Normal and frictional interactions of purified human statherin adsorbed on molecularly-smooth solid substrata

Human salivary statherin was purified from parotid saliva and adsorbed to bare hydrophilic (HP) mica and STAI-coated hydrophobic (HB) mica in a series of Surface Force Balance experiments that measured the normal (F _n) and friction forces (F _s*) between statherin-coated mica substrata. Readings were taken both in the presence of statherin solution (HP and HB mica) and after rinsing (HP mica). F _n measurements showed, for both substrata, monotonic steric repulsion that set on at a surface separation D ～ 20 nm, indicating an adsorbed layer whose unperturbed thickness was ca 10 nm. An additional longer-ranged repulsion, probably of electrostatic double-layer origin, was observed for rinsed surfaces under pure water. Under applied pressures of ～ 1 MPa, each surface layer was compressed to a thickness of ca 2 nm on both types of substratum, comparable with earlier estimates of the size of the statherin molecule. Friction measurements, in contrast with F _n observations, were markedly different on the two different substrata: friction coefficients, μ ≡ ?F _s*/?F _n, on the HB substratum (μ ≈ 0.88) were almost an order of magnitude higher than on the HP substratum (μ ≈ 0.09 and 0.12 for unrinsed and rinsed, respectively), and on the HB mica there was a lower dependence of friction on sliding speed than on the HP mica. The observations were attributed to statherin adsorbing to the mica in multimer aggregates, with internal re-arrangement of the protein molecules within the aggregate dependent on the substratum to which the aggregate adsorbed. This internal re-arrangement permitted aggregates to be of similar size on HP and HB mica but to have different internal molecular orientations, thus exposing different moieties to the solution in each case and accounting for the very different friction behaviour.     

Specific and non-specific interactions in bacterial adhesion to solid substrata

Abstract Based on a literature review, a hypothesis is forwarded on the mechanism of initial bacterial adhesion to solid substrata, which accounts both for the role of specific microscopic surface components as well as for the role of non-specific macroscopic surface properties (surface free energy, zeta potential or hydrophobicity). Three distinct regions in the adhesion process are suggested in which at large and intermediate separation distances adhesion is mediated by the macroscopic surface properties as surface free energy and surface charge, respectively. At small separation distances specific short-range interactions can occur, leading to a strong and irreversible bonding, provided the water film present in between the interaction surfaces can be removed. A major role of hydrophobic groups, supposed to be associated with bacterial surface appendages is suggested to be its dehydrating capacity, enabling the removal of the vicinal water film yielding small areas of direct contact between protruberant parts of the cell surface and the substratum.     

Lubrication and load-bearing properties of human salivary pellicles adsorbed ex vivo on molecularly smooth substrata

In a series of Surface Force Balance experiments, material from human whole saliva was adsorbed to molecularly smooth mica substrata (to form an 'adsorbed salivary film'). Measurements were taken of normal (load bearing, F (n)) and shear (frictional, F (s)*) forces between two interacting surfaces. One investigation involved a salivary film formed by overnight adsorption from undiluted, centrifuged saliva, with the adsorbed film rinsed with pure water before measurement. Measurements were taken under pure water and 70?mM NaNO(3). In a second investigation, a film was formed from and measured under a solution of 7% filtered saliva in 10?mM NaNO(3). F (n) results for both systems showed purely repulsive layers, with an uncompressed thickness of 35-70?nm for the diluted saliva investigation and, prior to the application of shear, 11?nm for the rinsed system. F (s)* was essentially proportional to F (n) for all systems and independent of shear speed (in the range 100-2000?nm s(-1)), with coefficients of friction μ?～?0.24 and μ?～?0.46 for the unrinsed and rinsed systems, respectively. All properties of the rinsed system remained similar when the pure water measurement environment was changed to 70?mM NaNO(3). For all systems studied, shear gave rise to an approximately threefold increase in the range of normal forces, attributed to the ploughing up of adsorbed material during shear to form debris that stood proud of the adsorbed layer. The results provide a microscopic demonstration of the wear process for a salivary film under shear and may be of particular interest for understanding the implications for in vivo oral lubrication under conditions such as rinsing of the mouth cavity. The work is interpreted in light of earlier studies that showed a structural collapse and increase in friction for an adsorbed salivary film in an environment of low ionic strength.     

