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PubMed2003

Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion.

Integrin-mediated cell adhesion to proteins adsorbed onto synthetic surfaces anchors cells and triggers signals that direct cell function. In the case of fibronectin (Fn), adsorption onto substrates of varying properties alters its conformation/structure and its ability to support cell adhesion. In the present study, self-assembled monolayers (SAMs) of alkanethiols on gold were used as model surfaces to investigate the effects of surface chemistry on Fn adsorption, integrin binding, and cell adhesion. SAMs presenting terminal CH(3), OH, COOH, and NH(2) functionalities modulated adsorbed Fn conformation as determined through differences in the binding affinities of monoclonal antibodies raised against the central cell-binding domain (OH > COOH = NH(2) > CH(3)). Binding of alpha(5)beta(1) integrin to adsorbed Fn was controlled by SAM surface chemistry in a manner consistent with antibody binding (OH > COOH = NH(2) > CH(3)), whereas alpha(V) integrin binding followed the trend: COOH >> OH = NH(2) = CH(3), demonstrating alpha(5)beta(1) integrin specificity for Fn adsorbed onto the NH(2) and OH SAMs. Cell adhesion strength to Fn-coated SAMs correlated with alpha(5)beta(1) integrin binding (OH > COOH = NH(2) > CH(3)), and experiments with function-perturbing antibodies demonstrated that this receptor provides the dominant adhesion mechanism in this cell model. This work establishes an experimental framework to analyze adhesive mechanisms controlling cell-surface interactions and provides a general strategy of surface-directed control of adsorbed protein activity to manipulate cell function in biomaterial and biotechnological applications.

Animals↗
PubMed1991

Substrate-associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells.

Differentiated mouse BC3H1 myogenic cells secrete substrate-associated macro-molecules (SAM) which restrict the proliferation of undifferentiated cells and promote both cell shape changes and expression of predominantly the vascular smooth muscle (VSM)-specific isoform of the contractile protein alpha-actin. While we previously reported that high cell density was required for stimulating maximal expression of VSM alpha-actin in BC3H1 cells (Strauch and Reeser: Journal of Biological Chemistry 264:8345-8355, 1989), the permissive effect of SAM on myoblast cytodifferentiation was not at all dependent on the formation of cell to cell contacts. This observation suggests that biogenesis of an extracellular matrix rather than the formation of physical contacts between cells may be the rate-limiting step for induction of VSM alpha-actin expression at high cell density. The biologically active moieties in SAM that promote cytodifferentiation also are expressed by mouse embryonic fibroblast cell lines and are distinctly different from a class of adheron-like macromolecules released by differentiated BC3H1 myocytes directly into the culture medium. While SAM was cell growth restrictive, reconstituted particulate material (RPM) prepared from myocyte-conditioned medium promoted the adhesion and proliferation of growth-arrested myoblasts. SAM and RPM are composed of different polypeptide subunits which collectively may establish microenvironmental conditions that are permissive for BC3H1 myogenic cell differentiation.

Actins↗
PubMed2001

What is the value added by human intervention in protein structure prediction?

This article presents results of blind predictions submitted to the CASP4 protein structure prediction experiment. We made two sets of predictions: one using the fully automated SAM-T99 server and one using the improved SAM-T2K method with human intervention. Both methods use iterative hidden Markov model-based methods for constructing protein family profiles, using only sequence information. Although the SAM-T99 method is purely sequence based, the SAM-T2K method uses the predicted secondary structure of the target sequence and the known secondary structure of the templates to improve fold recognition and alignment. In this article, we try to determine what aspects of the SAM-T2K method were responsible for its significantly better performance in the CASP4 experiment in the hopes of producing a better automatic prediction server. The use of secondary structure prediction seems to be the most valuable single improvement, though the combined total of various human interventions is probably at least as important.

Adenosine Triphosphatases↗
PubMed1998

3-Hydroxy-3-methylglutaryl-coenzyme A reductase kinase and sucrose-phosphate synthase kinase activities in cauliflower florets: Ca2+ dependence and substrate specificities.

