Dansyl Cadaverine Research Articles

Bacillus amyloliquefaciens modulate autophagy pathways to control Rhizoctonia solani infection in rice

The necrotrophic fungus Rhizoctonia solani significantly threatens rice harvests and agricultural productivity by causing sheath blight disease. This study investigates the potential of the plant growth-promoting rhizobacteria Bacillus amyloliquefaciens (SN13) as a biocontrol agent in the sensitive rice variety Swarna against R. solani infection. Disease incidence analysis reveals untreated rice plants suffer from R. solani infection, while SN13 treatment effectively suppresses fungal growth. In detached leaf assays, SN13 mitigates R. solani-induced damage, and physio-biochemical analyses indicate improved growth in SN13-treated rice plants. Notably, treatment with chloroquine, an autophagy inhibitor, increases disease incidence, whereas SN13 treatment enhances the formation of autophagosomes stained with Mono Dansyl Cadaverine (MDC) dye, as observed through confocal microscopy, suggesting the involvement of autophagy in plant defense against R. solani. Gene expression analysis reveals alterations in ATG and defence-related genes (BZ1, 5H5, and 8A1), affirming that SN13 activates autophagy and bolsters plant resilience. Metabolite analysis using GC-MS indicates the accumulation of defence signalling molecules such as gluconic acid, arabitol, glucopyranoside, ribose, xylopyranose, and arabinofuranoside. Overall, this study demonstrates the role of SN13 in inducing the autophagy response and modulating crucial defense pathways to control R. solani infection in rice var Swarna.

Fluorescent polymer nanofibers based on polycaprolactone and dansyl derivatives for development of latent fingerprints

AbstractThis work provides a convenient low‐cost strategy for fast fabrication of dansyl‐based fluorescent nanofibers for development of latent fingerprints aiming forensic applications. Polycaprolactone (PCL) and dansyl cadaverine (DnsCad) or dansylglycine (DnsGly) nanofibers are prepared by electrospinning technique and characterized by SEM, FTIR, thermal analysis, and fluorescence spectroscopy. The electrospun PCL/DnsCad and PCL/DnsGly nanofibers are fluorescent with greenish blue and yellowish green emission, respectively. Such nanofibers are useful for enhancement of the visual contrast of latent fingerprints on metallic surfaces when exposed to UV light. These findings are particularly important for development of latent fingerprints on cartridge cases.

Signature Ions in MS/MS Spectra for Dansyl-Aminohexyl-QQIV Adducts on Lysine.

Bacterial transglutaminase was used to label human plasma proteins with fluorescent tags. Protein lysines were modified with dansyl-epsilon-aminohexyl-Gln-Gln-Ile-Val-OH (dansylQQIV), while protein glutamines were modified with dansyl cadaverine. Labeled proteins included human butyrylcholinesterase, apolipoprotein A-1, haptoglobin, haptoglobin-related protein, immunoglobulin heavy chain, and hemopexin. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector and Proteome Discoverer searches of mass spectrometry data. The MS/MS fragmentation spectra from dansylQQIV-modified peptides gave intense peaks at 475.2015, 364.1691, 347.1426, 234.0585, and 170.0965 m/z. These signature ions are useful markers for identifying modified peptides. Human butyrylcholinesterase retained full activity following modification by dansylQQIV or dansyl cadaverine.

Characteristic fragment ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine

Glutamine residues susceptible to transglutaminase-catalyzed crosslinking can be identified by incorporation of dansyl cadaverine or biotin cadaverine. Bacterial transglutaminase and human transglutaminase 2 were used to modify residues in beta-casein with dansyl cadaverine. Bacterial transglutaminase was used to modify residues in human butyrylcholinesterase with biotin cadaverine. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector searches of mass spectrometry data. The MS/MS spectra from modified casein included intense peaks at 336.2, 402.2, and 447.2 for fragments of dansyl cadaverine adducts on glutamine. The MS/MS spectra from modified butyrylcholinesterase included intense peaks at 329.2, 395.2, and 440.2 for fragments of biotin cadaverine adducts on glutamine. No evidence for transglutaminase-catalyzed adducts on glutamic acid, aspartic acid, or asparagine was found. Consistent with expectation, it was concluded that bacterial transglutaminase and human transglutaminase 2 specifically modify glutamine. The characteristic ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine are useful markers for modified peptides.

