Half-maximal Saturation Research Articles

Development of a noncompetitive idiometric nanobodies phage immumoassay for the determination of fumonisin B1

ABSTRACT Here we demonstrate a noncompetitive idiometric nanobodies phage immunoassay for the determination of FB1, utilizing two types of anti-idiotypic nanobody, that is specific for different epitopes within the hypervariable region of the primary mAb. Three alphatype anti-idiotypic nanobodies were obtained from a naive nanobodies phage-display library. A noncompetitive idiometric immunoassay was established with a combination of betatype anti-idiotypic nanobody and phage-displayed alphatype anti-idiotypic nanobody. The half-maximal saturation of the signal value (SC50) was 0.68 ng/mL, with the limit of detection (LOD) was 0.19 ng/mL. When the noncompetitive assay was compared to the competitive ELISA (LOD = 3.41 ng/mL), an over 17-fold improvement in sensitivity was observed. A correlation (R 2) was 0.988 between the data of the noncompetitive assay and LC–MS/MS for the determination of FB1 in cereal samples. The noncompetitive idiometric immunoassay would have a broad utility for monitoring small molecules in food and environment.

Colistin-Functionalized Nanoparticles for the Rapid Capture of Acinetobacter baumannii.

Gold nanoparticles (AuNPs) were functionalized for rapid binding of Acinetobacter baumannii (A. baumannii), a Gram-negative bacterium. AuNPs were functionalized with colistin (Col), a polycationic antibiotic, using a two-step self-assembly process, in which heterobifunctional polyethylene glycol (PEG) was used as a linker. Colistin was successfully conjugated to the AuNPs (Col-PEG-AuNP), as validated by dynamic light scattering (DLS) and proton nuclear magnetic resonance (H1 NMR). High angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images, acquired simultaneously with X-ray energy dispersive spectroscopy (EDS) data, confirmed binding of Col-PEG-AuNPs to the cell envelope of A. baumannii. Results generated from a binding assay indicated that Col-PEG-AuNP complexation with A. baumannii occurred rapidly and reached half-maximum saturation in approximately 7 minutes, on average, for all A. baumannii strains evaluated. Quantitative measurement of the kinetics of Col-PEG-AuNP binding to A. baumannii is essential to inform the design of colistin-functionalized magnetic nanoparticles for magnetic separation of nanoparticle-bound A. baumannii.

Oxidative stress based response of a transcriptional regulator, OhrR, from Burkholderia thailandensis (LB252)

Bacteria face a burst of oxidative species during host invasion. As a protective mechanism, they possess several proteins involved in inorganic peroxide sensing. However, much has to be discovered about the sensor‐responder proteins involved in organic hydroperoxide (OHP) resistance, which is important since OHPs are more toxic. Burkholderia thailandensis, a soil dwelling bacterium, contains genes ohrR and ohr, that are involved in OHP detoxification, with the former encoding an organic hydroperoxide reductase regulator (OhrR), a MarR family transcriptional regulator, regulating expression of the gene encoding organic hydroperoxide reductase (Ohr). EMSAs revealed specificity of B. thailandensis OhrR for the promoter region of ohr, with half‐maximal saturation of 3.6 nM. SDS‐PAGE demonstrated formation of reversible, oligomeric species of OhrR on treatment with organic and inorganic oxidants. Conformational changes were indicated by reduced thermal stability of oxidized protein compared to its reduced form (Tm 65.3 °C). Attenuated DNA binding in the presence of oxidants further confirmed structural changes in oxidized OhrR by virtue of disulfide bond formation between redox‐active cysteines. B. thailandensis shares 80% genetic homology with its pathogenic homologues B. pseudomallei and B. mallei, making characterizing its protein machinery involved in peroxide resistance even more crucial.

Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia

The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC3), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC3 to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC3 or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC3 and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an EC 50 of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC3 to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC3/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC3 and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System: INSIGHTS INTO THE DISTINCT BINDING PROPERTIES OF HIGH AND LOW MOLECULAR WEIGHT HYALURONAN

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan.

