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PubMed2003

Surface and capillary transitions in an associating binary mixture model.

We investigate the phase diagram of a two-component associating fluid mixture in the presence of selectively adsorbing substrates. The mixture is characterized by a bulk phase diagram that displays peculiar features such as closed loops of immiscibility. The presence of the substrates may interfere with the physical mechanism involved in the appearance of these phase diagrams, leading to an enhanced tendency to phase separate below the lower critical solution point. Three different cases are considered: a planar solid surface in contact with a bulk fluid, while the other two represent two models of porous systems, namely, a slit and an array on infinitely long parallel cylinders. We confirm that surface transitions, as well as capillary transitions for a large surface area to volume ratio, are stabilized in the one-phase region. Applicability of our results to experiments reported in the literature is discussed.

Journal Article↗

PubMed1988

Specific killing of human endothelial cells by antibody-conjugated glucose oxidase.

Conjugates of antibody with glucose oxidase obtained via the carbohydrate moiety of the enzyme via oxidation with periodate are suggested as a tool for selective killing of the target cell. The conjugate was separated from uncoupled enzyme by repeated precipitation with ammonium sulfate (42% saturation). Purity of the conjugate was estimated by gel filtration on Toya Pearl TP-65. The glucose oxidase conjugated with rabbit antibody against mouse IgG bound specifically to plastic-adsorbed mouse immunoglobulins and to cultured human endothelial cells pretreated with mouse anti-endothelial antiserum. Glucose oxidase targeted to the cells generates hydrogen peroxide in the presence of glucose. This hydrogen peroxide killed the endothelial cells even in the absence of a halide-peroxidase system and in the presence of catalase. Features of the conjugate (specificity, effective cytotoxicity, high stability) make it suitable for prospective in vivo application and for immunoselective segregation of heterogeneous cell populations.

Animals↗

PubMed2007

Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise.

O-GlcNAcase catalyzes the cleavage of beta-O-linked 2-acetamido-2-deoxy-beta-d-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitors of this enzyme are O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-methyl-alpha-d-glucopyranoso[2,1-d]-Delta2'-thiazoline (NAG-thiazoline). Derivatives of these inhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomal beta-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline derivatives showing high selectivities. The molecular basis for this difference in selectivities is addressed as is how well these inhibitors mimic the O-GlcNAcase-stabilized transition state (TS). Using a series of substrates, ground state (GS) inhibitors, and transition state mimics having analogous structural variations, we describe linear free energy relationships of log(KM/kcat) versus log(KI) for PUGNAc and NAG-thiazoline. These relationships suggest that PUGNAc is a poor transition state analogue, while NAG-thiazoline is revealed as a transition state mimic. Comparative X-ray crystallographic analyses of enzyme-inhibitor complexes reveal subtle molecular differences accounting for the differences in selectivities between these two inhibitors and illustrate key molecular interactions. Computational modeling of species along the reaction coordinate, as well as PUGNAc and NAG-thiazoline, provide insight into the features of NAG-thiazoline that resemble the transition state and reveal where PUGNAc fails to capture significant binding energy. These studies also point to late transition state poise for the O-GlcNAcase catalyzed reaction with significant nucleophilic participation and little involvement of the leaving group. The potency of NAG-thiazoline, its transition state mimicry, and its lack of traditional transition state-like design features suggest that potent rationally designed glycosidase inhibitors can be developed that exploit variation in transition state poise.

Acetylglucosamine↗

PubMed2003

Subset-specific, uniform activation among V gamma 6/V delta 1+ gamma delta T cells elicited by inflammation.

The V gamma 6/V delta 1(+) cells, the second murine gamma delta T cell subset to arise in the thymus, express a nearly invariant T cell receptor (TCR), colonize select tissues, and expand preferentially in other tissues during inflammation. These cells are thought to help in regulating the inflammatory response. Until now, V gamma 6/V delta 1(+) cells have only been detectable indirectly, by expression of V gamma 6-encoding mRNA. Here, we report that 17D1, a monoclonal antibody, which detects the related epidermis-associated V gamma 5/V delta 1(+) TCR, will also bind the V gamma 6/V delta 1(+) cells if their TCR is first complexed to an anti-C delta antibody. Features of this special condition for recognition suggest the possibility that an alternate structure exists for the V gamma 6/V delta 1 TCR, which is stabilized upon binding to the anti-C delta antibody. Using the 17D1 antibody as means to track this gamma delta T cell subset by flow cytometry, we discovered that the response of V gamma 6/V delta 1(+) cells during inflammation often far exceeds that of other subsets and that the responding V gamma 6/V delta 1(+) cells display a strikingly uniform activation/memory phenotype compared with other gamma delta T cell subsets.