Plant 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR; EC 1.1.1.34) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) and synthetic peptides designed from the known phosphorylation sites of plant HMGR (SAMS*: KSHMKYNRSTKDVK), rat acetyl-CoA carboxylase (SAMS: HMRSAMSGLHLVKRR), spinach SPS (SP2: GRRJRRISSVEJJDKK), and spinach NADH:nitrate reductase (NR6: GPTLKRTASTPFJNTTSK) were used to characterize kinase activities from cauliflower (Brassica oleracea L. ) inflorescences. The three major peaks of protein kinase activity resolved by anion-exchange FPLC are homologs of those observed previously in spinach leaves and thus are designated PKI, PKIV, and PKIII, listed in order of elution. PKIV was the most active in terms of phosphorylation and inactivation of recombinant Nicotiana HMGR and was also strictly Ca2+ dependent. The novel aspects are that PKIII has not been detected in previous cauliflower studies, that SAMS* is a more specific peptide substrate to identify potential HMGR kinases, and that the major HMGR kinase in cauliflower is Ca2+ dependent. Of the three major kinases that phosphorylated the SP2 peptide only PKI (partially Ca2+ sensitive) and PKIII (Ca2+ insensitive) inactivated native spinach leaf SPS. Cauliflower extracts contained endogenous SPS that was inactivated by endogenous kinase(s) in an ATP-dependent manner and this may be one of the substrate target proteins for PKI and/or PKIII. The substrate specificity of the three kinase peaks was studied using synthetic peptide variants of the SP2 sequence. All three kinases had a strong preference for peptides with a basic residue at P-6 (as in SP2 and SAMS*; SAMS has a free amino terminus at this position) or a Pro at P-7 (as in NR6). This requirement for certain residues at P-6 or P-7 was not recognized in earlier studies but appears to be a general requirement. In plant HMGR, a conserved His residue at P-6 is involved directly in catalysis and this may explain why substrates reduced HMGR phosphorylation in vitro.

AMP-Activated Protein Kinases↗
PubMed1999

Chemical Force Microscopy Study of Adhesion and Friction between Surfaces Functionalized with Self-Assembled Monolayers and Immersed in Solvents.

Adhesive and frictional forces between surfaces modified with self-assembled monolayers (SAMs) and immersed in solvents were measured with chemical force microscopy as functions of surface functionality and solvent. Si/SiO2 substrates were modified with SAMs of alkylsiloxanes (SiCl3(CH2)n-X), and gold-coated AFM tips were modified with SAMs of alkylthiolates (HS-(CH2)n-X). SAMs of alkylsiloxanes terminated in a methyl or oxidized vinyl group; SAMs of alkanethiolates terminated in a methyl or carboxyl group. Adhesive and frictional forces were measured in hexadecane, ethanol, 1,2-propanediol, 1,3-propanediol, and water. The work of adhesion (W) was calculated with the Johnson-Kendall-Roberts theory of adhesive contact. The JKR values agreed well with values derived from the Fowkes-van Oss-Chaudhury-Good surface tension model and from contact angle results. Calculated values of W for all combinations of contacting surfaces and solvents spanned two orders of magnitude. W correlated with the surface tension of the solvent for hydrophobic/hydrophobic interactions; hydrophilic/hydrophilic and hydrophobic/hydrophilic interactions were more complex. Friction forces were fit to a modified form of Amonton's law. For any solvent, friction coefficients were largest for the hydrophilic/hydrophilic contacting surfaces. The friction coefficient for any contacting pair was largest in hexadecane. In polar solvents, friction coefficients scaled with solvent polarity only for hydrophobic/hydrophobic contacting pairs. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

The 6-(10-Mercaptodecoxyl)quinoline Self-Assembled Monolayer on Gold: Spectroscopy and Wettability Investigation.