Escherichia coli Lipopolysaccharide Modulates Biological Activities of Human-β-Defensin Analogues but Not Non-Ribosomally Synthesized Peptides.

Human-β-defensins (HBD1-3) are antibacterial peptides containing three disulphide bonds. In the present study, the effect of Escherichia coli lipopolysaccharide (LPS) on the antibacterial activities of HBD2-3, C-terminal analogues having a single disulphide bond, Phd1-3, and their corresponding myristoylated analogues MPhd1-3 were investigated. The effect of LPS on the activities of linear amphipathic peptides melittin, LL37 and non-ribosomally synthesized peptides, polymyxin B, alamethicin, gramicidin A, and gramicidin S was also examined. The antibacterial activity of HBD 2-3, Phd1-3, and MPhd1-3 in the presence of LPS against E. coli and Staphylococcus aureus was inhibited. While LPS inhibited the antibacterial activity of LL37, the inhibition of melittin activity was partial. The hemolytic activity exhibited by MPhd1, MPhd3, melittin, and LL37 was inhibited in the presence of LPS. HBD2-3, Phd1-3, and MPhd1-3 also showed endotoxin neutralizing activity. The antibacterial and hemolytic activities of polymyxin B, alamethicin, gramicidin A, and gramicidin S were not inhibited in the presence of LPS. Fluorescence assays employing dansyl cadaverine showed that HBD2-3 and defensin analogues bind to LPS more strongly as compared to alamethicin, gramicidin A, and gramicidin S. Electron microscopy images indicated that peptides disintegrate the structure of LPS. The inhibition of the antibacterial activity of native defensins and analogues in the presence of LPS indicates that the initial interaction with the bacterial surface is similar. The native defensin sequence or structure is also not essential, although cationic charges are necessary for binding to LPS. Hydrophobic interaction is the main driving force for association of non-ribosomally synthesized polymyxin B, alamethicin, gramicidin A, and gramicidin S with LPS. It is likely that these peptides rapidly insert into membranes and do not interact with the bacterial cell surface, whereas cationic peptides such as β-defensin and their analogues, melittin and LL37, first interact with the bacterial cell surface and then the membrane. Our results suggest that evaluating interaction of antibacterial and hemolytic peptides with LPS is a compelling way of elucidating the mechanism of bacterial killing or hemolysis.

Paenibacillus lentimorbus induces autophagy for protecting tomato from Sclerotium rolfsii infection

During biotic stress, plants use several mechanisms to protect themselves that include the production of reactive oxygen species (ROS), induction of pathogenesis-related proteins and cell death. Some plant growth promoting rhizobacteria (PGPR) are known to act as bio-control agents that protect crops against pathogens. The biocontrol activity of PGPR Paenibacillus lentimorbus (B-30488) against Sclerotium rolfsii showed previously where several defense-related genes were upregulated with ROS induction in tomato. We further evaluate the other possibility, i.e. role of autophagy in enhancing defense in tomato using PGPR. Confocal microscopy revealed the presence of an acidotropic dye Mono Dansyl Cadaverine (MDC) stained autophagosomes in B-30488 treated healthy and infected plants. These autophagosomes almost disappeared in plants treated with an autophagy inhibitor chloroquine. The results were also confirmed by ultrastructural analysis of leaf tissues using transmission electron microscopy. Enhanced expression of autophagy-related genes was also monitored in B-30488 primed fungal infected tissues as compared to control by qRT-PCR. Results of ROS accumulation, fluorescence, confocal and transmission electron microscopy and gene expression analysis revealed induction of autophagy using B-30488 as a biocontrol agent suggesting a role in enhancing disease resistance in tomato. Overall, the present study indicated a role of B-30488 as a biocontrol in enhancing disease resistance in tomato and also assists a better understanding of fungal pathogenesis that is expected to be useful in developing new strategies for disease control.

Engineered glycated amino dendritic polymers as specific nonviral gene delivery vectors targeting the receptor for advanced glycation end products.