Animals↗

PubMed2006

Expression and characterization of a recombinant single-domain monoclonal antibody against MUC1 mucin in tobacco plants.

A promising alternative to conventional antibodies is the single-domain antibody fragment of the Camelidae (V(HH)), which (because of features such as small length, high expression, solubility, and stability) is preferred to other antibody derivatives. In this report, a recombinant single-domain antibody (V(HH)) against MUC1 mucin in the tobacco plant, which may be considered as a suitable and economical alternative expression system, was produced. This antibody was expressed under the control of a strong constitutive promoter, CaMV35S, and NOS terminator. A plant high-expression sequence (Kozak sequence) was linked at the 5' end for overexpression of the V(HH) gene. The constructed cassette (pBIV(HH)) was transferred to agrobacterium, and the VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. Transgenic lines were selected on kanamycin (100 mg/L) and maintained in soil, and subsequent generations were obtained. The presence and expression of the transgene was confirmed in the transformants by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Tobacco transgenic lines leave expressed V(HH) at levels varying from 1.12% to 1.63% of the total soluble protein. This report examines the transformation and expression of recombinant single-domain antibody (V(HH)) against antigen-associated tumor in tobacco plants.

Antibodies, Monoclonal↗

PubMed2006

Functional coassembly of KCNQ4 with KCNE-beta- subunits in Xenopus oocytes.

The KCNQ gene family comprises voltage-gated potassium channels expressed in epithelial tissues (KCNQ1, KCNQ5), inner ear structures (KCNQ1, KCNQ4) and the brain (KCNQ2-5). KCNQ4 is expressed in inner and outer hair cells of the inner ear where it influences electrical excitability and cell survival. Accordingly, loss of function mutations of the KCNQ4 gene cause hearing loss in humans and functional k.o.-mice show progressive degeneration of outer hair cells (OHCs). However, characteristic electrophysiological features of the native KCNQ4- carried current I(K,n) in OHCs are not recapitulated by expression of KCNQ4 channels in heterologous expression systems. This might suggest modulation of KCNQ4 by interacting KCNE Beta-subunits, which are known to modify the properties of the closely related KCNQ1. The present study explored whether transcripts of the KCNE isoforms could be identified in OHC mRNA and whether the subunits modulate KCNQ4 function. RT-PCR indeed yielded transcripts of all five KCNEs in OHCs. Coexpression of the KCNE- Beta-subunits with human KCNQ4 in the Xenopus laevis oocyte expression system revealed that all KCNEs modulate KCNQ4 voltage dependence, protein stability and ion selectivity of hKCNQ4 in Xenopus oocytes. The deafness-associated Jervell and Lange- Nielsen syndrome (JLNS) mutation KCNE1(D76N) impairs KCNQ4-function whereas the Romano-Ward syndrome (RWS) mutant KCNE1(S74L), which shows normal hearing in patients, does not impair KCNQ4 channel function. In conclusion, KCNEs are presumably coexpressed with KCNQ4 in hair cells from the organ of Corti and might regulate KCNQ4 functional properties, effects that could be important under physiological and pathophysiological conditions.

Animals↗

PubMed1999

Regulation by molluscan myosins.

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.

Adenosine Triphosphatases↗

PubMed1980

Transmission and scanning electron microscopy of whole glioma cells cultured in vitro.

Twenty-six lines of malignant glioma cells have been established in our laboratories. The cell lines have all been derived from low-differentiated human astrocytomas which did not differ in their histopathological classification. A representative selection of these lines was cultured on formvar-covered, carbon stabilized, gold, EM grids. After glutaraldehyde and osmium fixation, they were dehydrated, critical-point dried and studied as whole-cell preparations in the transmission electron microscopy (TEM) mode. The specimens were then coated with 10-nm of a gold/palladium alloy and the same cells identified and examined in the scanning electron microscopy (SEM) mode. This combined TEM and SEM method makes it possible to relate intracellular structures to surface details, such as ruffles, endocytotic vacuoles, filopodia and microvilli. The study also revealed a large spectrum of dissimilarities between the studied lines, which underlines the fact that no essential structural features of a tumor type can be drawn from studies on occasional lines of cells derived from it.

Astrocytoma↗

PubMed2005

Mice selectively bred for open-field thigmotaxis: life span and stability of the selection trait.

