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PubMed2003

Structural and human cellular assessment of a novel microsphere-based tissue engineered scaffold for bone repair.

The limitations of current grafting materials have driven the search for synthetic alternatives for the regeneration of trabecular bone. A variety of biodegradable polymer foams composed of 85/15 poly(lactide-co-glycolide) (PLAGA) have been evaluated for such uses. However, structural limitations may restrict the clinical use of these scaffolds. We have developed a novel sintered microsphere scaffold with a biomimetic pore system equivalent to the structure of trabecular bone. By modifying processing parameters, several different sintered microsphere structures were fabricated. Optimization of the structure dealt with modifications to sphere diameter and heating time. Compressive testing illustrated a trend between microsphere diameter and modulus, where increased microsphere diameter resulted in decreased modulus. In addition, evaluation of the pore system showed a positive correlation between sphere diameter and pore diameter. Mercury porosimetry showed increased median pore size with an increased microsphere diameter. Heating time modifications showed that compressive modulus was dependent on the period of heating with longer heating times resulting in higher moduli. It was also shown that heating time did not affect the pore structure. Analysis of the structural data indicated that the microsphere matrix sintered for 4h at a temperature of 160 degrees C with a microsphere diameter of 600-710 microm resulted in an optimal, biomimetic structure with range in pore diameter of 83-300 microm, a median pore size of 210 microm, 35% porosity, and a compressive modulus of 232 MPa. An in vitro evaluation of human osteoblasts seeded onto the sintered matrix indicated that the structure was capable of supporting the attachment and proliferation of cells throughout its pore system. Immunofluorescent staining of actin showed that the cells were proliferating three-dimensionally through the pore system. The stain for osteocalcin was used and showed that cells maintained phenotypic expression for this bone specific protein. Through this work, it was shown that an osteoconductive PLAGA scaffold with a pore system used as a reverse template to the structure of trabecular bone could be fabricated through the sintered microsphere method.

Actins↗
PubMed1993

Structure of the hydrophobic core in the transition state for folding of chymotrypsin inhibitor 2: a critical test of the protein engineering method of analysis.

Chymotrypsin inhibitor 2 (CI2) unfolds and refolds according to a simple two-state kinetic mechanism. The single rate-determining transition state may thus be studied by kinetics of both unfolding and refolding. This has allowed the direct testing of some facets of the protein engineering procedure (phi-value analysis). The structure of the hydrophobic core of CI2 in the transition state was analyzed from kinetic and thermodynamic measurements of guanidinium chloride-induced unfolding of 11 mutants and of their rates of refolding. In all cases, the strengths of the interactions measured from refolding kinetics in water are in excellent agreement with those measured from unfolding kinetics in guanidinium chloride solutions and extrapolated to zero molar denaturant. Changes in the free energies of unfolding on mutation, as well as other equilibrium properties calculated from the rate constants, are also in excellent agreement with those measured directly from equilibrium studies. These data provide further evidence for application of the principle of microscopic reversibility to aspects of protein folding in the presence of denaturant and the validity of extrapolation to the absence of denaturant. The edges of the hydrophobic core of CI2 are significantly weakened in the transition state, and, in many cases, the interactions are totally lost. The center of the core remains partially intact; the interaction energy is lowered by about 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

PEG-grafted chitosan as an injectable thermoreversible hydrogel.

PEG-grafted chitosan was formulated such that its solution undergoes a thermally reversed phase transition from an injectable free-flowing solution at low temperature to a gel at body temperature. Aqueous solutions of PEG-grafted chitosan can be prepared at physiological pH values, thereby allowing safe incorporation of bioactive molecules. This injectable thermoreversible hydrogel is potentially suitable for a wide range of biomedical applications, particularly in sustained in vivo drug release and tissue engineering. An aqueous solution of PEG-grafted chitosan polymer is injectable at low temperatures but forms a gel at body temperature.

Chitosan↗
PubMed1998

Stabilization of recombinant Drosophila acetylcholinesterase.