A self-assembled monolayer (SAM) with an exposed basic group has been generated by the adsorption of a novel quinoline derivative 6-(10-mercaptodecoxyl)quinoline (MDQ) on a gold surface. The structure and surface properties of the SAMs of MDQ were characterized by ellipsometry, reflection absorption FTIR (RA-IR), and contact angle titration. It was found that the quinoline moiety of MDQ forms an ordered layer on the top of the film and the alkyl chain exhibits a packing density similar to that of a monolayer of decanethiol. The molecular orientation of MDQ in SAMs was evaluated by RA-IR spectroscopy which indicates that the alkyl chain exhibits a tilting angle of 24 +/- 4 degrees with respect to the surface normal and a twisting angle of 50 +/- 5 degrees around its axis. Contact angle titration in the pH range between 2 and 12 illustrates that the surface of SAM of MDQ is less hydrophilic for acidic solution than for basic solution, which is in contrast with the previous reports on SAMs that basic groups are located at the ends of the molecules. Copyright 1999 Academic Press.

Journal Article↗
PubMed1994

S-adenosyl-L-methionine synthetase and methionine metabolism deficiencies in cirrhosis.

Methionine metabolism impairment in human liver disease has been related with an alteration in SAM-synthetase. This deficiency is produced by a post-translational event since human liver cirrhosis presents normal levels of SAM-synthetase mRNA in spite of a more than 50% diminution in its activity. A series of different experiments on the structure and activity of this enzyme have provided strong evidence that SAM-synthetase is regulated by reduced/oxidized glutathione ratio. Restoration of glutathione levels by the addition of S-adenosyl-methionine or glutathione esters in various experimental conditions (buthionine sulfoximine and carbon tetrachloride intoxication) resulted in a normalization of the SAM-synthetase diminution caused by the toxics and an attenuation of the morfological alteration produced in the liver, including fiber production. This findings might have pharmacological implications in the treatment of liver diseases, since the possible beneficial effect of long term administration of SAM could include a reduction of fiber production.

Animals↗
PubMed1987

S-adenosylmethionine and transmethylation reactions in trichomonads.

S-Adenosylmethionine (SAM) levels in trichomonads, a range of trypanosomatids and mouse liver were measured using HPLC techniques. The concentrations were found to be similar in each with the exception of Herpetomonas muscarum ingenoplastis, which contained approximately ten-fold more. Living trichomonads were found to incorporate exogenous L-methionine into intracellular SAM and its methyl carbon was also detected in lipids and nucleic acids, presumably through its involvement in transmethylation reactions. Norleucine and cycloleucine inhibited L-methionine uptake and incorporation into living Trichomonas vaginalis. Both the rates of incorporation of exogenous L-methionine into intracellular SAM and its involvement in transmethylation reactions were greater for Trichomonas vaginalis than for Tritrichomonas foetus. The results suggest that Trichomonas vaginalis and other trichomonads contain enzymes equivalent to SAM synthetase (EC 2.5.1.6) and SAM-dependent methyltransferases (EC 2.1.1).

Animals↗
PubMed1993

Nonlinear protein binding and enzyme heterogeneity: effects on hepatic drug removal.

The kinetics of substrate removal by the liver and the resulting nonlinear changes in unbound fraction along the flow path at varying input drug concentrations were examined by a model simulation study. Specifically, we varied the binding association constant, KA, and the Michaelis-Menten constants (Km and Vmax) to examine the steady state drug removal (expressed as hepatic extraction ratio E) and changes in drug binding for (i) unienzyme systems and (ii) simple, parallel metabolic pathways; zonal metabolic heterogeneity was also added as a variable. At low KA, E declined with increasing input drug concentration, due primarily to saturation of enzymes; only small differences in binding were present across the liver. At high KA, a parabolic profile for E with concentration was observed; changes in unbound fraction between the inlet and the outlet of the liver followed in parallel fashion. Protein binding was the rate-determining step at low input drug concentrations, whereas enzyme saturation was the rate-controlling factor at high input drug concentration. Heterogeneous enzymic distribution modulated changes in unbound fraction within the liver and at the outlet. Despite marked changes in unbound fraction occurring within the liver for different enzymic distributions, the overall transhepatic differences were relatively small. We then investigated the logarithmic average unbound concentration and the length averaged concentration as estimates of substrate concentration in liver in the presence of nonlinear drug binding. Fitting of simulated data, with and without assigned random error (10%), to the Michaelis-Menten equation was performed; fitting was repeated for simulated data obtained with presence of a specific inhibitor of the high-affinity, anteriorly distributed pathway. Results were similar for both concentration terms: accurate estimates were obtained for anterior, high affinity pathways; an overestimation of parameters was observed for the lower affinity posteriorly distributed pathways. Improved estimations were found for posteriorly distributed pathways upon inhibition with specific inhibitors; with added random error, however, the improvement was much decreased. We applied the method for fitting of several sets of metabolic data obtained from rat liver perfusion studies performed with salicylamide (SAM) (i) without and (ii) with the presence of 2,6-dichloro-4-nitrophenol (DCNP), a SAM sulfation inhibitor. The fitted results showed that SAM sulfation was a high-affinity high-capacity pathway; SAM glucuronidation was of lower affinity but comparable capacity as the sulfation pathway, whereas SAM hydroxylation was of lower affinity and lower capacity.