In 2 experiments, the authors examined 69 mice selectively bred for high or low levels of open-field (OF) thigmotactic behavior (high open-field thigmotaxis [HOFT] and low open-field thigmotaxis [LOFT], respectively). They found that the strains differed in defecation during the 60-min exposure to the OF. Furthermore, the strains differed with regard to their life spans: The more thigmotactic HOFT mice lived longer than the LOFT mice. The strains were not differentiated by food intake or excretion. The strain difference in thigmotaxis was not age dependent, and it persisted in the home-cage condition as well. Neither the location (center or wall) of the starting point nor the shape (circular or square) of the OF arena affected the difference in wall-seeking behavior between the two strains. The authors concluded that the difference in thigmotaxis (or emotionality) between the HOFT and LOFT mice is a stable and robust feature of these animals.

Age Factors↗

PubMed2001

Non-peptidic inhibitors of human chymase. Synthesis, structure-activity relationships, and pharmacokinetic profiles of a series of 5-amino-6-oxo-1,6-dihydropyrimidine-containing trifluoromethyl ketones.

Chymase possesses a wide variety of actions, including promotion of angiotensin II production and histamine release from mast cells. However, due to a lack of effective inhibitors featuring both high inhibitory activity and high metabolic stability, the pathophysiological role of chymase has not been fully elucidated. We designed non-peptidic inhibitors based on the predicted binding mode of the peptidic chymase inhibitor Val-Pro-Phe-CF3 and demonstrated that the Val-Pro unit is replaceable with a (5-amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)acetyl moiety. Structure-activity relationship studies revealed that phenyl substitution at the 2-position of the pyrimidinone ring is indispensable for high activity. The most potent compound 1h (Ki = 0.0506 microM) is superior in potency to the parent peptidic inhibitor Val-Pro-Phe-CF3 and has good selectivity for chymase over other proteases. The related analogue 1e was orally absorbed and maintained high plasma levels for at least 2h. These results suggest that the derivatives reported here could be developed as agents for treatment of chymase-induced disease.

Animals↗

PubMed2000

Characterization of structural features that mediate the tethering of Caenorhabditis elegans protein kinase A to a novel A kinase anchor protein. Insights into the anchoring of PKAI isoforms.

Caenorhabditis elegans protein kinase A (PKAI(CE)) is tethered to organelles in vivo. A unique A kinase anchor protein (AKAP(CE)) avidly binds the RI-like regulatory subunits (R(CE)) of PKAI(CE) and stringently discriminates against RIIalpha and RIIbeta subunits, the preferred ligands for classical AKAPs. We elucidated structural features that stabilize AKAP(CE).R(CE) complexes and confer atypical R isoform specificity on the anchor protein. Three large aliphatic amino acids (Leu(236), Ile(248), and Leu(252)) in the tethering domain of AKAP(CE) (residues 236-255) are crucial for ligation of R(CE). Their side chains apparently generate a precisely configured hydrophobic binding pocket that accommodates an apolar surface on R(CE) dimers. Basic residues (His(254)-Arg(255)-Lys(256)) at the C terminus of the tethering site set an upper limit on affinity for R(CE.) A central dipeptide (Phe(243)-Ser(244)) contributes critical and distinctive properties of the tethering site. Ser(244) is essential for selective binding of R(CE) and exclusion of RII isoforms. The aromatic hydrophobic character of Phe(243) ensures maximal R(CE) binding activity, thereby supporting a "gatekeeper" function of Ser(244). Substitution of Phe(243)-Ser(244) with Leu-Val generated an RII-specific AKAP. R(CE) and RII subunits contain similar dimerization domains. AKAP-binding domains of R(CE) (residues 23-47) and RII differ markedly in size, amino acid sequence, and docking specificity. Four hydrophobic residues (Cys(23), Val(27), Ile(32), and Cys(44)) in R(CE) are crucial for avid binding with AKAP(CE), whereas side chains from Leu(20), Leu(35), Val(36), Ile(40), and Ile(41) have little impact on complex formation. Tyr(26) is embedded in the docking domain, but its aromatic ring is required for R(CE)-R(CE) dimerization. Residues 236-255 in AKAP(CE) also constitute a binding site for mammalian RIalpha. RIalpha (PKAIalpha) is tightly sequestered by AKAP(CE) in vitro (K(D) = approximately 10 nM) and in the environment of intact cells. The tethering domain of AKAP(CE) provides a molecular module for manipulating intracellular localization of RI and elucidating functions of anchored PKAI in eukaryotes.