The uses of pure and stable acetylcholinesterase can range from simple basic research to applications in environment quality assessment. In order to satisfy some of these needs its recombinant expression is routinely performed. Affinity-purified recombinant Drosophila melanogaster acetylcholinesterase proved to be instable; an apparent cause of this seemed to be the presence of contaminants with protease activity as evidenced by SDS-PAGE. The elimination of these accompanying products was achieved by anion-exchange, hydrophobic interaction, and cibacron blue affinity chromatography applied downstream from procainamide affinity chromatography. The utilization of a parallel affinity acting via an engineered histidine tail permitted the elimination of the copurified proteases as well. Despite the elimination of the contaminants, the apparently pure extracts were still unstable. It is shown that such instability can be counterbalanced by provoking protein-protein interactions, either between enzyme molecules or with other molecules such as bovine serum albumin. Another way to reduce instability is the addition of a reversible inhibitor or polyethylene glycol 3350.

Acetylcholinesterase↗
PubMed2003

Amikacin-induced acute renal injury in rats: protective role of melatonin.

It is well established that some agents such as aminoglycosides generate free oxygen radicals, leading to an increased oxireductase production, which in turn increases tissue toxicity. The aim of this study is to test whether melatonin, the chief secretory product of the pineal gland and a highly effective antioxidant and free radical scavenger, reduces the nephrotoxicity caused by amikacin (AK). Herein, we investigated the physiologic and pharmacological role of melatonin in influencing AK-induced nephrotoxicity. For this, pinealectomized (Px) and sham operated (non-Px) rats were used. Both AK and melatonin were administered to all groups. We investigated the effects of melatonin on AK-induced changes in levels of malondialdehyde (MDA), a lipid peroxidation product, glutathione (GSH), an antioxidant whose levels are influenced by oxidative stress, and blood urea nitrogen (BUN) and serum creatine (Cr) levels. Morphologic changes in the kidney were also examined by using light microscopy. MDA levels were found to be higher in Px than in non-Px AK-treated animals. Melatonin administration to Px rats reduced MDA levels. In relative to non-Px rats, Px animals treated with AK had significantly lower GSH concentrations while melatonin administration elevated GSH levels in the kidney; however, this stimulatory effect of melatonin was not observed in non-Px AK-treated rats. Treatment with AK alone resulted in significantly higher plasma Cr and BUN levels. Repeated administration of melatonin prevented the AK-induced elevation of plasma Cr and BUN levels. Morphologic damage to renal tubules as a result of AK was more severe in the renal cortex than in the medulla. The damage to the kidney induced by AK was reversed by melatonin in the Px rats. In conclusion, these results show that physiologic melatonin concentrations are important in reducing AK-induced renal damage, while pharmacologic concentrations of melatonin did not add to the beneficial effect.

Amikacin↗
PubMed2001

An integrated study of threonine-pathway enzyme kinetics in Escherichia coli.

We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions.

Aspartate Kinase↗
PubMed1992

Initiator methionine tRNA is essential for Ty1 transposition in yeast.

The yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism similar to that of retroviral reverse transcription and integration. Ty1 RNA contains a putative minus strand primer binding site (-PBS) that is complementary to the 3' acceptor stem of the initiator methionine tRNA (tRNA(iMet)). Here we demonstrate that the tRNA(iMet) is used as a primer for Ty1 reverse transcription. Mutations in the Ty1 element that alter 5 of 10 nucleotides that are complementary to the tRNA(iMet) abolish Ty1 transposition, even though they are silent with regard to Ty1 protein coding. We have constructed a yeast strain lacking wild-type tRNA(iMet) that is dependent on a mutant derivative of tRNA(iMet) that has an altered acceptor stem sequence, engineered to restore homology with the Ty1 -PBS mutant. The compensatory mutations made in the tRNA(iMet) alleviate the transposition defect of the Ty1 -PBS mutant. The mutant and wild-type tRNA(iMet) are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1 -PBS. Thus, complementarity between the Ty1 -PBS and tRNA(iMet) is essential for transposition but is not necessary for packaging of the tRNA inside virus-like particles.

Base Sequence↗
PubMed2000

Restoration of cognitive and motor functions by ciliary neurotrophic factor in a primate model of Huntington's disease.