The receptor for advanced glycation end products (RAGE) is involved in diabetes or angiogenesis in tumors. Under pathological conditions, RAGE is overexpressed and upon ligand binding and internalization stimulates signaling pathways that promote cell proliferation. In this work, amino dendritic polymers PEI 25 kDa and alkylated derivatives of PAMAM-G2 were engineered by the nonenzymatic Maillard glycation reaction to generate novel AGE-containing gene delivery vectors targeting the RAGE. The glycated dendritic polymers were easily prepared and retained the capability to bind and protect DNA from endonucleases. Furthermore, while glycation decreased the transfection efficiency of the dendriplexes in CHO-k1 cells which do not express RAGE, glycated dendriplexes acted as efficient transfection reagents in CHO-k1 cells which stably express recombinant RAGE. In addition, preincubation with BSA-AGEs, a natural ligand of the RAGE, or dansyl cadaverine, an inhibitor of the RAGE internalization, blocked transfection, confirming their specificity toward RAGE. The results were confirmed in NRK and RAW264.7 cell lines, which naturally express the receptor. The glycated compounds retain their transfection efficiency in the presence of serum and promote in vivo transfection in a mouse model. Accordingly, RAGE is a suitable molecular target for the development of site-directed engineered glycated nonviral gene vectors.

Alendronate promotes bone formation by inhibiting protein prenylation in osteoblasts in rat tooth replantation model

Bisphosphonates (BPs) are a major class of antiresorptive drug, and their molecular mechanisms of antiresorptive action have been extensively studied. Recent studies have suggested that BPs target bone-forming cells as well as bone-resorbing cells. We previously demonstrated that local application of a nitrogen-containing BP (N-BP), alendronate (ALN), for a short period of time increased bone tissue in a rat tooth replantation model. Here, we investigated cellular mechanisms of bone formation by ALN. Bone histomorphometry confirmed that bone formation was increased by local application of ALN. ALN increased proliferation of bone-forming cells residing on the bone surface, whereas it suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment. In vitro studies revealed that pulse treatment with ALN promoted osteocalcin expression. To track the target cells of N-BPs, we applied fluorescence-labeled ALN (F-ALN) in vivo and in vitro. F-ALN was taken into bone-forming cells both in vivo and in vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiation in vitro and it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formation in vivo. DC also blocked the inhibition of Rap1A prenylation by ALN in the osteoblastic cells. These data suggest that local application of ALN promotes bone formation by stimulating proliferation and differentiation of bone-forming cells as well as inhibiting osteoclast function. These effects may occur through endocytic incorporation of ALN and subsequent inhibition of protein prenylation.

Highly selective fluorescent chemosensor for Fe3+ imaging in living cells

Abstract 2-Hydroxybenzyl dansyl cadaverine (DNSCH) was designed as a fluorescent chemosensor for Fe 3+ . DNSCH exhibited significant FL quenching with increasing concentrations of Fe 3+ . The presence of 15 M equiv of Fe 3+ significantly quenched the FL intensity of DNSCH. The binding constant of DNSCH towards Fe 3+ was determined and found to be 1.5 ± 0.4 × 10 −4 M −1 . DNSCH was employed as an intracellular chemosensor for Fe 3+ .

Poly(amidoamine) Conjugates Containing Doxorubicin Bound via an Acid‐Sensitive Linker

Poly(amidoamine)s with amino pendant groups were prepared by hydrogen-transfer polyaddition of primary and secondary amines to bis-acrylamines. Dansyl cadaverine (DC) doxorubicin (Dox) were bound to the polymers via a cis-aconityl spacer to give conjugates containing 3 microg of DC per mg of polymer and 28 to 35 microg of Dox per mg of polymer. Release of DC and Dox at physiological and acidic pH varied from 0 to 35% over 48 h and was pH dependent. Although the ISA1Dox conjugate (IC(50) = 6 microg Dox x mL(-1)) presented similar toxicity as the parent polymer without Dox, ISA23Dox showed increased toxicity (IC(50) = 10 microg Dox x mL(-1)). These results suggest that ISA23Dox is able to release biologically active Dox in vitro and that this conjugate might be suitable for further development.