Imatinib inhibits vascular smooth muscle proteoglycan synthesis and reduces LDL binding in vitro and aortic lipid deposition in vivo

The ‘response to retention’ hypothesis of atherogenesis proposes that proteoglycans bind and retain low-density lipoproteins (LDL) in the vessel wall. Platelet-derived growth factor (PDGF) is strongly implicated in atherosclerosis and stimulates proteoglycan synthesis. Here we investigated the action of the PDGF receptor inhibitor imatinib on PDGF-mediated proteoglycan biosynthesis in vitro, lipid deposition in the aortic wall in vivo and the carotid artery ex vivo. In human vSMCs, imatinib inhibited PDGF mediated 35S-SO4 incorporation into proteoglycans by 31% (P < 0.01) and inhibited PDGF-mediated size increases in both chemically cleaved and xyloside associated glycosaminoglycan (GAG) chains by 19%, P < 0.05 and 27%, P < 0.05, respectively. Imatinib decreased PDGF stimulation of the 6:4 position sulphation ratio of disaccharides. The half maximal saturation value for LDL binding for proteoglycans from PDGF stimulated cells in the presence of imatinib was approximately 2.5-fold higher than for PDGF treatment alone. In high fat fed ApoE−/– mice, imatinib reduced total lipid staining area by ∼31% (P < 0.05). Carotid artery lipid accumulation in imatinib treated mice was also reduced. Furthermore, we demonstrate that imatinib inhibits phosphorylation of tyrosine 857, the autophosphorylation site of the PDGF receptor, in vSMCs. Thus imatinib inhibits GAG synthesis on vascular proteoglycans and reduces LDL binding in vitro and in vivo and this effect is mediated via the PDGF receptor. These findings validate a novel mechanism to prevent cardiac disease.

Three conformational antibodies specific for different PSMA epitopes are promising diagnostic and therapeutic tools for prostate cancer

The prostate specific membrane antigen (PSMA) represents an attractive antigen for antibody-based diagnostic and therapeutic intervention in prostate cancer, since it is highly restricted to the prostate and overexpressed in all tumor stages. The present work describes the in vitro characterization of the three anti-PSMA monoclonal antibodies (mAbs) 3/A12, 3/E7, and 3/F11 in comparison to the mAb J591. The mAbs were tested for saturation and competitive binding on C4-2 prostate cancer cells by flow cytometry. Immunohistochemical staining was conducted on frozen prostate normal and cancer tissues as well as on lymph node metastases. Similarly, potential crossreactivities were tested on a broad panel of human normal tissues. The anti-PSMA mAbs showed a strong binding to C4-2 cells with mean half-maximal saturation concentrations of about 14 nM for 3/A12, 17 nM for 3/E7, 9 nM for 3/F11, and 16 nM for J591. Competitive binding studies revealed that our three mAbs bind to different extracellular PSMA epitopes. The mAbs showed comparable staining of epithelial cells for all tested normal and tumorous prostate tissues. Extraprostatic staining was observed on secretory cells of the salivary glands and on the brush border of the duodenal columnar epithelium. J591 additionally showed positive staining of the normal breast epithelium. Due to their specific binding characteristics, the anti-PSMA mAbs 3/A12, 3/E7, and 3/F11 show great promise for diagnostic and therapeutic applications in prostate cancer.

Monosaccharide transport across membranes of human spermatozoa. I. Development of a radiochemical method of measuring monosaccharide uptake by spermatozoa.

A method for determination of the monosaccharide uptake into human spermatozoa is described. Intracellular monosaccharide concentrations were calculated on the basis of determination of intracellular radioactivity after incubation of the cells with labelled monosaccharide and using a mathematical procedure to approximate the intracellular space of spermatozoa. The D-fructose uptake depends on the extracellular D-fructose concentration in a hyperbolic manner. Half maximal saturation is present at 4,1 mM. This corresponds closely with the lowest limit of D-fructose concentration in human fertile semen.

Effects of Cardiac Troponin C Mutants on TnC-TnI interaction and its modulation by PKA phosphorylation