Huntington's disease (HD) is an inherited disorder characterized by cognitive impairments, motor deficits, and progressive dementia. These symptoms result from progressive neurodegenerative changes mainly affecting the neostriatum. This pathology is fatal in 10 to 20 years and there is currently no treatment for HD. Early in the course of the disease, initial clinical manifestations are due to striatal neuronal dysfunction, which is later followed by massive neuronal death. A major therapeutic objective is therefore to reverse striatal dysfunction prior to cell death. Using a primate model reproducing the clinical features and the progressive neuronal degeneration typical of HD, we tested the therapeutic effects of direct intrastriatal infusion of ciliary neurotrophic factor (CNTF). To achieve a continuous delivery of CNTF over the full period of evaluation, we took advantage of the macroencapsulation technique. Baby hamster kidney (BHK) cells previously engineered to produce human CNTF were encapsulated into semipermeable membranes and implanted bilaterally into striata. We show here that intracerebral delivery of low doses of CNTF at the onset of symptoms not only protects neurons from degeneration but also restores neostriatal functions. CNTF-treated primates recovered, in particular, cognitive and motor functions dependent on the anatomofunctional integrity of frontostriatal pathways that were distinctively altered in this HD model. These results support the hypothesis that CNTF infusion into the striatum of HD patients not only could block the degeneration of neurons but also alleviated motor and cognitive symptoms associated with persistent neuronal dysfunction.

Animals↗
PubMed2000

Genetic susceptibility to renal ischemia reperfusion injury revealed in a murine model.

BACKGROUND: The development of genetically engineered mice has led to increased use of mouse models to study renal ischemic reperfusion injury (IRI). We hypothesized that susceptibility to IRI could result from strain differences due to genetic factors. METHODS: Our study compared recovery subsequent to renal IRI in NIH Swiss, C57BL/6, and BALB/c mice. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were evaluated postischemia. We also conducted reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of renal cytokines and adhesion molecules postischemia. RESULTS: At 48 hr postischemia, renal dysfunction in NIH Swiss mice was significantly reduced, compared with other groups (P<0.01). BUN measurements confirmed renal protection at 48 hr in the NIH Swiss group. RT-PCR analysis of mRNA postischemia demonstrated that, between strains, there was little difference in mRNA expression for renal cytokines and adhesion molecules. CONCLUSIONS: NIH Swiss mice appear to be resistant in susceptibility to renal IRI. Early expression of pro-inflammatory genes was not associated with resistance to IRI, thus genetic factors could be important in outcome after renal IRI.

Animals↗
PubMed2002

Inserting a nuclear targeting signal into a replication-competent Moloney murine leukemia virus affects viral export and is not sufficient for cell cycle-independent infection.

The effects of inserting reported nuclear localization signals (NLSs) into the Moloney murine leukemia virus (Mo-MuLV) integrase (IN) protein, within a replication-competent viral construct, were studied. In contrast to the virus harboring IN fused to the simian virus 40 (SV40) large T antigen NLS (SV40 NLS) (J. A. Seamon, M. Adams, S. Sengupta, and M. J. Roth, Virology 274:412-419, 2000), a codon-modified SV40 NLS was stably expressed during viral propagation. Incorporation of the codon-modified SV40 NLS into IN, however, altered the packaging of the Gag-Pol precursor in the virus; viral particles contained decreased levels of reverse transcriptase (RT) and IN. In addition, the virus showed delayed kinetics of viral DNA synthesis upon infection. A panel of infectious MuLVs containing alternative IN-NLS fusions was generated and assayed for cell cycle-independent infection. Viral infection with the NLS-tagged proteins, however, remained dependent on passage of the cells through mitosis. This finding has direct implications for engineering murine-based retroviral vectors for gene therapy.

Amino Acid Sequence↗
PubMed2006

Cell proliferation inhibition and alterations in retinol esterification induced by phytanic acid and docosahexaenoic acid.