99mTc-NC100668, a new tracer for imaging venous thromboemboli: pre-clinical biodistribution and incorporation into plasma clots in vivo and in vitro

(99m)Tc-NC100668 is a new radiotracer being developed to aid the diagnosis of thromboembolism. The structure of NC100668 is similar to a region of human alpha(2)-antiplasmin, which is a substrate for factor XIIIa (FXIIIa). The purpose of this study was to confirm the uptake of (99m)Tc-NC100668 into forming plasma clot and to establish the biodistribution of (99m)Tc-NC100668 in Wistar rats. The in vitro plasma clot uptake of (99m)Tc-NC100668 and other compounds with known affinities to FXIIIa was measured using a plasma clot assay. The biodistribution and blood clot uptake of radioactivity of (99m)Tc-NC100668 in normal Wistar rats and those bearing experimentally induced deep vein thrombi were investigated. The in vitro uptake of (99m)Tc-NC100668 was greater than that for [(14)C]dansyl cadaverine, a known substrate of FXIIIa in the plasma clot assay. The biodistribution of (99m)Tc-NC100668 in male and female Wistar rats up to 24 h p.i. showed that radioactivity was rapidly excreted, predominantly into the urine, with very little background tissue retention. In vivo the uptake and retention of (99m)Tc-NC100668 into the blood clot was greater than could be accounted for by non-specific accumulation of the radiotracer within the blood clot. (99m)Tc-NC100668 was retained by plasma clots in vitro and blood clots in vivo. No significant tissue retention which could interfere with the ability to image thrombi in vivo was observed. This evidence suggests that (99m)Tc-NC100668 might be useful in the detection of thromboembolism.

Signaling Mechanisms Underlying Slit2-Induced Collapse of Xenopus Retinal Growth Cones

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.

Penetration behavior and subsurface grafting of dansyl cadaverine and polyethylene glycol (PEG) derivatives in poly(ethylene- co-acrylic acid) (EAA) film

Dansyl cadaverine and polyethylene glycol (PEG) derivatives were grafted on the surface of EAA film and in its subsurface region through formation of amides and esters, respectively. A two-step reaction was conducted. First, EAA film was activated with PCl 5 at room temperature. Second, the acid chloride was reacted with dansyl cadaverine or a PEG derivative to form a modified film. ATR-FTIR spectroscopy and fluorometry were employed to analyze the modified films after each step. It was found that dichloromethane yielded the highest grafting efficiency, with the dansyl cadaverine penetrating throughout the ATR-FTIR analysis region (∼400 nm) in a few minutes. As the grafting depth increased with time, so did the amount of fluorescence intensity of grafted dansyl cadaverine. ATR-FTIR spectra for PEG grafting indicated that the acid chloride peak decreased with time, while the ester peak increased. However, hydrolysis occurred at later times, consuming the acid chloride groups within the film. A marked decrease of static water contact angle was observed for EAA grafted with PEG99 (PEG that contains 99 ethylene glycol repeat units), almost 40° lower than that of neat EAA (∼99°). For other PEG-grafted films, the surface hydrophilicity was also improved.

Role of Actin DNase-I-Binding Loop in Myosin Subfragment 1-Induced Polymerization of G-actin: Implications for the Mechanism of Polymerization

Proteolytic cleavage of actin between Gly 42 and Val 43 within its DNase-I-binding loop (D-loop) abolishes the ability of Ca-G-actin to spontaneously polymerize in the presence of KCl. Here we show that such modified actin is assembled into filaments, albeit at a lower rate than unmodified actin, by myosin subfragment 1 (S1) carrying the A1 essential light chain but not by S1(A2). S1 titration of pyrene-G-actin showed a diminished affinity of cleaved actin for S1, but this could be compensated for by using S1 in excess. The most significant effect of the cleavage, revealed by measuring the fluorescence of pyrene-actin and light-scattering intensities as a function of actin concentration at saturating concentrations of S1, is strong inhibition of association of G-actin-S1 complexes into oligomers. Measurements of the fluorescence of dansyl cadaverine attached to Gln 41 indicate substantial inhibition of the initial association of G-actin-S1 into longitudinal dimers. The data provide experimental evidence for the critical role of D-loop conformation in both longitudinal and lateral, cross-strand actin-actin contact formation in the nucleation reaction. Electron microscopic analysis of the changes in filament-length distribution during polymerization of actin by S1(A1) and S1(A2) suggests that the mechanism of S1-induced polymerization is not substantially different from the nucleation-elongation scheme of spontaneous actin polymerization.