We are considering several cardiac TnC mutants as potential therapeutic strategies for cardiomyopathies. To judge their potential to improve in situ function it is important to understand how these mutants affect TnC-TnI interaction and its modulation by PKA-mediated phosphorylation of TnI Ser 23, Ser 24. In this study, we are characterizing two cardiac TnC mutations, Leu48Glu (L48Q) and Ile61Glu (I61Q), with increased and decreased (respectively) Ca2+ binding affinity. In previous studies we showed these mutations resulted in increased (L48Q) and decreased (I61Q) Ca2+ sensitivity of steady state force in skinned rat trabeculae. To determine if these changes in Ca2+ sensitivity were due to altered TnC-TnI interactions we generated a structural marker by attaching IANBD to Cys84 in the N-lobe of cTnC. Half-maximal IANBD fluorescence saturation of Ca2+ binding occurred at pCa7.42 for L48Q cTnC, 7.38 for wild-type (WT) cTnC and 7.30 for I61Q cTnC. In both the absence and presence of saturating Ca2+ (0.6 μM TnC) IANBD fluorescence increased with increasing TnI and saturated at different [TnI] in the order L48Q, WT, I61Q. Fluorescence half-maximal saturation occurred at 0.26μM(saturating Ca2+) and 0.25 μM (no Ca2+)TnI for L48Q cTnC, 0.78 μM and 0.49μM for WT cTnC , and 1.45 μM and 0.69 μM I61Q cTnC according to the exponential function fit of the data. However, preliminary experiments suggest that when PKA phosphorylated [cTnI] was titrated to cTnC, in both the absence and presence of saturating Ca2+, IANBD fluorescence enhancement with L48Q may be impaired. The data thus far suggest that single amino acid mutations that alter Ca2+ binding affinity of TnC can influence interaction with TnI and its modulation by PKA mediated phosphorylation of Ser 23, Ser 24. Supported by HL65497 to MR.

Photosynthetic features of non-Kranz type C4 versus Kranz type C4 and C3 species in subfamily Suaedoideae (Chenopodiaceae).

The objective of this study was to characterise photosynthesis in terrestrial non-Kranz (NK) C4 species, Bienertia sinuspersici Akhani and Suaeda aralocaspica (Bunge) Freitag & Schütze (formerly Borszczowia aralocaspica), compared with closely related Kranz type C4 Suaeda eltonica Iljin and Suaeda taxifolia Standley, and C3 species Suaeda heterophylla Bunge and Suaeda maritima Dumort in subfamily Suaedoideae (Chenopodiaceae). Traditional Kranz type C4 photosynthesis has several advantages over C3 photosynthesis under certain environmental conditions by suppressing photorespiration. The different photosynthetic types were evaluated under varying levels of CO2 and light at 25°C. Both NK and Kranz type species had C4 type CO2 compensation points (corrected for dark-type respiration) and half maximum saturation of photosynthesis at similar levels of atmospheric CO2 (average of 145 µbar for the C4 species v. 330 µbar CO2 for C3 species) characteristic of C4 photosynthesis. CO2 saturated rates of photosynthesis per unit chlorophyll was higher in the C3 (at ~2.5 current ambient CO2 levels) than the C4 species, which is likely related to their higher Rubisco content. The amount of Rubisco as a percentage of total protein was similar in NK and Kranz type species (mean 10.2%), but much lower than in the C3 species (35%). Light saturated rates of CO2 fixation per unit leaf area at 25°C and 340 µbar CO2 were higher in the Kranz species and the NK C4 S. aralocaspica than in the C3 species. In response to light at 340 µbar CO2, there was a difference in rates of photosynthesis per unit Rubisco with NK > Kranz > C3 species. There were no significant differences between the three photosynthetic types in maximum quantum yields, convexity of light response curves, and light compensation points at 25°C. The water use efficiency (CO2 fixed per water transpired) at 340 µbar CO2, 25°C and 1000 µmol quanta m-2 s-1 was on average 3-fold higher in the C4 (NK and Kranz) compared with the C3 species. The results show that the NK species have several C4 traits like the Kranz type species in subfamily Suaedoideae.

Analysis of Nucleotide Binding to P97 Reveals the Properties of a Tandem AAA Hexameric ATPase

p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5′-O-(thiotriphosphate) (ATPγS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPγS can saturate all 6 nucleotide binding sites in D1, only 3–4 of the 6 D2 sites can bind ATPγS simultaneously. ATPγS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

Iodohydroxybenzylpindolol binding to beta-adrenergic receptors of diabetic and control rat adipocytes' crude membrane.

We have characterized the binding of [125I]-Iodohydroxybenzylpindolol to rat adipocytes' crude membrane. The binding was rapid, reaching equilibrium within 19 min. Binding of [125I]-Iodohydroxybenzylpindolol was saturable with 9 nM. Half-maximal saturation provided an estimated Kd of 2.42 nM. The order for 125IHYP displacement by various compounds was (-)-propranolol (-)-isoproterenol greater than (-)-epinephrine greater than (-)-norepinephrine. [125I]-Iodohydroxybenzylpindolol binding was more sensitive to L-propranolol inhibition in crude membrane prepared from diabetic rat fat cells than in those prepared from controls. The basal cAMP level was identical in diabetic and control rat crude membrane. Isoproterenol increased the cAMP level in diabetic more than in control rats.

