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PubMed1985

Clonal stability and heterogeneity of hybridomas: analysis by multiparameter flow cytometry.

Flow cytometry of cellular DNA and RNA content was employed to determine DNA ploidy, proliferation, and RNA content of hybridoma cultures shortly after cell fusion and sequentially thereafter. The parental mouse myeloma cell line P3UI was characterized by tetraploid DNA content, S-phase 50%, and high RNA, the parental mouse spleen cells by diploid DNA content, low proliferation, and low RNA content. Hybridoma cultures studied as early as 21 days after fusion were found to contain the sum of the parental cells' DNA content (hexaploid), or if less, more than that of the myeloma parental cells. Only one clone of 35 tested was shown to be hexaploid and the rest hypertetraploid or hyperpentaploid. Hybrid cell cultures were frequently found to contain a variable mixture of unfused parental cells. The high proliferation of hybridoma cells determined by flow cytometry indicates that these cells would eventually overgrow the parental cells. Flow cytometry also enabled an accurate estimation of the effect of various doses of dexamethasone added to HAT medium immediately after cell fusion on hybridoma formation. Cultures treated with 10(-5) mM of the hormone had a higher DNA ploidy than cultures grown in the presence of 10(-3) mM dexamethasone. No parental cells were observed in the hybridoma cultures studied with this hormone. Sequential DNA/RNA measurements of hybridoma clones showed a decrease in DNA ploidy over time with high dexamethasone doses and a minimal increase or no change with low hormone dose. Flow cytometry is suggested to be a useful technique for evaluating the effects of various agents on DNA ploidy and proliferation and on stability of fused cells.

Animals↗
PubMed2005

Microarray immunoassay for phenoxybenzoic acid using polymer encapsulated Eu:Gd2O3 nanoparticles as fluorescent labels.

Currently, detection in microarray bioanalysis is based mainly on the use of organic dyes. To overcome photobleaching and spectral overlaps we applied a new type of fluorophore, crystalline europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles, as labels in immunoassay microarrays. The Eu:Gd2O3 nanoparticles synthesized by spray pyrolysis offer narrow red emission, large Stokes shift, photostable laser-induced fluorescence with a long lifetime (1 ms). The amino functionalization of the particles was achieved by poly(L-lysine) (PL) encapsulation. The formation of a stable PL shell was confirmed by TEM analysis, colloidal stability studies, and quantification of the surface reactive amino groups. The PL-encapsulated particles were covalently conjugated to antibodies and successfully applied as reporters in a competitive fluorescence microimmunoassay for phenoxybenzoic acid (PBA), a generic biomarker of human exposure to pyrethroid insecticides. Microarrays were fabricated by microcontact printing of BSA-PBA in line patterns (10 x 10 microm). Confocal fluorescence microscopy combined with internal standard (fluorescein) calibration was used for quantitative measurements. The microarray immunoassay demonstrated a limit of detection of 1.4 microg L(-1) PBA. This work suggests the potential application of lanthanide oxide nanoparticles as fluorescent probes in microarray and biosensor technology, immunodiagnostics, and high-throughput screening.

Antibodies↗
PubMed2000

Primary lumbosacral stability after open posterior and endoscopic anterior fusion with interbody implants: a roentgen stereophotogrammetric analysis.

STUDY DESIGN: After posterior stabilization of the spondylolytic lumbosacral level, mobility of the fused vertebrae could be studied before and after an additional anterior endoscopic interbody fusion using roentgen stereophotogrammetric analysis. OBJECTIVE: To determine the in vivo primary lumbosacral stability of additional anterior interbody fusion after transpedicular screw fixation. SUMMARY OF BACKGROUND DATA: In vitro studies indicate a significant decrease in segmental motion after pedicle screw fixation and additional anterior fusion. Roentgen stereophotogrammetric studies demonstrate the adequacy of transpedicular lumbar instrumentation in posterolateral fusions. There are no studies examining the effect of additional anterior interbody fusion after posterior instrumentation in vivo. METHODS: In this study, 15 patients with low-grade spondylolisthesis at L5-S1 underwent a two-stage open posterior and endoscopic anterior lumbar fusion using carbon fiber (Brantigan I/F) cages. At surgery, tantalum markers were implanted into the fifth lumbar (L5) and the first sacral (S1) vertebra. All the patients were examined by roentgen stereophotogrammetric analysis after the first and second surgical procedures. RESULTS: After implantation of the posterior pedicle system only, the mean intervertebral mobility determined by roentgen stereophotogrammetric analysis was 0.23 mm in the transverse (x), 0.54 mm in the vertical (y), and 1.2 mm in the sagittal (z) axes. After additional anterior endoscopic fusion with carbon cages, the remaining translation between the fused segment L5/S1 decreased to 0.17 mm in the x, 0.16 mm in the y, and 0.44 mm in the z axes. CONCLUSION: Anterior endoscopic lumbosacral fusion significantly increases the primary stability of the posterior fusion with a pedicle system in two axes of motion.