Animals↗
PubMed1985

Color-coded Doppler imaging of systolic flow patterns in hypertrophic cardiomyopathy.

We studied 11 patients with hypertrophic cardiomyopathy by color Doppler echocardiography (Group I: 6 patients with outflow obstruction, and Group II: 5 patients without outflow obstruction) to assess systolic structure and function as observed by cross-sectional echocardiography in relation to the flow dynamics. The structure and function included systolic anterior motion of mitral valve (SAM), midsystolic aortic valve closure (AoC), systolic cavity obliteration and the presence and timing of mitral incompetence. Their occurrence and timing was related to presence of aortic systolic flow and presence of turbulence. While all patients in Group I had SAM and turbulence, none of the patients in Group II had SAM nor turbulence. Early mitral incompetence appearing before SAM and turbulence, occurred in all patients of Group I and in none of Group II. Midsystolic aortic valve closure was only present in Group I and blood flow was unilaterally directed so that only 60% of aortic cross-sectional area showed blood flow. We conclude that mitral incompetence in hypertrophic cardiomyopathy in early systole is common when outflow gradient is present and is independent of mitral incompetence of mid- and late systole. During SAM, turbulence in the subaortic area and mid and late mitral incompetence occurred simultaneously. The midsystolic aortic valve closure was related to the unilaterally directed blood flow through the aortic cross-sectional area.

Adult↗
PubMed1999

Inhibition of thiopurine S-methyltransferase activity by impurities in commercially available substrates: a factor for differing results of TPMT measurements.

OBJECTIVES: Thiopurine S-methyltransferase (TPMT) activity, when measured in red blood cells (RBC) with a recently published TPMT activity assay using 6-thioguanine (6-TG) as substrate, could not be reproduced in another laboratory. We investigated factors which could influence the results of the TPMT activity measurement. METHODS: We tested twelve 6-TG and four 6-mercaptopurine (6-MP) compounds from different suppliers as substrates and determined the enzyme kinetic parameters Km and Vmax. Furthermore, we studied the influence of different 6-TG compounds on the affinity of the methyl donor S-adenosyl-L-methionine (SAM) to the TPMT enzyme. RESULTS: All 6-TG products were of equal purity (declared >98% by the supplier): this was ascertained by HPLC. However, the rate of methylation obtained following incubation with 6-TG from different suppliers ranged from 10% to 100% when incubated with the same RBC lysate. The lowest apparent Km value for a 6-TG was 22.3 micromol x l(-1), while the product with the highest methylation rate showed a Km of 156 micromol x l(-1). From these results we assume that there is a contaminant in some 6-TG products, which acts as a strong inhibitor of TPMT activity. Compounds possibly used for the synthesis of 6-TG (guanine, pyridine, 6-chloroguanine) did not affect the methylation rate. Thioxanthine, which is known to be a strong inhibitor of TPMT when added to the assay system to give a 2% contamination, reduced TPMT activity from 100% to 72%. Using 6-MP from different suppliers as substrate resulted in Km values ranging from 110 to 162 micromol x l(-1) and Vmox values ranging from 54 to 68 nmol 6-MMP x g(-1)Hb x h(-1). The Km value for the methyl donor SAM was similar to and independent from the thiopurine substrates tested (range 4.9-11 micromol-l(-1) SAM). In contrast to other investigators, we found non-enzymatic S-methylation, which was negligible under our assay conditions (3% with 128 micromol x l(-1) SAM), but could become relevant in experiments using higher SAM concentrations. CONCLUSIONS: TPMT enzyme activity determined with 6-TG as substrate may be strongly inhibited by a contaminant in some of the 6-TG lots distributed.