We investigated the effects of two natural dietary retinoid X receptor (RXR) ligands, phytanic acid (PA) and docosahexaenoic acid (DHA), on proliferation and on the metabolism of retinol (vitamin A) in both cultured normal human prostate epithelial cells (PrECs) and PC-3 prostate carcinoma cells. PA and DHA inhibited the proliferation of the parental PC-3 cells and PC-3 cells engineered to overexpress human lecithin:retinol acyltransferase (LRAT) in both the absence and presence of retinol. A synthetic RXR-specific ligand also inhibited PC-3 cell proliferation, whereas all-trans retinoic acid (ATRA) did not. PA and DHA treatment increased the levels of retinyl esters (REs) in both PrECs and PC-3 cells and generated novel REs that eluted on reverse-phase HPLC at 54.0 and 50.5 min, respectively. Mass spectrometric analyses demonstrated that these novel REs were retinyl phytanate (54.0 min) and retinyl docosahexaenoate (50.5 min). Neither PA nor DHA increased LRAT mRNA levels in these cells. In addition, we demonstrate that retinyl phytanate was generated by LRAT in the presence of PA and retinol; however, retinyl docosahexaenoate was produced by another enzyme in the presence of DHA and retinol.

Cell Division↗
PubMed2006

Murine B16 melanomas expressing high levels of the chemokine stromal-derived factor-1/CXCL12 induce tumor-specific T cell chemorepulsion and escape from immune control.

The chemokine, stromal-derived factor-1/CXCL12, is expressed by normal and neoplastic tissues and is involved in tumor growth, metastasis, and modulation of tumor immunity. T cell-mediated tumor immunity depends on the migration and colocalization of CTL with tumor cells, a process regulated by chemokines and adhesion molecules. It has been demonstrated that T cells are repelled by high concentrations of the chemokine CXCL12 via a concentration-dependent and CXCR4 receptor-mediated mechanism, termed chemorepulsion or fugetaxis. We proposed that repulsion of tumor Ag-specific T cells from a tumor expressing high levels of CXCL12 allows the tumor to evade immune control. Murine B16/OVA melanoma cells (H2b) were engineered to constitutively express CXCL12. Immunization of C57BL/6 mice with B16/OVA cells lead to destruction of B16/OVA tumors expressing no or low levels of CXCL12 but not tumors expressing high levels of the chemokine. Early recruitment of adoptively transferred OVA-specific CTL into B16/OVA tumors expressing high levels of CXCL12 was significantly reduced in comparison to B16/OVA tumors, and this reduction was reversed when tumor-specific CTLs were pretreated with the specific CXCR4 antagonist, AMD3100. Memory OVA-specific CD8+ T cells demonstrated antitumor activity against B16/OVA tumors but not B16/OVA.CXCL12-high tumors. Expression of high levels of CXCL12 by B16/OVA cells significantly reduced CTL colocalization with and killing of target cells in vitro in a CXCR4-dependent manner. The repulsion of tumor Ag-specific T cells away from melanomas expressing CXCL12 confirms the chemorepellent activity of high concentrations of CXCL12 and may represent a novel mechanism by which certain tumors evade the immune system.

Animals↗
PubMed2006

[Cloning of NEP1-40 gene and expression of its protein].

OBJECTIVE: To clone the genes of nogo-66 and NEP1-40 from spinal cord of rat and to realize the expression of its protein in vitro. METHODS: The nogo-66 and NEP1-40 genes were cloned from the spinal cord of juvenile rat by use of RT-PCR techniques, and the objective genes were bonded to T vector through gene coupled action, recombinant plasmid were sequencing, and the genes were cloned into PQE30-GST vector, then the recombinant plasmids were induced by isopropyl thiogalactoside (IPTG) to express the proteins. The two proteins were purified by Ni-column and detected by using Western-blot test. RESULTS: The Nogo-66 and NEP1-40 genes were successfully cloned from rat, which were 215 bp and 137 bp for each one when add the enzyme site. No gene mutations were detected in the two genes after sequencing. The expression plasmids were cut by the two enzyme (BamH I and Hind III), the target bands were seen on the results of electrophoresis. The expression plasmids were induced by IPTG and got the purified GST fusion protein nogo-66 and NEP1-40, which relative molecular weight were 33.2 x 10(3) and 30.3 x 10(3) respectively. The results of Western-blot test confirmed that the antigenicity of the two proteins was precise. CONCLUSION: Nogo-66 and NEP1-40 proteins can be expressed in a high efficiency in vitro using genetic engineering, so it provides a good basis for further research on its function and vaccine for spinal injury.