A Kinase Anchor Proteins↗

PubMed1986

Aniridic glaucoma: the results of gonio-surgery to prevent and treat this problem.

Repetitive gonioscopy of children with congenital aniridia confirms the presence of an angle abnormality which can be progressive and cause glaucoma. This abnormality features obstruction of the trabecular meshwork by variable mixtures of anterior migration of the peripheral iris and thickening of the uveal meshwork associated with a vascular net over exposed trabecular meshwork adjacent to the anterior edge of the iris. Preliminary results of prophylactic gonio-surgery in 28 eyes of 16 children with an average age of 4 years was reported. This surgery was performed without complication and produced a permanent exposure of the trabecular meshwork to the anterior chamber for an average of 8 circumferential hours, if two procedures were performed. Preliminary results suggest a stabilization of eye pressures at least through childhood and encourage the continuation of these prophylactic operations on selected eyes with congenital aniridia. Therapeutic goniotomy for established acquired glaucoma in congenital aniridia cannot be relied on, but may be a benefit for early detected cases or for glaucoma associated with aniridia in infancy.

Child↗

PubMed1988

Genetic stability of Ross River virus during epidemic spread in nonimmune humans.

We have examined the rate of evolution of Ross River virus, a mosquito-borne RNA virus, during epidemic spread through tens of thousands of nonimmune humans over a period of 10 months. Two regions of the Ross River virus genome were sequenced: the E2 gene (1.2 kb in length), which encodes the major neutralization determinant of the virus, and 0.4 kb of the 3'-untranslated region. In the E2 gene, a single nucleotide change was selected which led to a predicted amino acid change at residue 219. No changes were selected in the 3'-untranslated region. By comparison with rates of evolution reported for non-arthropod-borne RNA viruses, the rate for Ross River virus is surprisingly low. We identify three features of the Ross River virus replication and transmission cycle which may limit the rate of evolution of arthropod-borne viruses in the field.

Alphavirus↗

PubMed1995

Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis.

Argingipain, so termed due to its peptide cleavage specificity at arginine residue, is a unique extracellular cysteine proteinase produced by the anaerobic rod Porphyromonas gingivalis, which is known as a major pathogenic factor of the progressive periodontal disease (T. Kadowaki, M. Yoneda, K. Okamoto, K. Maeda, and K. Yamamoto (1994) J. Biol. Chem. 269, 21371-21378). The catalytic specificity and functional importance of this enzyme prompted us to elucidate its structural features. A DNA fragment for argingipain was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the NH2-terminal amino acid sequence of the purified enzyme. Although the extracellular mature enzyme was shown to have an apparent molecular mass of 44 kDa in gels, the nucleotide sequence of the isolated gene revealed a single gene coding for a 109-kDa precursor of argingipain. The deduced amino acid sequence exhibited no significant similarity to the sequences of representative members of the cysteine protease family. The precursor contained four functional domains: the NH2-terminal signal peptide required for the inner membrane transport; the NH2-terminal prosequence, which is assumed to stabilize the precursor structure; the proteinase domain; and the COOH-terminal hemagglutinin domain, which appears to be essential for extracellular secretion of the proteinase domain. Experiments involving the addition of the argingipain inhibitors to the culture medium of P. gingivalis suggested that the maturation of argingipain occurs intracellularly via an autocatalytic cleavage of the pro-argingipain propeptide.

Adhesins, Bacterial↗

PubMed1990

Secretory IgA, IgG, and IgM immunoglobulins isolated simultaneously from colostral whey by selective thiophilic adsorption.

In order to evaluate the collective contribution of immunoglobulins to gastrointestinal and immune system development in newborn infants, it would be advantageous to develop a simple and rapid procedure for the selective and quantitative removal of all immunoglobulin classes from colostrum and milk. Toward this end, the major immunoglobulin classes typically present in colostrum (IgM, sIgA, and IgG) have been isolated simultaneously by selective removal from a porcine colostral whey model system using a single-step, preparative scale procedure termed thiophilic adsorption. At neutral pH, the salt-promoted thiophilic affinity of immunoglobulins for the synthetic sulfone-thioether ligands exploits a common (as yet unknown) structural feature of immunoglobulins. The adsorption and recovery procedures operate efficiently under mild buffer conditions permitting the subsequent comparative biochemical analyses of individual immunoglobulin classes for structural and functional heterogeneity. Thus, 1 liter columns of thiophilic adsorbent (T-gel) were employed to obtain pure, structurally intact immunoglobulins from 1 liter batches of porcine colostral whey. The identity and purity of the isolated immunoglobulins were determined under both structure-stabilizing ('native') conditions and denaturing conditions using high-performance size-exclusion chromatography, sucrose density gradient centrifugation, immunodiffusion techniques, SDS-polyacrylamide gradient gel electrophoresis with immunoblotting identification procedures, and two-dimensional electrophoresis. This is the first procedure known to us that allows for the simultaneous one-step isolation (under homologous conditions) of various immunoglobulins with preserved quaternary structure and apparent biological activity.