DNA inhibits catalysis by the carboxyltransferase subunit of acetyl‐CoA carboxylase: Implications for active site communication

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in the synthesis of long-chain fatty acids. The crystal structure of the Escherichia coli carboxyltransferase component of ACC revealed an alpha(2)beta(2) subunit composition with two active sites and, most importantly, a unique zinc domain in each alphabeta pair that is absent in the eukaryotic enzyme. We show here that carboxyltransferase binds DNA. Half-maximal saturation of different single-stranded or double-stranded DNA constructs is seen at 0.5-1.0 muM, and binding is cooperative and nonspecific. The substrates (malonyl-CoA and biocytin) inhibit DNA:carboxyltransferase complex formation. More significantly, single-stranded DNA, double-stranded DNA, and heparin inhibit the reaction catalyzed by carboxyltransferase, with single-stranded DNA and heparin acting as competitive inhibitors. However, double-inhibition experiments revealed that both DNA and heparin can bind the enzyme in the presence of a bisubstrate analog (BiSA), and the binding of BiSA has a very weak synergistic effect on the binding of the second inhibitor (DNA or heparin) and vice versa. In contrast, DNA and heparin can also bind to the enzyme simultaneously, but the binding of either molecule has a strong synergistic effect on binding of the other. An important mechanistic implication of these observations is that the dual active sites of ACC are functionally connected.

Estimation of dose requirements for sustained in vivo activity of a therapeutic human anti‐CD20 antibody

We evaluated the dose requirements for sustained in vivo activity of ofatumumab, a human anti-CD20 antibody under development for the treatment of B cell-mediated diseases. In a mouse xenograft model, a single dose, resulting in an initial plasma antibody concentration of 5 microg/ml, which was expected to result in full target saturation, effectively inhibited human B-cell tumour development. Tumour growth resumed when plasma concentrations dropped below levels that are expected to result in half-maximal saturation. Notably, tumour load significantly impacted antibody pharmacokinetics. In monkeys, initial depletion of circulating and tissue residing B cells required relatively high-dose levels. Re-population of B-cell compartments, however, only became detectable when ofatumumab levels dropped below 10 microg/ml. We conclude that, once saturation of CD20 throughout the body has been reached by high initial dosing, plasma concentrations that maintain target saturation on circulating cells (5-10 microg/ml) are probably sufficient for sustained biological activity. These observations may provide a rationale for establishing dosing schedules for maintenance immunotherapy following initial depletion of CD20 positive (tumour) cells.

Molecular Basis of RNA Recognition by the Embryonic Polarity Determinant MEX-5

Embryonic development requires maternal proteins and RNA. In Caenorhabditis elegans, a gradient of CCCH tandem zinc finger (TZF) proteins coordinates axis polarization and germline differentiation. These proteins govern expression from maternal mRNAs by an unknown mechanism. Here we show that the TZF protein MEX-5, a primary anterior determinant, is an RNA-binding protein that recognizes linear RNA sequences with high affinity but low specificity. The minimal binding site is a tract of six or more uridines within a 9-13-nucleotide window. This sequence is remarkably abundant in the 3'-untranslated region of C. elegans transcripts, demonstrating that MEX-5 alone cannot specify mRNA target selection. In contrast, human TZF homologs tristetraprolin and ERF-2 bind with high specificity to UUAUUUAUU elements. We show that mutation of a single amino acid in each MEX-5 zinc finger confers tristetraprolin-like specificity to this protein. We propose that divergence of this discriminator residue modulates the RNA-binding specificity in this protein class. This residue is variable in nematode TZF proteins, but is invariant in other metazoans. Therefore, the divergence of TZF proteins and their critical role in early development is likely a nematode-specific adaptation.