Animals↗
PubMed1991

Inducible transformation of fibroblasts using a metallothionein-v-myc gene construct.

An inducible oncogene construct has been engineered by coupling the MC29 v-myc oncogene to the sheep metallothionein promoter. Transfection of this plasmid, which also contains the neomycin resistance gene, into Rat-1 cells, has resulted in the isolation of independent clones resistant to G418. Certain of these clones were found to exhibit inducible transformation in response to ZnSO4. Transformation was graded with increasing ZnSO4 levels and was reversible when ZnSO4 was removed from the media. By analyzing v-myc mRNA levels, the inducible alterations in cellular morphology and growth were found to be associated with increased v-myc expression. The metallothionein promoter exhibited negligible constitutive expression of v-myc and none of the clones isolated exhibited spontaneous transformation. Our results show that the use of a metallothionein promoter v-myc construct facilitates the study of inducible fibroblast transformation.

Animals↗
PubMed1989

Drug products of recombinant DNA technology.

Presented are the steps in creating a recombinant DNA molecule, examples of recombinant drug products, a description of DNA fingerprinting methods for diagnosing diseases, a discussion of the patenting of recombinant drugs, and a look to the future of this revolutionary biotechnology. Constructing a recombinant DNA molecule involves cutting the DNA into fragments with restriction endonucleases and rejoining the fragments in novel arrangements with ligase. Propagating the molecule in a microorganism, or cloning, is necessary to increase the number of gene copies to facilitate detection of the gene of interest and to produce the protein it encodes. Recombinant DNA drug products have been developed that represent the communicator, structural, and modifier classes of proteins. Recombinant communicator proteins include interferons alfa-2a and alfa-2b and granulocyte-macrophage colony-stimulating factor (immune system modulators); epidermal growth factor and erythropoietin (tissue repair promoters); and human insulin, growth hormone, and atrial peptide (metabolism modulators). Recombinant structural proteins include hepatitis B virus vaccine and CD4 protein, and recombinant modifier proteins include tissue plasminogen activator and superoxide dismutase (agents that split or splice organic molecules). In the future, gene defects associated with genetic diseases will be unraveled, leading to the production of new therapeutic agents designed to counteract or actually reverse those defects. Recombinant protein drugs will be further tailored to enhance their activity and specificity. These advances are so novel and momentous that patent protection has been extended not only to recombinant drugs but to the recombinant microorganisms in which they are manufactured. In cloning genes, investigators directly use the protein designs that occur naturally. Basic research will soon lead to the engineering of novel proteins with specified functions.

Cloning, Molecular↗
PubMed2005

Luteinising hormone-releasing hormone analogue reverses the cell adhesion profile of EGFR overexpressing DU-145 human prostate carcinoma subline.

Cetrorelix, a luteinising hormone-releasing hormone (LHRH) analogue, has been shown to limit growth of the human androgen-independent prostate cell line DU-145, although other inhibitory actions may also be affected. Both growth and invasion of DU-145 cells are linked to autocrine epidermal growth factor receptor (EGFR) signalling. Invasiveness requires not only cells to migrate to conduits, but also reduced adhesiveness between tumour cells to enable separation from the tumour mass. Thus, we investigated whether Cetrorelix alters the DU-145 cell-cell adhesion and if this occurs via altered EGFR signalling. Pharmacologic levels of Cetrorelix limited the invasiveness of a highly invasive DU-145 subline overexpressing full-length EGFR (DU-145 WT). Extended exposure of the cells to Cetrorelix resulted in increased levels of the cell-cell adhesion complex molecules E-cadherin, alpha- and beta-catenin, and p120. Puromycin blocked the increases in E-cadherin and beta-catenin levels, suggesting that de novo protein synthesis is required. The Cetrorelix effect appears to occur via transmodulation of EGFR by a protein kinase C (PKC)-dependent mechanism, as there were no changes in DU-145 cells expressing EGFR engineered to negate the PKC transattenuation site (DU-145 A654); downregulation of EGFR signalling produced a similar upregulation in adhesion complex proteins, further suggesting a role for autocrine signalling. Cetrorelix increased the cell-cell adhesiveness of DU-145 WT cells to an extent similar to that seen when autocrine EGFR signalling is blocked; as expected, DU-145 A654 cell-cell adhesion also was unaffected by Cetrorelix. The increased adhesiveness is expected as the adhesion complex molecules moved to the cells' periphery. These data offer direct insight into the possible crosstalk pathways between the LHRH and EGFR receptor signalling. The ability of Cetrorelix to downregulate EGFR signalling and subsequently reverse the antiadhesiveness found in metastatic prostate cancer highlights a novel potential target for therapeutic strategies.