Adsorption↗

PubMed2002

Insights into Lou Gehrig's disease from the structure and instability of the A4V mutant of human Cu,Zn superoxide dismutase.

Mutations in human superoxide dismutase (HSOD) have been linked to the familial form of amyotrophic lateral sclerosis (FALS). Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is one of the most common neurodegenerative disorders in humans. In ALS patients, selective killing of motor neurons leads to progressive paralysis and death within one to five years of onset. The most frequent FALS mutation in HSOD, Ala4-->Val, is associated with the most rapid disease progression. Here we identify and characterize key differences in the stability between the A4V mutant protein and its thermostable parent (HSOD-AS), in which free cysteine residues were mutated to eliminate interferences from cysteine oxidation. Denaturation studies reveal that A4V unfolds at a guanidine-HCl concentration 1M lower than HSOD-AS, revealing that A4V is significantly less stable than HSOD-AS. Determination and analysis of the crystallographic structures of A4V and HSOD-AS reveal structural features likely responsible for the loss of architectural stability of A4V observed in the denaturation experiments. The combined structural and biophysical results presented here argue that architectural destabilization of the HSOD protein may underlie the toxic function of the many HSOD FALS mutations.

Amyotrophic Lateral Sclerosis↗

PubMed2003

Differential helix stabilities and sites pre-organized for tertiary interactions revealed by monitoring local nucleotide flexibility in the bI5 group I intron RNA.

The local environment at adenosine residues in the bI5 group I intron RNA was monitored as a function of Mg(2+) using both the traditional method of dimethyl sulfate (DMS) N1 methylation and a new approach, selective acylation of 2'-amine substituted nucleotides. These probes yield complementary structural information because N1 methylation reports accessibility at the base pairing face, whereas 2'-amine acylation scores overall residue flexibility. 2'-Amine acylation robustly detects RNA secondary structure and is sensitive to higher order interactions not monitored by DMS. Disruption of RNA structure due to the 2'-amine substitution is rare and can be compensated by stabilizing folding conditions. Peripheral helices that do not interact with other parts of the RNA are more stable than both base paired helices and tertiary interactions in the conserved catalytic core. The equilibrium state of the bI5 intron RNA, prior to assembly with its protein cofactor, thus features a relatively loosely packed core anchored by more stable external stem-loop structures. Adenosine residues in J4/5 and P9.0 form structures in which the nucleotide is constrained but the N1 position is accessible, consistent with pre-organization to form long-range interactions with the 5' and 3' splice sites.

Acylation↗

PubMed2005

Characterization of the 13-cis-retinoic acid/cyclodextrin inclusion complexes by phase solubility, photostability, physicochemical and computational analysis.

13-cis-Retinoic acid (13-cis-RA) is a synthetic retinoid commonly used in the treatment of severe acne. It has also been found to possess potential chemopreventive activity. It has extremely low aqueous solubility and high photo-sensitivity. This study investigated the effects of the complexation of 13-cis-RA with alpha-cyclodextrin (alpha-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on its phase solubility. HP-beta-CD was found to be more effective in increasing the aqueous solubility of 13-cis-RA compared to alpha-CD. Phase solubility studies indicated that the solubility of 13-cis-RA was increased dramatically by the formation of inclusion complex with HP-beta-CD. The solubility was further enhanced by pH adjustment. The photostability of the selected inclusion complex of 13-cis-RA:HP-beta-CD was then evaluated. Complexation with HP-beta-CD was found to delay the photo-degradation of 13-cis-RA in aqueous solution. The physicochemical properties of the solid inclusion complex were characterized by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and X-ray diffractometry (XRD). Molecular modeling with MMFF94s force field (SYBYL V6.6) was utilized to predict the preferred orientation of 13-cis-RA in the CD cavity and the main structural features responsible for the enhancement of its solubility and photostability. The energy scores estimated from the computational analysis were found capable of reflecting the stability constants of the cyclodextrin complexes obtained in the phase solubility studies. The results showed that HP-beta-CD was a proper excipient for increasing solubility and stability of 13-cis-RA.

Calorimetry, Differential Scanning↗