The Deinococcus radiodurans-Encoded HU Protein Has Two DNA-Binding Domains

Deinococcus radiodurans can reconstitute its genome from double-strand breaks, most likely due to unusually efficient DNA repair and recombination. Factors that may contribute to such processes include the histone-like protein HU. The D. radiodurans-encoded HU (DrHU), which binds preferentially to DNA recombination intermediates, contains a 47-amino acid extension preceding the fold characteristic of HU proteins. Here we use electrophoretic mobility shift assays and DNA footprinting to show that the DrHU N-terminal domain significantly modulates DNA binding. The truncated DrHU (deltaDrHU), comprising only the conserved DNA-binding fold, has a site size of approximately 11 bp in contrast to full-length DrHU which does not stably engage DNA shorter than approximately 50 bp. Unlike wild-type DrHU, deltaDrHU distinguishes between linear DNA and DNA with nicks or gaps. DeltaDrHU also binds preferentially to four-way junction (4WJ) DNA, with half-maximal saturation of 1.4 +/- 0.4 nM compared to 20 +/- 2 nM for 37 bp duplex DNA. However, in contrast to full-length protein which binds the junction arms, deltaDrHU primarily protects the junction crossover. Evidently, the DrHU N-terminus changes the mode of binding to both 4WJ DNA, duplex DNA, and DNA with nicks or gaps, thereby resulting in DrHU binding preferentially only to 4WJ structures. Combined with Western blots that confirm the presence of the extended form of DrHU in vivo, our data provide mechanistic insight into discrimination between 4WJ DNA and other distorted DNA constructs and suggest that an in vivo role of DrHU may be to stabilize DNA junctions.

Loop 1 of Transducer Region in Mammalian Class I Myosin, Myo1b, Modulates Actin Affinity, ATPase Activity, and Nucleotide Access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.

The Saccharomyces cerevisiae High Mobility Group Box Protein HMO1 Contains Two Functional DNA Binding Domains

High mobility group box (HMGB) proteins are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as 4-way junctions. HMO1 is one of 10 Saccharomyces cerevisiae HMGB proteins, and it is required for normal growth and plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease. Using electrophoretic mobility shift assays, we have shown here that HMO1 binds 26-bp duplex DNA with K(d) = 39.6 +/- 5.0 nm and that its divergent box A domain participates in DNA interactions, albeit with low affinity. HMO1 has only modest preference for DNA with altered conformations, including DNA with nicks, gaps, overhangs, or loops, as well as for 4-way junction structures and supercoiled DNA. HMO1 binds 4-way junctions with half-maximal saturation of 19.6 +/- 2.2 nm, with only a modest increase in affinity in the absence of magnesium ions (half-maximal saturation 6.1 +/- 1.1 nm). Whereas the box A domain contributes modest structure-specific binding, the box B domain is required for high affinity binding. HMO1 bends DNA, as measured by DNA cyclization assays, facilitating cyclization of 136-, 105-, and 87-bp DNA, but not 75-bp DNA, and it has a significantly longer residence time on DNA minicircles compared with linear duplex DNA. The unique DNA binding properties of HMO1 are consistent with global roles in the maintenance of chromatin structure.

Fenofibrate modifies human vascular smooth muscle proteoglycans and reduces lipoprotein binding

Vascular disease in type 2 diabetes is associated with an up-regulation of atherogenic growth factors, which stimulate matrix synthesis including proteoglycans. We have examined the direct actions of fenofibrate on human vascular smooth muscle cells (VSMCs) and have specifically investigated proteoglycan synthesis and binding to LDL. Proteoglycans synthesised by human VSMCs treated with fenofibrate (30 micromol/l) were assessed for binding to human LDL using a gel mobility shift assay, metabolically labelled with [(35)S]-sulphate and quantitated by cetylpyridinium chloride. They were then assessed for electrophoretic mobility by SDS-PAGE, for size by gel filtration, for sulphation pattern by fluorophore-assisted carbohydrate electrophoresis, and for glycosaminoglycan (GAG) composition by enzyme digestion. Proteoglycans synthesised in the presence of fenofibrate showed an increase in the half-maximum saturation concentration of LDL from 36.8+/-12.4 microg/ml to 77.7+/-17 microg/ml under basal conditions, from 24.9+/-4.6 microg/ml to 39.1+/-6.1 microg/ml in the presence of TGF-beta1, and from 9.5+/-4.4 microg/ml to 31.1+/-3.4 microg/ml in the presence of platelet-derived growth factor/insulin. Fenofibrate treatment in the presence of TGF-beta1 inhibited the incorporation of [(35)S]-sulphate into secreted and cell-associated proteoglycans synthesised by human VSMCs by 59.2% (p<0.01) and 39.8% (p<0.01) respectively. The changes in sulphate incorporation following treatment with fenofibrate were associated with a concentration-related increase in the electrophoretic mobility due to a reduction in GAG length. There was no change in the sulphation pattern; however, there was an alteration in the disaccharide composition of the GAGs. Fenofibrate modifies the structure of vascular proteoglycans by reducing the length of the GAG chains and GAG composition, resulting in reduced binding to human LDL, a mechanism which may lead to a reduction of atherosclerosis and cardiovascular disease in people with diabetes treated with fenofibrate.

Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.