Lubrication and load-bearing properties of human salivary pellicles adsorbed ex vivo on molecularly smooth substrata

In a series of Surface Force Balance experiments, material from human whole saliva was adsorbed to molecularly smooth mica substrata (to form an ‘adsorbed salivary film’). Measurements were taken of normal (load bearing, F _n) and shear (frictional, F _s*) forces between two interacting surfaces. One investigation involved a salivary film formed by overnight adsorption from undiluted, centrifuged saliva, with the adsorbed film rinsed with pure water before measurement. Measurements were taken under pure water and 70 mM NaNO₃. In a second investigation, a film was formed from and measured under a solution of 7% filtered saliva in 10 mM NaNO₃. F _n results for both systems showed purely repulsive layers, with an uncompressed thickness of 35–70 nm for the diluted saliva investigation and, prior to the application of shear, 11 nm for the rinsed system. F _s* was essentially proportional to F _n for all systems and independent of shear speed (in the range 100–2000 nm s^?1), with coefficients of friction  μ ～ 0.24 and μ ～ 0.46 for the unrinsed and rinsed systems, respectively. All properties of the rinsed system remained similar when the pure water measurement environment was changed to 70 mM NaNO₃. For all systems studied, shear gave rise to an approximately threefold increase in the range of normal forces, attributed to the ploughing up of adsorbed material during shear to form debris that stood proud of the adsorbed layer. The results provide a microscopic demonstration of the wear process for a salivary film under shear and may be of particular interest for understanding the implications for in vivo oral lubrication under conditions such as rinsing of the mouth cavity. The work is interpreted in light of earlier studies that showed a structural collapse and increase in friction for an adsorbed salivary film in an environment of low ionic strength.     

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18–116 erg cm⁻². To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm⁻² for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm⁻². The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Y _s <50 erg cm⁻²), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (ΔF _adh) show that ΔF _adh decreases with increasingY _s , and equals zero around 45 erg cm⁻² for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.     

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18-116 erg cm-2. To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm-2 for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm-2. The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Ys less than 50 erg cm-2), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (delta Fadh) show that delta Fadh decreases with increasing Ys, and equals zero around 45 erg cm-2 for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.     

Location of cellular adhesions to solid substrata

Micromanipulation and time lapse cinemicrography were used to locate the adhesions to glass and polystyrene formed in culture by a variety of different cell types. These adhesions were observed to be confined to relatively narrow areas near the cell margins, principally in the vicinity of ruffling activity. During mitosis and after certain treatments, cell-substratum adhesion was found to be restricted to many tiny points connected to the cell body by narrow, nearly invisible retraction fibers. The relationship of these phenomena to cell locomotion, contact inhibition, and contact retraction is discussed.     

Expression,purification, phosphorylation and characterization of recombinant human statherin

This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.     

Detection in human saliva of different statherin and P-B fragments and derivatives

Statherin is a multifunctional polypeptide specific of human saliva involved in oral calcium homeostasis, phosphate buffering and formation of protein networks. Salivary P-B peptide is usually included into the basic proline-rich protein family but it shows some similarities with statherin and its specific biological role is still undefined. In this study, various fragments and derivatives of statherin and P-B peptide were consistently detected by RP-HPLC ESI-IT MS in 23 samples of human saliva. They were: statherin mono- and non-phosphorylated, statherin Des-Phe(43) (statherin SV1), statherin Des-Thr(42),Phe(43), statherin Des-Asp(1), statherin Des(6-15) (statherin SV2), statherin Des(1-9), statherin Des(1-10), statherin Des(1-13) and P-B Des(1-5). Statherin SV3 (statherin Des(6-15), Phe(43)) was detected only in one sample. Identity of the fragments was confirmed either by MS/MS experiments or by enzymatic digestion or by Edman sequencing. Detection of the fragments suggests that statherin and P-B peptide are submitted to post-translational proteolytic cleavages that are common to other classes of salivary proteins.     