Derivatization of phosphopeptides with mercapto- and amino-functionalized conjugate groups by phosphate elimination and subsequent Michael addition

Kinetics of the beta-elimination of the phosphate group from H-Tyr-Ser(PO3H2)-Phe-OH and H-Tyr-Thr(PO3H2)-Phe-OH and subsequent addition of thiols and amines to the dehydroalaninyl and beta-methyldehydroalaninyl residues formed, were followed by RP HPLC under alkaline conditions in the absence and presence of Ba2+ ions. By this reaction sequence, the phosphoserinyl peptide was conjugated with mono-N-(2-mercaptoethyl)amide of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (4), a mercapto-functionalized pentapeptide, H-His-Gly-Gly-His-Gly-NH(CH2)4SH, and an amino-functionalized fluorescent dye, 5-dimethylaminonaphthalene-1-[N-(5-aminopentyl)]sulfonamide (dansyl cadaverine). The beta-methyldehydroalanine residue was, in turn, observed to be a poor Michael acceptor.

More than one type of transglutaminase in invertebrates? A second type of transglutaminase is involved in shrimp coagulation

Coagulation (clot formation) forms a physical barrier to prevent the loss of body fluid and dissemination of microbes into the haemocoel after injury or infection. Its quickness and efficiency are essential for the survival of invertebrates that rely solely on innate immunity. Transglutaminase (TG) catalyses intermolecular or intramolecular ε-(γ-glutamyl) lysine bond formation, resulting in a protein polymerisation, and plays a role in blood coagulation and post-translational protein remodelling. In the present study, we cloned a TG from shrimp ( Penaeus monodon) haemocyte cDNA. It was assigned as shrimp transglutaminase II (STG II). The STG II cDNA consists of a coding region of 2,274 bp. The deduced protein has 757 amino acid residues with a calculated molecular mass of 85,000 Da and an isoelectric point of 5.48. RT-PCR results showed a significant level of STG II expression in haemocytes but not in hepatopancreas, in contrast to shrimp STG I (AY074924.1). The genetic distance between STG II and STG I is much larger than the distance between STG II and the TG of the kuruma shrimp ( Marsupenaeus japonicus). Evidence based on tissue distribution and genetic distance suggests that no less than two types of shrimp TG exist that are encoded at different chromosomal locations. The recombinant STG II (rSTG II) incorporated a TG-specific substrate, dansylcadaverine (DCA), into clottable proteins (CP) in a calcium dependent manner. Other haemocyte- or plasma-derived TG substrate is not required for CP polymerisation but may be necessary for stable clot formation. The rSTG II catalysed clottable proteins into a long chain under transmission electron microscopy (TEM) observation. In conclusion, STG II is characterized as a haemocyte TG and is involved in coagulation.

Role of Mechanical Stress on the Transglutaminase -Mediated Crosslinking of Erythrocyte Membrane Proteins.

The life-span of erythrocytes is relatively constant within species and it may be determine by the number of times the cell passes through the circulatory system — enduring repeated cycles of mechanical stress. The intrinsic transglutaminase is the major enzyme capable of catalyzing covalent γ-glutamyl-ε-lysine crosslinks that enhance erythrocyte membrane rigidity. Here we report the cloning of mouse erythrocyte transglutaminase (TG) and the role of mechanical stress on the TG-mediated crosslinking of erythrocyte membrane proteins. TG was PCR cloned from reticulocyte cDNA by using consensus primers. Northern blot analysis of the polyA+ mRNA revealed the TG transcript to be 4.6 kb. Recombinant TG was expressed in yeast and the enzymatic crosslinking was detected by labeling glutamine (Q) acyl-donors in inside out vesicles (IOVs) with fluorescent dansylcadaverine (DNC), which serves as the acyl-acceptor. Crosslinking of spectrin/ankyrin, protein 4.1, and band 3 regions (known Q-donors) by TG required CaCl2 at 0.1–0.25 mM and was regulated by both GTP (optimal at 0.01–0.05 mM) and ATP (optimal at 0.5–2.0 mM) with inhibitory effects at higher concentrations. Protein 4.2 (P4.2), a TG pseudo-enzyme, and the cytoplasmic domain of band 3 (cdb3) were also produced recombinantly which allowed for an in-vitro model to test molecular interactions with TG. Specifically, the crosslinking of cdb3 by TG and the binding of P4.2 and TG to cdb3 were examined. A rise in [CaCl2] to near 1 mM showed increased activity of TG (~75% of maximal activity) together with a slight decrease in TG binding affinity to cdb3 (~25% decrease). The crosslinking of cdb3 by TG was inhibited by P4.2, however the presence of P4.2 facilitated the binding of TG to cdb3. The analysis of endogenous TG activity in intact erythrocytes subjected to mechanical stress by hypo-osmolarity (equi-biaxial stretch) or shear stress (anisotropic extension) was also performed. Hypo-osmotically stressed DNC-loaded erythrocytes in the presence of 2 mM [CaCl2] produced a profile with a less intense crosslinking as compared to that of IOVs. In addition, two novel Q-donors (Q1 and Q2) that required mechanical deformation of the membrane to be crosslinked were discovered. Mechanical shearing of erythrocytes in a viscometer produced a similar profile, again with Q2 being the most crosslinked Q-donor. The ratio of Q2/band 3 crosslinking under shear, however, was two fold greater than that under hypo-osmotically induced stress, indicating that varying degrees of crosslinking may be induced by different modes of mechanical stress. Interestingly, Q2 also became crosslinked in IOVs prepared from erythrocytes previously hyposmotically incubated in the presence of extracellular calcium. The importance of mechanical deformation and calcium influx in the binding of Q2 to membrane-associated proteins was further supported by the findings that calcium introduced into erythrocytes using ionophore A23187 alone was not sufficient to induce the crosslinking of Q2. Together these experiments support our hypothesis that periodic mechanical stress may serve as an inherent molecular timer. It is likely that the activation of TG by transient increases in near-membrane calcium concentrations may lead to the accumulation of crosslinks of proteins at the membrane and an eventual entrapment of less deformable erythrocytes in the sinusoids in the spleen followed by subsequent phagocytosis through the recognition of band 3 auto-antibodies.