Biocompatible Materials↗
PubMed2003

WEB-THERMODYN: Sequence analysis software for profiling DNA helical stability.

WEB-THERMODYN analyzes DNA sequences and computes the DNA helical stability, i.e. the free energy required to unwind and separate the strands of the double helix. A helical stability profile across a selected DNA region or the entire sequence is generated by sliding-window analysis. WEB-THERMODYN can predict sites of low helical stability present at regulatory regions for transcription and replication and can be used to test the influence of mutations. The program can be accessed at: http://wings.buffalo.edu/gsa/dna/dk/WEBTHERMODYN/.

Chromosomes, Fungal↗
PubMed1996

Alternate drug delivery routes for A-71623, a potent cholecystokinin-A receptor agonist tetrapeptide.

A-71623 (BOC-Trp-Lys(epsilon-N-2-methylphenylaminocarbonyl)- Asp-(N-methyl)-Phe-NH2) is a tetrapeptide which has high affinity and selectivity for cholecystokinin receptors; it is a potent appetite suppresser in animal studies. Because of its low (< 1%) oral bioavailability, studies were performed to assess the feasibility of delivery of A-71623 by pulmonary, sublingual, and transdermal routes of administration. The pKa was determined to be 4.2 by spectrophotometric titration; aqueous solubility is increased by increasing pH and by increasing ethanol content. The solubility of A-71623 in ethanol/propellant mixtures was investigated; solubility ranged from 1.0 to 2.5 mg/mL in mixtures of ethanol, propellant 11 (trichlorofluoromethane), and propellant 12 (dichlorodifluoromethane). The log apparent octanol/water partition coefficient was 2.8 at pH 5 and 1.0 at pH 8. Maximum stability at 70 degrees C was seen in the range of pH values of 5.5-7.5; hydrolysis of the N-terminal BOC group appears to be the primary route of degradation. Increasing ethanol content increases the stability; Arrhenius analysis indicated a t90 of 150 days under ambient conditions in 25% ethanol. Intratracheal delivery of 3 mumol/kg A-71623 in 50% ethanol to rats showed rapid and efficient absorption of drug from the lungs, with a Cmax of 2.7 microM and an AUC of 85 microM*min. Similar studies in dogs showed bioavailabilities of 59% and 46% for 2 and 3 mumol/kg intratracheal doses, respectively, relative to intravenous administration. Sublingual administration of 1 mumol/kg A-71623 in a vehicle of 80% ethanol/2% Klucel/2.5% peppermint oil gave high prolonged plasma levels of A-71623, with a Cmax of 0.37 microM, indicating high bioavailability and favorable partitioning and distribution effects from the sublingual cavity for this formulation. Transdermal delivery was examined by in vitro diffusion through human skin; the permeability coefficient of A-71623 in 40% ethanol was 2.6 x 10(-5) cm/hr, suggesting that transdermal delivery of up to 2 mg/day may be feasible. In conclusion, the results provide preliminary indications that delivery of efficacious doses of A-71623, and perhaps other CCK analogs, by alternate routes of delivery is probably feasible.

Administration, Cutaneous↗
PubMed2004

Insight into the structural role of carotenoids in the photosystem I: a quantum chemical analysis.

The structural stabilization role of carotenoids in the formation of photosynthetic pigment-protein complexes is investigated theoretically. The pi-pi stacking and CH-pi interactions between beta-carotenes and their surrounding chlorophylls (and/or aromatic residues) in Photosystem I (PS1) from the cyanobacterium Synechococcus elongatus were studied by means of the supermolecular approach at the level of the second-order Møller-Plesset perturbation method. PS1 features a core integral antenna system consisting of 22 beta-carotenes intertwined with 90 chlorophyll molecules. The binding environments of all 22 beta-carotenes were systematically analyzed. For 21 out of the 22 cases, one or more chlorophyll molecules exist within van der Waals' contacts of the beta-carotene molecule. The calculated strengths of pi-pi stacking interactions between the conjugated core of beta-carotene and the aromatic tetrapyrrole rings of chlorophyll are substantial, ranging from -3.54 kcal/mol for the perpendicular-positioned BCR4004...CHL1217 pair to -16.01 kcal/mol for the parallel-oriented BCR4007...CHL1122 pair. A strong dependence of the pi-pi stacking interaction energies on the intermolecular configurations of the two interacting pi-planes is observed. The parallel-oriented beta-carotene and chlorophyll pair is energetically much more stable than the perpendicular-positioned pair. The larger the extent of pi-pi overlapping, the stronger the interaction strength. In many cases, the beta-ring ends of beta-carotene molecules are found to interact with the tetrapyrrole rings of chlorophyll via CH-pi interactions. For the latter interactions, the calculated interaction strengths vary from -7.03 to -11.03 kcal/mol, depending on the intermolecular configuration. This work leads to the conclusion that pi-pi stacking and CH-pi interactions between beta-carotene and their surrounding chlorophylls and aromatic residues play an essential role in binding beta-carotenes in PS1 from S. elongatus. Consequently, the molecular basis of the structural stabilization function of carotenoids in formation of the photosynthetic pigment-protein complexes is established.