Structure and sequence determination of the gene encoding human salivary statherin

Human statherin (STT) is a low-Mr (43 amino acids) acidic phosphoprotein secreted mainly by salivary glands. It acts as an inhibitor of precipitation of Ca.phosphate salts in the oral cavity. DNA (12.2 kb) was isolated from human genomic phage lambda libraries as a series of overlapping clones, and the nucleotide sequence of the STT-encoding gene (STT) was determined. The transcribed region spans 6.5 kb and contains six exons and five introns. Upstream DNA (1.6 kb) was also sequenced and a number of possible regulatory elements were identified. The exon-intron boundaries of the STT gene roughly coincide with the protein-coding regions of the mRNA and with the functional domains of STT. This pattern of organization has been seen in a variety of eukaryotic genes and is consistent with the domain theory of gene evolution.     

Improved elution of isoamylase adsorbed on raw starch and the preservation of purified enzyme

The isoamylase from Pseudomonas amyloderamosa can be recovered by adsorption—elution on raw starch. In this process, the elution of adsorbed isoamylase was significantly affected by elution design. When the raw starch-enzyme suspension was packed into a funnel-type glass filter instead of being stirred in a flask to elute the adsorbed isoamylase, the recovery was increased from 53.6% to about 81% and the concentration of isoamylase was also increased 42-fold in the eluted solution. The addition of 1% potassium sorbate into the eluted enzyme solution gave a retention of more than 80% of the recovered isoamylase activity in a 5-month storage at 4°C.     

Separation of human colostral macrophages and neutrophils on gelatin and collagen-serum substrata

A new technique for separation of human colostral macrophages and neutrophils was developed. Macrophages attached more readily to acid soluble collagen-serum or gelatin-serum substrata than neutrophils. Most of the adherent neutrophils were removed during the first 5 minutes incubation in 3 mM EDTA, thereafter, complete detachment of macrophages followed. Neutrophils and macrophages were enriched more than 80% in nonadherent cell populations and detached cell populations, respectively. These separated colostral leukocytes retained their viability and phagocytic activities. Therefore, functional studies of these purified human colostral leukocytes are possible.     

Normal and malignant cells, including neurons, deposit plasminogen activator on the growth substrata

The results of four different assay methods showed that both normal and malignant plasminogen activator-secreting cells deposited substantial amounts of this protease on tissue-culture substrata, including collagen coatings. The cells studied were Rous sarcoma virus (RSV)-transformed vole fibroblasts, a malignant neural cell line (NG108-15) capable of neurite formation, and normal mouse-regenerating sensory neurons. Deposited plasminogen activator was detected by a fibrin overlay assay at sites from which cells growing on coverslips had been gently dislodged, showing that active enzyme is left beneath cells and in the immediate pericellular area. For neuronal cells, fibrinolytic zones were detected not only at the previous positions of cell bodies but also along the terrain conditioned by neurite extension, suggesting that a trail of plasminogen activator is left behind during growth cone movement. Substratum-bound enzyme could be solubilized in buffers containing sodium dodecyl sulfate (SDS) or Triton X-100 and demonstrated by zymography following electrophoresis or assayed for amidolytic activity with a chromogenic substrate (Kabi S-2251). The results suggest that plasminogen activator may be considered a component of substrate-adhesion material. Secretory proteases deposited directly on matrix molecules would seem strategically positioned to participate in local degradation of components of the extracellular environment.