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PubMed2006

Atomic force microscopy reveals DNA bending during group II intron ribonucleoprotein particle integration into double-stranded DNA.

The mobile Lactococcus lactis Ll.LtrB group II intron integrates into DNA target sites by a mechanism in which the intron RNA reverse splices into one DNA strand while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3' end to prime reverse transcription of the inserted intron RNA. These reactions are mediated by an RNP particle that contains the intron-encoded protein and the excised intron lariat RNA, with both the protein and base pairing of the intron RNA used to recognize DNA target sequences. Here, computational analysis indicates that Escherichia coli DNA target sequences that support Ll.LtrB integration have greater predicted bendability than do random E. coli genomic sequences, and atomic force microscopy shows that target DNA is bent during the reaction with Ll.LtrB RNPs. Time course and mutational analyses show that DNA bending occurs after reverse splicing and requires subsequent interactions between the intron-encoded protein and the 3' exon, which lead to two progressively larger bend angles. Our results suggest a model in which RNPs bend the target DNA by maintaining initial contacts with the 5' exon while engaging in subsequent 3' exon interactions that successively position the scissile phosphate for bottom-strand cleavage at the DNA endonuclease active site and then reposition the 3' end of the cleaved bottom strand to the reverse transcriptase active site for initiation of cDNA synthesis. Our findings indicate that bendability of the DNA target site is a significant factor for Ll.LtrB RNP integration.

Bacterial Proteins↗

PubMedDate not supplied

Effects of conditioners on surface hardness of hair fibers: an investigation using atomic force microscopy.

Conditioners are known to have a prophylactic effect on hair damage caused by cosmetic chemical treatments or mechanical grooming procedures. They are known to impart softness and smoothness to hair by moisturizing the fiber. Since the amount of conditioners deposited on the fiber is very small in quantity, it is conceivable that mainly the surface is moisturized. This is especially true of polymeric conditioners, which deposit preferentially on the surface of the fiber, rather than penetrate into the cortex. Therefore, this study strictly investigates whether cationic polymeric conditioners impart softness to the surface cuticle cell as a result of their hydrophilicity, with no regard to its applicability to cosmetic effects. Such softening can be detected by indentation of the surface and can be quantified by measuring the depth of the indent in real time. Atomic force microscopy (AFM), equipped with nano-indentation capability, is ideally suited for this purpose. In this work it was used to determine changes in the microhardness (micromechanical properties) of the hair fiber surface as a result of fiber/conditioner/moisture interactions. In a preliminary study, we observed that the scale faces of hair treated with Polyquaternium 10 (PQ-10) conditioner gave deeper indents, while scale edges yielded shallower ones in comparison to cuticle cells of untreated hair. This suggests that the conditioner softens the scale face and hardens the scale edges. However, because of significant amounts of conditioner residues left on the scale face, this conclusion was rather ambiguous. Therefore, the study was repeated in which multiple indentations were made on the surface cuticle cells of a larger number of the same hair fibers before and after multiple applications of the conditioner. This reduces errors due to fiber-to-fiber variation in pre-existing microhardness differences in surface cuticle cells. Also, the larger number of fibers investigated in the current work allowed for a statistical outcome. This latter study has led to a rather definite conclusion that the scale face is indeed softened by polymeric conditioners such as Polyquaternium-10 (PQ-10). These studies will ultimately help in the development of conditioners with suitable moisturizing and softening effect on hair.

Hair Preparations↗

PubMed2002

Atomic force microscopy reveals kinks in the p53 response element DNA.

p53 is a 53 kDa nuclear phosphoprotein. Its function as a tumor suppressor critically lies in its ability to recognize its target DNA response elements as a tetramer. Here, we report the structural theme intrinsic to the response element DNA that governs this recognition phenomenon. The intrinsic flexibility or dynamic bending between two distinctly different, but naturally occurring p53 response elements has been compared by ring closure. Results show that DNA binding sites containing helically phased d(CATG.CATG) tetra-nucleotide sequences at the centers of quasi-dyad symmetry in each half-response site are more intrinsically flexible (i.e. preferentially bent under axial stress) than their d(CTTG.CTTG) counterparts. Intriguingly, p53 binding sites containing these more flexible d(CATG.CATG) sequence elements also exhibit a stronger tendency for tetrameric binding of the p53 DNA binding domain peptide. Examination of the shapes of DNA microcircles obtained by circularization of oligomers constructed from such flexible p53 target DNA sequences in tandem using MacMode atomic force microscopy directly revealed sequence-specific kinks in solution. The tetra-nucleotide sequence d(CATG.CATG) is highly conserved in most functional p53 response elements. Consequently, we propose that the sequence-specific kinks originating from d(CATG.CATG) sequences could be a common structural theme in p53 response elements and as evident from the results reported here, could be a determinant of binding site recognition by the p53 protein and the subsequent stability of the p53-DNA complex.