Cell Adhesion↗
PubMed2005

Osteoblastic phenotype expression of MC3T3-E1 cells cultured on polymer surfaces.

BACKGROUND: Current efforts in bone tissue engineering have as one focus the search for a scaffold material that will support osteoblast proliferation, matrix mineralization, and, ultimately, bone formation. The goal is to develop a bone substitute that is functionally equivalent to autograft bone. Previously published reports have shown that osteoblasts exhibit varying rates and degrees of proliferation and mineralization when grown on different surfaces. METHODS: This study presents a histologic and biomolecular analysis of MC3T3-E1 murine preosteoblast cells grown on poly(lactide-co-glycolide) (PLGA) versus poly(-caprolactone) (PCL), two commonly studied scaffold polymers. MC3T3-E1 cells were cultured on slides coated with either PLGA or PCL, and on uncoated glass slides as control, with six slides in each group. After 6 weeks in culture, the cells were stained for osteocalcin, alkaline phosphatase activity, and matrix mineralization. In addition, to assess the effects of the surface material on phenotypic expression at the molecular level, MC3T3-E1 cells were cultured on polymer-coated 24-well plates for 4 days, and analyzed by reverse transcription polymerase chain reaction for the expression of osteocalcin and alkaline phosphatase. RESULTS: The results showed that three groups of slides stained positively for osteocalcin at 6 weeks. However, markedly less alkaline phosphatase activity and mineralization were observed on the cells grown on PCL. Real-time polymerase chain reaction assays subsequently revealed decreased expression of both markers by cells cultured on PCL compared with PLGA. CONCLUSIONS: These results suggest that PCL does not support the full expression of an osteoblastic phenotype by MC3T3-E1 cells. PCL, therefore, is less desirable as a scaffold polymer in bone tissue engineering in so far as supporting bone formation is concerned. However, because PCL has favorable handling characteristics and strength, modifications of PCL may prompt further investigation.

3T3 Cells↗
PubMed2006

Engineering DNA-mediated colloidal crystallization.

DNA is a powerful and versatile tool for nanoscale self-assembly. Several researchers have assembled nanoparticles and colloids into a variety of structures using the sequence-specific binding properties of DNA. Until recently, however, all of the reported structures were disordered, even in systems where ordered colloidal crystals might be expected. We detail the experimental approach and surface preparation that we used to form the first DNA-mediated colloidal crystals, using 1 mum diameter polystyrene particles. Control experiments based on the depletion interaction clearly indicate that two standard methods for grafting biomolecules to colloidal particles (biotin/avidin and water-soluble carbodiimide) do not lead to ordered structures, even when blockers are employed that yield nominally stable, reversibly aggregating dispersions. In contrast, a swelling/deswelling-based method with poly(ethylene glycol) spacers resulted in particles that readily formed ordered crystals. The sequence specificity of the interaction is demonstrated by the crystal excluding particles bearing a noninteracting sequence. The temperature dependence of gelation and crystallization agree well with a simple thermodynamic model and a more detailed model of the effective colloidal pair interaction potential. We hypothesize that the surfaces yielded by the first two chemistries somehow hinder the particle-particle rolling required for annealing ordered structures, while at the same time not inducing a significant mean-force interaction that would alter the self-assembly phase diagram. Finally, we observe that particle crystallization kinetics become faster as the grafted-DNA density is increased, consistent with the particle-particle binding process being reaction, rather than diffusion limited.

Avidin↗