A Sensitive Fluorometric Assay for Tissue Transglutaminase

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (λexc 260 nm, λem 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5′N′N′-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 μU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).

Monolayer-Protected Clusters with Fluorescent Dansyl Ligands

Monolayer-protected Au cluster (MPC) molecules with mixed monolayers of alkanethiolate and ω-carboxylalkanethiolate ligands were functionalized with the fluorescent label dansyl cadaverine. The emission intensity per MPC-attached dansyl group varies with (a) the number of dansyl sites per MPC (i.e., loading, 4 to 24), (b) with the linker chainlength between Au core and dansyl site, and (c) with chainlengths of alkanethiolates in the surrounding MPC monolayer coating. Emission intensity per MPC-attached dansyl group increases with loading and with the length of core−dansyl linker chains when the surrounding alkanethiolate monolayers have chain lengths equal to or greater than the linker. These results are interpreted in terms of distance-dependent energy transfer from the excited dansyl label to the metal-like MPC core and various steric constraints on thermal fluctuations of linker chains that influence the distance of approach of the label to the core. Correction for self-absorption by the highly colored...

Pellicle precursor proteins: acidic proline-rich proteins, statherin, and histatins, and their crosslinking reaction by oral transglutaminase.

Previous studies have demonstrated that whole saliva and pellicle formed in vitro from oral fluid contain covalently crosslinked salivary proteins. The purpose of this study was to determine which salivary proteins can act as substrates for transglutaminase, an enzyme responsible for the covalent crosslink reaction between a glutamine residue and a lysine residue. Transglutaminase was prepared from the pellet fraction of human whole saliva. Dansyl cadaverine (N-dansyl-1,5-diaminopentane) was used to study the reactivity of glutamine residues in acidic large and small proline-rich proteins, statherin, and the major histatins, whereas a glutamine-containing dansylated peptide was used to study the reactivity of lysine residues in these proteins. Crosslink formation was measured fluorometrically after the addition of fluorescent probe to the salivary protein substrate and transglutaminase. The covalent attachment of the fluorescent probe to salivary proteins was confirmed by SDS-PAGE. It was found that almost all of the lysines present in the acidic PRPs and statherin, and some of the lysines present in histatins, could participate in the crosslink reaction. Glutamine reactivity was also observed, but a maximum of only 14% of glutamine residues present in acidic PRPs and statherin participated in the crosslink formation. These results demonstrate that primary pellicle precursor proteins, acidic proline-rich proteins, statherin, and the major histatins are capable of undergoing crosslink reactions catalyzed by oral transglutaminase. This may enable other proteins in the oral cavity to be incorporated into the acquired enamel pellicle.