Base Sequence↗

PubMed1994

Atomic force microscopy of embedment-free sections of cells and tissues.

The atomic force microscope (AFM) was applied for the first time to embedment-free biological sections. Aldehyde-fixed tissues (kidney, liver etc.) of mice were postfixed with osmium tetroxide and cut into 500-700 nm thick sections after embedding of the tissue block in polyethylene glycol (PEG); the sections mounted on glass slides were deembedded and critical point-dried. The AFM images were collected in air from those tissue sections in a dynamic force mode. Solid-height-mode images, which were comparable to transmission electron microscope (TEM) images, seemed to provide us more useful information than the solid-mode images which resembled scanning electron microscope (SEM) images. Internal structures including chromatin fibers in the nuclei and cytoplasmic organelles were clearly recognizable by AFM. Minute surface structures including the end-feet of renal podocytes were demonstrated. We confirm that a vertical resolution of 0.1 nm and a lateral resolution of 5-10 nm are attainable with the dynamic force mode of AFM using thin sections of tissues. The present paper proposes the usefulness of AFM for observation of biological materials without metal coating in a non-vacuous environment.

Animals↗

PubMed2006

Electrochemistry of conductive polymers 39. Contacts between conducting polymers and noble metal nanoparticles studied by current-sensing atomic force microscopy.

Electrical properties of contacts formed between conducting polymers and noble metal nanoparticles have been examined using current-sensing atomic force microscopy (CS-AFM). Contacts formed between electrochemically prepared pi-conjugated polymer films such as polypyrrole (PPy), poly(3-methylthiophene) (P3MeT), as well as poly(3,4-ethylenedioxythiophene) (PEDOT) and noble metal nanoparticles including platinum (Pt), gold (Au), and silver (Ag) have been examined. The Pt nanoparticles were electrochemically deposited on a pre-coated PPy film surface by reducing a platinum precursor (PtCl62-) at a constant potential. Both current and scanning electron microscopic images of the film showed the presence of Pt islands. The Au and Ag nanoparticles were dispersed on the P3MeT and PEDOT film surfaces simply by dipping the polymer films into colloid solutions containing Au or Ag particles for specified periods (5 to approximately 10 min). The deposition of Au or Ag particles resulted from either their physical adsorption or chemical bonding between particles and the polymer surface depending on the polymer. When compared with PPy, P3MeT and PEDOT showed a stronger binding to Au or Ag nanoparticles when dipped in their colloidal solutions for the same period. This indicates that Au and Ag particles are predominantly linked with the sulfur atoms via chemical bonding. Of the two, PEDOT was more conductive at the sites where the particles are connected to the polymer. It appears that PEDOT has better aligned sulfur atoms on the surface and is strongly bonded to Au and Ag nanoparticles due to their strong affinity to gold and silver. The current-voltage curves obtained at the metal islands demonstrate that the contacts between these metal islands and polymers are ohmic.

Journal Article↗

PubMed2004

Classification of hyper-variable Corynebacterium glutamicum surface-layer proteins by sequence analyses and atomic force microscopy.