Enzyme Inhibitors↗
PubMed2003

Rescue of shoot apical meristems of chrysanthemum by culturing on root tips.

A new method to regenerate plants from leaf primordia-free shoot apical meristem domes (LP-free SAMs) was developed by establishing the meristem dome on the cut surface of root tips. Ten days after culture, the viable rate of LP-free SAMs of chrysanthemum 'Piato' attached to chrysanthemum root tips was >40%. Shoot regeneration was not observed from LP-free SAMs without the root tips. When LP-free SAMs of chrysanthemum were transferred to root tips of either petunia, cabbage, or carnation, the highest shoot regeneration rate was observed with cabbage root tips. Microscopic observation documented that the LP-free SAM temporarily adhered to the cut surface of the root tip of cabbage.

Botany↗
PubMed2002

The shoot apical meristem of Sinapis alba L. expands its central symplasmic field during the floral transition.

The shoot apical meristem (SAM) is functionally subdivided into zones with distinct tasks. During vegetative growth the peripheral zone of the meristem gives rise to leaf primordia that develop into dorsiventral leaves under the influence of signals from the central zone. During the floral transition the function of the SAM is altered and its peripheral zone starts to form floral structures in a specific pattern. This requires alterations in the signal networks that coordinate the activities of the peripheral and central zone of the SAM. These signal networks are partly housed in the symplasmic space of the SAM. Dye-coupling experiments demonstrate that in the superficial layer of the Sinapis alba meristem this space is radially subdivided. The cells of the central zone are coupled into a symplasmic field, which is shielded from the peripheral zone by the positional closing of plasmodesmata. In the vegetative meristems, most of these central symplasmic fields have a triangular geometry and are relatively small in size. Plants that are induced to flower by exposure to a single long day alter the geometry as well as the size of their central symplasmic field. After two subsequent days under short photoperiod the central symplasmic fields exhibit a circular form. Simultaneously, their size strongly increases both in an absolute sense and relative to the enlarging meristem. The geometric change in the fields is hypothesized to be due to recruitment of extra initial cells, required to support the increase in phyllotactic complexity. The proportional increase in field size is interpreted as an adjustment in the balance between the central and peripheral zone of the SAM, accompanying the shift from leaf production to flower formation.

Cell Division↗
PubMed1997

Effects of chronic cigarette smoke inhalation on the development of senile lung in senescence-accelerated mouse.

The senescence-accelerated mouse (SAM) manifests most of the features of function and morphology in the senile lung with aging. However, little is known about the effects of age and cigarette smoke on alterations of the lung in SAM. In the present study, we examined the effects of chronic cigarette smoke inhalation and age on the function and morphology of lungs in two strains of SAM, SAMP2 (senescence-prone strain) and SAMR1 (senescence-resistant strain), from 6 months of age (young) and 18 months of age (aged). After 4 weeks of cigarette smoke inhalation, a small but significant airspace along with a leftward shift of the pressure-volume (P-V) curve was observed in young SAMP2, but not in SAMR1. However, the airspace size of young SAMP2 with cigarette inhalation was smaller than that in aged SAM with air inhalation, suggesting that the effect of age may be greater than that of the small burder of tobacco smoke on the lung alterations in SAMP2. In the aged SAM, there were no differences in function and structure between tobacco-exposed and air-exposed mice. Because the changes in the lungs of young SAMP2 exposed to cigarette smoke were partly simulated with age-related alterations in human lung, and because age-dependent changes of lungs were clearly investigated in SAMP2, this strain may be an interesting animal model for investigating the effects of age and/or cigarette smoke on alterations in lung structure and function.

