue light. In particular, freshly prepared Tll0078 protein showed slow but reversible aggregation, indicative of light-induced conformational changes in the protein structure. Tll0078 is far more stable as to heat treatment than Slr1694, as judged from flavin fluorescence. The slr1694-disruptant showed phototactic motility away from the light source (negative phototaxis), while the wild type Synechocystis showed positive phototaxis toward the source. Yeast two-hybrid screening with slr1694 showed self-interaction of Slr1694 (PixD) with itself and interaction with a novel PatA-like response regulator, Slr1693 (PixE). These results were discussed in relation to the signaling mechanism of the "short" BLUF proteins in the regulation of cyanobacterial phototaxis.     

A comparison of two predator-prey models with Holling's type I functional response

In this paper, we analyze a laissez-faire predator-prey model and a Leslie-type predator-prey model with type I functional responses. We study the stability of the equilibrium where the predator and prey coexist by both performing a linearized stability analysis and by constructing a Lyapunov function. For the Leslie-type model, we use a generalized Jacobian to determine how eigenvalues jump at the corner of the functional response. We show, numerically, that our two models can both possess two limit cycles that surround a stable equilibrium and that these cycles arise through global cyclic-fold bifurcations. The Leslie-type model may also exhibit super-critical and discontinuous Hopf bifurcations. We then present and analyze a new functional response, built around the arctangent, that smoothes the sharp corner in a type I functional response. For this new functional response, both models undergo Hopf, cyclic-fold, and Bautin bifurcations. We use our analyses to characterize predator-prey systems that may exhibit bistability.     

Biochemical and functional characterization of high-affinity urotensin II receptors in rat cortical astrocytes

The urotensin II (UII) gene is primarily expressed in the central nervous system, but the functions of UII in the brain remain elusive. Here, we show that cultured rat astrocytes constitutively express the UII receptor (UT). Saturation and competition experiments performed with iodinated rat UII ([(125)I]rUII) revealed the presence of high- and low-affinity binding sites on astrocytes. Human UII (hUII) and the two highly active agonists hUII(4-11) and [3-iodo-Tyr9]hUII(4-11) were also very potent in displacing [(125)I]rUII from its binding sites, whereas the non-cyclic analogue [Ser5,10]hUII(4-11) and somatostatin-14 could only displace [(125)I]rUII binding at micromolar concentrations. Reciprocally, rUII failed to compete with [(125)I-Tyr0,D-Trp8]somatostatin-14 binding on astrocytes. Exposure of cultured astrocytes to rUII stimulated [(3)H]inositol incorporation and increased intracellular Ca(2+) concentration in a dose-dependent manner. The stimulatory effect of rUII on polyphosphoinositide turnover was abolished by the phospholipase C inhibitor U73122, but only reduced by 56% by pertussis toxin. The GTP analogue Gpp(NH)p caused its own biphasic displacement of [(125)I]rUII binding and provoked an affinity shift of the competition curve of rUII. Pertussis toxin shifted the competition curve towards a single lower affinity state. Taken together, these data demonstrate that rat astrocytes express high- and low-affinity UII binding sites coupled to G proteins, the high-affinity receptor exhibiting the same pharmacological and functional characteristics as UT.     

Biochemical and functional characterization of Rapana thomasiana hemocyanin

• 1.1. The hemocyanin (Hc) of the marine gastropod mollusc Rapana thomasiana was collected from animals living on the west coast of the Black Sea and characterized for its biochemical and functional properties.
• 2.2. This Hc is very similar to other gastropod Hcs as far as amino acid composition, general structure and reactivity of the binuclear copper active site are concerned.
• 3.3. Some peculiarities in the dissociation-reassociation pattern are observed in comparison to other gastropod Hcs, in particular with respect to the ability to form sopramolecular aggregates.
• 4.4. Changes in pH disclose a strong reverse Bohr effect. Different R and T states are required to describe the oxygen binding curves at the different pHs.

     

Biochemical and functional changes during immunization of rats with angiotensin II

Angiotensin-converting enzyme (ACE) activity in serum and some brain areas, level of angiotensin I in the blood and drinking behaviour during immunization of rats against conjugate of angiotensin II with bovine serum albumin (BSA) were studied. The results show that an increase in antibodies against angiotensin II was correlated with elevated ACE activity in serum. There was a distinct tendency towards elevated level of angiotensin I in the blood. After a 6 month's immunization ACE-activity was reduced twofold to threefold in midbrain and hypothalamus-thalamus. During immunization water-uptake was increased by 40-45%.     

Mechanistic studies on type I and type II dehydroquinase with (6R)- and (6S)-6-fluoro-3-dehydroquinic acids

(6R)- and (6S)-6-Fluoro-3-dehydroquinic acids are shown to be substrates for type I and type II dehydroquinases. Their differential reactivity provides insight into details of the reaction mechanism and enables a novel enzyme-substrate imine to be trapped on the type I enzyme.     