The structural S-layer proteins of 28 different Corynebacterium glutamicum isolates have been analyzed systematically. Treatment of whole C. glutamicum cells with detergents resulted in the isolation of S-layer proteins with different apparent molecular masses, ranging in size from 55 to 66 kDa. The S-layer genes analyzed were characterized by coding regions ranging from 1,473 to 1,533 nucleotides coding for S-layer proteins with a size of 490-510 amino acids. Using PCR techniques, the corresponding S-layer genes of the 28 C. glutamicum isolates were all cloned and sequenced. The deduced amino acid sequences of the S-layer proteins showed identities between 69 and 98% and could be grouped into five phylogenetic classes. Furthermore, sequence analyses indicated that the S-layer proteins of the analyzed C. glutamicum isolates exhibit a mosaic structure of highly conserved and highly variable regions. Several conserved regions were assumed to play a key role in the formation of the C. glutamicum S-layers. Especially the N-terminal signal peptides and the C-terminal anchor sequences of the S-layer proteins showed a nearly perfect amino acid sequence conservation. Analyses by atomic force microscopy revealed a committed hexagonal structure. Morphological diversity of the C. glutamicum S-layers was observed in a class-specific unit cell dimension (ranging from 15.2 to 17.4 nm), which correlates with the sequence similarity-based classification. It could be demonstrated that differences in the primary structure of the S-layer proteins were reflected by the S-layer morphology.

Amino Acid Sequence↗

PubMed2007

Transformation of amyloid-like fibers, formed from an elastin-based biopolymer, into a hydrogel: an X-ray photoelectron spectroscopy and atomic force microscopy study.

Previous studies have revealed the propensity of elastin-based biopolymers to form amyloid-like fibers when dissolved in water. These are of interest when considered as "ancestral units" of elastin in which they represent the simplest sequences in the hydrophobic regions of the general type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu). We normally refer to these biopolymers based on elastin or related to elastin units as "elastin-like polypeptides". The requirement of water for the formation of amyloids seems quite interesting and deserves investigation, the water representing the natural transport medium in human cells. As a matter of fact, the "natural" supramolecular organization of elastin is in the form of beaded-string-like filaments and not in the form of amyloids whose "in vivo" deposition is associated with some important human diseases. Our work is directed, therefore, to understanding the mechanism by which such hydrophobic sequences form amyloids and any conditions by which they might regress to a non-amyloid filament. The elastin-like sequence here under investigation is the ValGlyGlyValGly pentapeptide that has been previously analyzed both in its monomer and polymer form. In particular, we have focused our investigation on the apparent stability of amyloids formed from poly(ValGlyGlyValGly), and we have observed these fibers evolving to a hydrogel after prolonged aging in water. We will show how atomic force microscopy can be combined with X-ray photoelectron spectroscopy to gain an insight into the spontaneous organization of an elastin-like polypeptide driven by interfacial interactions. The results are discussed also in light of fractal-like assembly and their implications from a biomedical point of view.

Amyloid↗

PubMed2005

Atomic force microscopy of a hybrid high-molecular-weight glutenin subunit from a transgenic hexaploid wheat.

The high-molecular-weight glutenin subunits (HMW-GS) of wheat gluten in their native form are incorporated into an intermolecularly disulfide-linked, polymeric system that gives rise to the elasticity of wheat flour doughs. These protein subunits range in molecular weight from about 70 K-90 K and are made up of small N-terminal and C-terminal domains and a large central domain that consists of repeating sequences rich in glutamine, proline, and glycine. The cysteines involved in forming intra- and intermolecular disulfide bonds are found in, or close to, the N- and C-terminal domains. A model has been proposed in which the repeating sequence domain of the HMW-GS forms a rod-like beta-spiral with length near 50 nm and diameter near 2 nm. We have sought to examine this model by using noncontact atomic force microscopy (NCAFM) to image a hybrid HMW-GS in which the N-terminal domain of subunit Dy10 has replaced the N-terminal domain of subunit Dx5. This hybrid subunit, coded by a transgene overexpressed in transgenic wheat, has the unusual characteristic of forming, in vivo, not only polymeric forms, but also a monomer in which a single disulfide bond links the C-terminal domain to the N-terminal domain, replacing the two intermolecular disulfide bonds normally formed by the corresponding cysteine side chains. No such monomeric subunits have been observed in normal wheat lines, only polymeric forms. NCAFM of the native, unreduced 93 K monomer showed fibrils of varying lengths but a length of about 110 nm was particularly noticeable whereas the reduced form showed rod-like structures with a length of about 300 nm or greater. The 110 nm fibrils may represent the length of the disulfide-linked monomer, in which case they would not be in accord with the beta-spiral model, but would favor a more extended conformation for the polypeptide chain, possibly polyproline II.