Aging↗
PubMed2004

Correlation study of scanning acoustic microscope reflection coefficients and image brightness intensities of micrographed osteons.

Scanning acoustic microscopy (SAM) of Haversian bone at high frequency clearly shows differences in the brightness (gray) levels of individual osteons, which were observed to correspond to the differences in reflection coefficients measured with the SAM technique: the darker the bony regions on the SAM micrographs, the lower the SAM reflection coefficient values, and vice versa. We studied the correlation between differences in brightness and the related reflection coefficients of osteons on undecalcified transverse sections of goat tibial diaphysis using a 400-MHz burst mode lens on the Olympus UH3 SAM. Results showed that there was a strong linear correlation between brightness intensities and the corresponding reflection coefficients, with a correlation coefficient r = 0.99 ( P << 0.001) using gray level 210 as cutoff. This result suggested that the image brightness intensity of osteonic bone could be measured retrospectively to provide information on the stiffness of the corresponding bone matrix.

Acoustics↗
PubMed2002

Embryonic shoot apical meristem formation in higher plants.

The shoot apical meristem (SAM) is essential for organ formation in higher plants. How the SAM is formed during plant development is poorly understood, however. In this review, we focus on several recent studies that provide new insights into the mechanism of SAM formation during embryogenesis. Recently, positive and negative regulators of the class I KNOX genes, which are thought to be necessary for SAM formation, have been identified; the Arabidopsis CUP-SHAPED COTYLEDON ( CUC) genes are required for the expression of a class I KNOX gene, SHOOT MERISSTEMLES( STM) during embryogenesis, and the Arabidopsis ASYMMETRIC LEAVES1 ( AS1), AS2, and several other genes negatively regulate KNOX gene expression in cotyledon primordia. Also, several genes that are involved in the formation of the adaxial-abaxial axis of cotyledons seem to regulate embryonic SAM formation.

Journal Article↗
PubMed1986

A sensitive high-performance liquid chromatographic procedure with fluorometric detection for the analysis of decarboxylated S-adenosylmethionine and analogs in urine samples.

A highly sensitive HPLC method for the determination of decarboxylated S-adenosylmethionine (dc-SAM) by fluorometric detection was developed. The reaction of dc-SAM and its analogs with chloroacetaldehyde leads to the corresponding 1,N6-etheno derivatives. These highly fluorescent derivatives were fully characterized through their proton nuclear magnetic resonance spectra and/or mass spectra. This derivatization procedure has been applied to the analysis of dc-SAM in rat and human urine. After a simple cation exchange column prepurification, the urine extracts were derivatized with chloroacetaldehyde and analyzed by reversed-phase HPLC with fluorometric detection. The method allowed the determination of subpicomole amounts of dc-SAM and was shown to be highly reproducible with the use of decarboxylated S-adenosylethionine as internal standard. The application of the method to the analysis of urine of rats treated with MDL 72175, a potent ornithine decarboxylase inhibitor, showed that the dc-SAM levels increased in a dose-related fashion.

Alkynes↗
PubMed1986

Increase of ATP level in human erythrocytes induced by S-adenosyl-L-methionine.

The effect of S-adenosyl-L-methionine (SAM) on the ATP level, the morphology and the deformability of human erythrocytes was investigated and compared with that of adenosine. (i) Upon incubation with SAM, the ATP level increased considerably in fresh cells (in both young and old cells in similar extent) and in stored (partially ATP-depleted) cells. But the incubation with adenosine increased ATP level to a lesser extent. (ii) The incubation of stored cells with SAM hardly affected (or rather decreased) the IMP level, while that with adenosine remarkably increased IMP (and ITP). (iii) The morphology and the deformability of stored erythrocytes were well conserved in spite of the treatment with SAM, as compared with the treatment with adenosine. The echinocytic transformation was induced in old cells to some extent by SAM, while did not in young cells.

Adenosine↗