Genome-wide analysis and phosphorylation sites identification of the 14-3-3 gene family and functional characterization of MeGRF3 in cassava

The biological functionality of many members of the 14-3-3 gene family is regulated via phosphorylation at multiple amino acid residues. The specific phosphorylation-mediated regulation of these proteins during cassava root tuberization, however, is not well understood. In this study, 15 different 14-3-3 genes (designated MeGRF1 – 15) were identified within the cassava genome. Based upon evolutionary conservation and structural analyses, these cassava 14-3-3 proteins were grouped into ε and non-ε clusters. We found these 15 MeGRF genes to be unevenly distributed across the eight cassava chromosomes. When comparing the expression of these genes during different developmental stages, we found that three of these genes (MeGRF9, 12 and 15) were overexpressed at all developmental stages at 75, 104, 135, 182 and 267 days post-planting relative to the fibrous root stage, whereas two (MeGRF5 and 7) were downregulated during these same points. In addition, the expression of most MeGRF genes changed significantly in the early and middle stages of root tuberization. This suggests that these different MeGRF genes likely play distinct regulatory roles during cassava root tuberization. Subsequently, 18 phosphorylated amino acid residues were detected on nine of these MeGRF proteins. A phosphomimetic mutation at serine-65 in MeGRF3 in Arabidopsis thaliana (Arabidopsis) slightly influenced starch metabolism in these plants, and significantly affected the role of MeGRF3 in salt stress responses. Together these results indicate that 14-3-3 genes play key roles in responses to abiotic stress and the regulation of starch metabolism, offering valuable insights into the functions of these genes in cassava.     

Biochemical and functional characterization of germ line KRAS mutations

Germ line missense mutations in HRAS and KRAS and in genes encoding molecules that function up- or downstream of Ras in cellular signaling networks cause a group of related developmental disorders that includes Costello syndrome, Noonan syndrome, and cardiofaciocutaneous syndrome. We performed detailed biochemical and functional studies of three mutant K-Ras proteins (P34R, D153V, and F156L) found in individuals with Noonan syndrome and cardiofaciocutaneous syndrome. Mutant K-Ras proteins demonstrate a range of gain-of-function effects in different cell types, and biochemical analysis supports the idea that the intrinsic Ras guanosine nucleotide triphosphatase (GTPase) activity, the responsiveness of these proteins to GTPase-activating proteins, and guanine nucleotide dissociation all regulate developmental programs in vivo.     

Biochemical characterization of Bacillus subtilis type II isopentenyl diphosphate isomerase, and phylogenetic distribution of isoprenoid biosynthesis pathways.

An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184,997-186,043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 micromol.mg(-1).min(-1), respectively, as diagnosed by 1H spectroscopy. FMN and divalent cations are required for catalytic activity; the highest rates were found with Ca2+. NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of (S)-alpha-hydroxyacid oxidizing enzymes including flavocytochrome b2 and L-lactate dehydrogenase and was shown to catalyse the formation of [2,3-13C2]lactate from [2,3-13C2]pyruvate, albeit at a low rate of 1 nmol.mg(-1).min(-1). Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy.     

Biochemical and functional characterization of cation dependent (Mr 46,000) goat mannose 6-phosphate receptor

The Mannose 6-phosphate receptor (MPR’s) proteins are important for transporting lysosomal enzymes from trans-golgi to the pre-lysosomal compartment. These are conserved in the vertebrates from fish to mammals. We have cloned the full length cDNA for the goat MPR 46 protein and compared its sequences to the other known vertebrate MPR 46 proteins. In the present study the full-length cDNA for the goat MPR 46 protein was expressed in MPR deficient cells. The expressed protein was purified on the multivalent phosphomannan gel in the presence of divalent metal ions. The apparent molecular mass of the expressed protein was found to be ∼46 kDa and also exhibits oligomeric nature as observed in the other species, by using an MSC1 antibody (that recognizes the MPR 46 from molluscs to mammals) as well as with a peptide specific antibody corresponding to amino acid residues (218–237) of the cytoplasmic tail of human MPR 46 protein. Furthermore the distribution of the expressed protein was visualized by immunofluorescence using MSC1 and LAMP1 antibody. Additionally in the goat MPR 46 expressing cells, the sorting function of the expressed protein to sort cathepsin D to lysosomes was studied by confocal microscopy using cathepsin D antiserum and LAMP1 antibody. The binding of goat MPR 46 to cathepsin D was shown in far Western blotting and the mannose 6-phosphate dependent binding was shown by co-immunoprecipitation.     

Biochemical characterization of arylsulfatase E and functional analysis of mutations found in patients with X-linked chondrodysplasia punctata.

X-linked chondrodysplasia punctata (CDPX) is a congenital disorder characterized by abnormalities in cartilage and bone development. Mutations leading to amino acid substitutions were identified recently in CDPX patients, in the coding region of the arylsulfatase E (ARSE) gene, a novel member of the sulfatase gene family. Transfection of the ARSE full-length cDNA, in Cos7 cells, allowed us to establish that its protein product is a 60-kD precursor, which is subject to N-glycosylation, to give a mature 68-kD form that, unique among sulfatases, is localized to the Golgi apparatus. Five missense mutations found in CDPX patients were introduced into wild-type ARSE cDNA by site-directed mutagenesis. These mutants were transfected into Cos7 cells, and the arylsulfatase activity and biochemical properties were determined, to study the effect of these substitutions on the ARSE protein. One of the mutants behaves as the wild-type protein. All four of the other mutations resulted in a complete lack of arylsulfatase activity, although the substitutions do not appear to affect the stability and subcellular localization of the protein. The loss of activity due to these mutations confirms their involvement in the clinical phenotype and points to the importance of these residues in the correct folding of a catalytically active ARSE enzyme.