Disulfides↗

PubMed2001

Quantitative Analysis of Fluid Interface-Atomic Force Microscopy.

Net repulsive interactions between n-hexadecane and a poly-styrene microsphere in aqueous solutions are measured with atomic force microscopy and interpreted using the augmented Young-Laplace equation. The true separation between probe and fluid interface is implicitly computed from the force-distance data, providing a more accurate description of drop or bubble deformation. Experiments and theoretical arguments demonstrate that a fluid interface stiffens with increasing deformation and is not accurately treated as a Hookean spring. The unexpected stability of the draining aqueous film between hydrophobic bodies in electrolyte solutions is explained primarily by the deformation of the oil drop in response to the applied normal force, as well as the increased hydrodynamic resistance due to the increased drainage area. Copyright 2001 Academic Press.

Journal Article↗

PubMed1992

Atomic force microscopy of DNA molecules.

DNA-cytochrome c complexes adsorbed on carbon-coated mica surfaces were directly imaged by atomic force microscopy in air using commercially available cantilevers, with a routine resolution of 6 nm. Images of M13 phage DNA and M13-DNA polymerase complex are also shown.

Cytochrome c Group↗

PubMed2002

Determination of the topography and biometry of chlorosomes by atomic force microscopy.

Isolated chlorosomes of several species of filamentous anoxygenic phototrophic bacteria (FAPB) and green sulfur bacteria (GSB) were examined by atomic force microscopy (AFM) to characterize their topography and biometry. Chlorosomes of Chloroflexus aurantiacus, Chloronema sp., and Chlorobium (Chl.) tepidum exhibited a smooth surface, whereas those of Chl. phaeobacteroides and Chl. vibrioforme showed a rough one. The potential artifactual nature of the two types of surfaces, which may have arisen because of sample manipulation or AFM processing, was ruled out when AFM images and transmission electron micrographs were compared. The difference in surface texture might be associated with the specific lipid and polypeptide composition of the chlorosomal envelope. The study of three-dimensional AFM images also provides information about the size and shape of individual chlorosomes. Chlorosomal volumes ranged from ca. 35 000 nm(3) to 247 000 nm(3) for Chl. vibrioforme and Chl. phaeobacteroides, respectively. The mean height was about 25 nm for all the species studied, except Chl. vibrioforme, which showed a height of only 14 nm, suggesting that GSB have 1-2 layers of bacteriochlorophyll (BChl) rods and GFB have approximately 4. Moreover, the average number of BChl molecules per chlorosome was estimated according to models of BChl rod organisation. These calculations yielded upper limits ranging from 34 000 BChl molecules in Chl. vibrioforme to 240 000 in Chl. phaeobacteroides, values that greatly surpass those conventionally accepted.

Journal Article↗

PubMed2001

Charge-dependent sidedness of cytochrome P450 forms studied by quartz crystal microbalance and atomic force microscopy.

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.

Adsorption↗

PubMed2005

Multidrug-resistant cancer cells contain two populations of P-glycoprotein with differently stimulated P-gp ATPase activities: evidence from atomic force microscopy and biochemical analysis.

Considerable interest exists about the localization of P-gp (P-glycoprotein) in DRMs (detergent-resistant membranes) of multidrug resistant cancer cells, in particular concerning the potential modulating role of the closely related lipids and proteins on P-gp activity. Our observation of the opposite effect of verapamil on P-gp ATPase activity from DRM and solubilized-membrane fractions of CEM-resistant leukaemia cells, and results from Langmuir experiments on membrane monolayers from resistant CEM cells, strongly suggest that two functional populations of P-gp exist. The first is located in DRM regions: it displays its optimal P-gp ATPase activity, which is almost completely inhibited by orthovanadate and activated by verapamil. The second is located elsewhere in the membrane; it displays a lower P-gp ATPase activity that is less sensitive to orthovanadate and is inhibited by verapamil. A 40% cholesterol depletion of DRM caused the loss of 52% of the P-gp ATPase activity. Cholesterol repletion allowed recovery of the initial P-gp ATPase activity. In contrast, in the solubilized-membrane-containing fractions, cholesterol depletion and repletion had no effect on the P-gp ATPase activity whereas up to 100% saturation with cholesterol induced a 58% increased P-gp ATPase activity, while no significant modification was observed for the DRM-enriched fraction. DRMs were analysed by atomic force microscopy: 40-60% cholesterol depletion was necessary to remove P-gp from DRMs. In conclusion, P-gp in DRMs appears to contain closely surrounding cholesterol that can stimulate P-gp ATPase activity to its optimal value, whereas cholesterol in the second population seems deprived of this function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Polymyxin B-lipid interactions in Langmuir-Blodgett monolayers of Escherichia coli lipids: a thermodynamic and atomic force microscopy study.

The dramatically increased frequency of antibiotic resistance has led to intensive efforts towards developing new families of antibiotics. Membrane-active antibiotic peptides such as polymyxin B (PxB) hold promise as the next generation of antibiotics, since they rarely spur the evolution of resistance. At low concentrations in the membrane, PxB forms vesicle-vesicle contacts and induces lipid exchange without leakage or fusion, a phenomenon that can explain its specificity towards gram-negative bacteria by contact formation between the two phospholipids interfaces in the periplasmatic space. In this work, the interaction of PxB and the nonantibiotic derivative polymyxin B nonapeptide (PxB-NP) with monolayers of Escherichia coli membrane lipids (ECL) has been studied by thermodynamic and structural methods. PxB inserts itself into ECL monolayers as a conformation that forms intermembrane contacts with vesicles injected underneath, and induces lipid exchange when the monolayer surface pressure is set at 32 mN/m (membrane equivalence pressure) or net transfer vesicle-to-monolayer at lower surface pressures. Thermodynamic analysis of the compression isotherms of mixed monolayers indicates that PxB inserts into the monolayer with an expansion of the mean molecular area, implying that peptide and lipids form nonideal mixtures. At low concentrations, corresponding to the membrane-membrane contact form of PxB, the mixed monolayers present positive excess energy values (deltaGm(Ex)), and atomic force microscopy (AFM) imaging reveals structures of approximately 120-nm diameter that protrude from the lipid surface approximately 0.7 nm. At concentrations of PxB above 4 mol %, thermodynamic analysis gives a very high deltaGm(Ex), corresponding to nonfavorable interactions, and AFM images show round structures of 20-30 nm diameter. PxB-NP behaves in a totally different way, in agreement with its inability to form vesicle-vesicle contacts and its lack of antibiotic effect. These results are discussed in the light of the mechanism of action of PxB on the membrane of gram-negative bacteria.

Anti-Bacterial Agents↗

PubMed2001

X-ray diffraction and atomic force microscopy analysis of twinned crystals: rhombohedral canavalin.

The structure of canavalin, the vicilin-class storage protein from jack bean, was refined to 1.7 A resolution in a highly twinned rhombohedral crystal of space group R3 and unit-cell parameters a = b = c = 83.0 A, alpha = beta = gamma = 111.1 degrees. The resulting R and R(free) were 0.176 and 0.245, respectively. The orthorhombic crystal structure (space group C222(1), unit-cell parameters a = 136.5, b = 150.3, c = 133.4 A) was also refined with threefold non-crystallographic symmetry restraints. R and R(free) were 0.181 and 0.226, respectively, for 2.6 A resolution data. No significant difference in the protein structure was seen between these two crystal forms, nor between these two and the hexagonal and cubic crystal forms reported elsewhere [Ko et al. (1993), Acta Cryst. D49, 478-489; Ko et al. (1993), Plant Physiol. 101, 729-744]. A phosphate ion was identified in the lumen of the C-terminal beta-barrel. Lattice interactions showed that the trimeric molecule could be well accommodated in both 'top-up' and 'bottom-up' orientations in a rhombohedral unit cell of the R3 crystal and explained the presence of a high twin fraction. The large inter-trimer stacking interface of the C222(1) crystal may account for its relative stability. Atomic force microscopy (AFM) investigations of the growth of three crystal forms of canavalin indicate the rhombohedral form to be unique. Unlike the other two crystal forms, it contains at least an order of magnitude more screw dislocations and stacking faults than any other macromolecular crystal yet studied, and it alone grows principally by generation of steps from the screw dislocations. The unusually high occurrence of the screw dislocations and stacking faults is attributed to mechanical stress produced by the alternate molecular orientations in the rhombohedral crystals and their organization into discrete domains or blocks. At boundaries of alternate domains, lattice strain is relieved by the formation of the screw dislocations.

Crystallization↗

PubMed1995

Atomic Force microscopy of neuron networks.

We imaged uncoated neuron networks by an atomic force microscope in the repulsive regime of contact mode. Images of granule cells and their axons have been clearly revealed with details smaller than 20 nm. The good stability of the sample and the mechanical reproducibility of the microscope allowed the imaging of a neuron culture area of several square microns. By combining tens of images, we were able to reconstruct a highly defined neuronal network. Furthermore, the images were very reproducible over repeated scanning acquisition, demonstrating the mechanical and thermal stability of the instrument-sample system.

Animal Population Groups↗

PubMed1999

Molecular self-recognition and adhesion via proteoglycan to proteoglycan interactions as a pathway to multicellularity: atomic force microscopy and color coded bead measurements in sponges.

During the emergence of multicellular organisms, molecular mechanisms evolved to allow maintenance of anatomical integrity and self-recognition. We propose that carbohydrates from proteoglycans, as the most peripheral cell surface, and matrix molecules might have provided these key adhesion and recognition functions. If so, the Porifera as the simplest metazoans alive today should retain, at least in part, proteoglycan adhesion and recognition mechanisms. Early work on cell adhesion of dissociated marine sponge cells provided important phenomenological evidence for cell sorting. Here is reviewed recent work on molecular mechanisms of cell recognition and adhesion mediated by cell surface proteoglycans purified from three marine sponge species, Microciona prolifera, Halichondria panicea, and Cliona celata. Biochemical characterization of isolated proteoglycans showed that each species expressed a unique type of primordial molecule named glyconectins. These proteoglycans displayed species-specific self-recognition and adhesion in color-coded bead, cell, and blotting assays. The specificity of homophilic proteoglycan to proteoglycan interactions in the Porifera approaches the binding selectivity of the evolutionarily advanced immunoglobulin superfamily system. Such xeno-selectivity may be a new paradigm for the molecular self-recognition, which was a fundamental requirement in the self/non-self discrimination during the emergence of multicellularity and further divergence of species. We have used atomic force microscopy (AFM) technology to directly measure intermolecular binding strength between individual pairs of ligand and receptor molecules in physiological solution. Homophilic glyconectin interactions were investigated by AFM after covalent attachment of the protein core to the sensor tip and to a flat surface, leaving the carbohydrates unmodified. AFM measurements of the binding strength between glyconectins indicated that one pair of molecules could theoretically hold the weight of 1,600 cells in physiological solution. These results provided the first essential and quantitative evidence that proteoglycan-proteoglycan binding can perform the adhesion function that we have assigned to it. Our investigations with purified proteoglycans from the marine sponge M. prolifera (glyconectin 1) using bead and cell adhesion assays have provided evidence that a new molecular mechanism of polyvalent and specific glycan-glycan binding between proteoglycans can mediate cell recognition and adhesion. Partial sequencing of the glycans has revealed two new cell adhesion carbohydrate structures: (3)GlcNAc(3OSO3)beta1-3Fuc and Pyr4,6Galbeta1-4GlcNAcbeta1-3Fuc.

Animals↗

PubMed1996

Atomic force microscopy study of the secretory granule lumen.

We have used an atomic force microscope to study the mechanical properties of the matrix found in the lumen of secretory granules isolated from mast cells. The matrices were insoluble and had an average height of 474 +/- 197 nm. The volume of these matrices increased reversibly about tenfold by decreasing the valency of the bathing external cation (La3+ < Ca2+ < Na+). The elastic (Young's) modulus was found to decrease by about 100-fold (4.3 MPa in La3+ to 37 kPa in Na+) upon a tenfold increase in the matrix volume. A swollen granule matrix had an elastic modulus similar to that of gelatin in water. The elastic modulus was inversely related to the change in the volume of the matrix, following a relationship similar to that predicted for the elasticity of weakly cross-linked polymers. Our results show that the matrix of these secretory granules have the mechanical properties of weak ion exchange resins, lending strong support to an ion exchange mechanism for the storage and release of cationic secretory products.

Animals↗