Biophysics↗
PubMed2004

Initial stability of ankle arthrodesis with three-screw fixation. A finite element analysis.

OBJECTIVE: Compare the initial stability at the fusion site of ankle arthrodesis fixed with two and three screws. DESIGN: Finite element models of ankle arthrodesis were developed from computed tomography images. Two-screw constructs were augmented with a third screw in different orientations and subjected to loads likely to affect the ankle postoperatively. BACKGROUND: More stable fixation seems to increase the chance of fusion, as it minimises the motion between the tibiotalar interfaces. METHODS: Non-linear elastic finite element analyses were performed in external torsion and dorsiflexion. The micromotions at the tibiotalar interface were computed to compare the two- and three-screw fixation in intact and flat-cut arthrodesis. RESULTS: Adding a third screw reduced the micromotions at the fusion site. Inserting the third screw anteriorly predicted lower peak micromotions than inserting the screw posteriorly, except for the intact arthrodesis tested in dorsiflexion. Three-screw intact arthrodesis predicted lower peak micromotions than flat-cut arthrodesis. CONCLUSIONS: Better stability was predicted for three-screw ankle arthrodesis. In flat-cut arthrodesis, a third screw inserted anteriorly performed better than a posterior screw. In intact arthrodesis, a posterior screw seemed a better option when flexion stability was the main concern. Even with three-screw fixation, the configuration of the first two-crossed screws may still be important to improve the stability at the fusion site.

Ankle Joint↗
PubMed2001

Stability studies of carbamate pesticides and analysis by gas chromatography with flame ionization and nitrogen-phosphorus detection.

As a result of thermal stability studies of carbamate pesticides, a method has been proposed for their direct determination by gas chromatography in the ranges 1-20 and 0.1-1 mg l(-1), using flame ionization and nitrogen-phosphorus detection, respectively. The method allows the determination of propham, propoxur, carbofuran, carbaryl, methiocarb, isopropoxyphenol and naphthol in powdered potato samples. The analytes were previously extracted with a light petroleum-dichloromethane (1:1, v/v) mixture and preconcentred by solid-phase extraction through a C8 cartridge. The recoveries obtained from spiked potato samples (n=4 replicates) at two concentration levels, 10 and 0.5 mg of pesticide per kg of sample, were in the ranges 72-115 and 50-73%, with relative standard deviations of 2-7 and 5-8%, respectively. The detection limits were 50-210 and 41-53 microg kg(-1) with flame ionization and nitrogen-phosphorus detection, respectively, and reaching the maximum residue levels, 0.05 mg kg(-1) for methiocarb and propoxur, set by the Real Decreto 280/1994 (based on the European directive).

Calibration↗
PubMed2001

The charge density distribution in a model compound of the catalytic triad in serine proteases.

Combined low temperature (28(1) K) X-ray and neutron diffraction measurements were carried out on the co-crystallised complex of betaine, imidazole, and picric acid (1). The experimental charge density was determined and compared with ab initio theoretical calculations at the B3LYP/6-311G(d,p) level of theory. The complex serves as a model for the active site in, for example, the serine protease class of enzymes, the so-called catalytic triad. The crystal contains three short strong N-H...O hydrogen bonds (HBs) with dN...O < 2.7 A. The three HBs have energies above 13 kcalmol(-1), although the hydrogen atoms are firmly localized in the "nitrogen wells". This suggests that low-barrier hydrogen bonding in catalytic enzyme reactions may be a sufficient, but not a necessary, condition for obtaining transition-state stabilization. Structural analysis (e.g., covalent N-H bond lengthening) indicates that the hydrogen bond between H3A and 08 of imidazole and betaine respectively (HB2) is slightly stronger than the bond between H1A and O1A of imidazole and picric acid (HB1), although HB1 is shorter than HB2: (dN...O(HB1)= 2.614(1) A, dN...O(HB2) = 2.684(1) A, dH...O(HB1) = 1.630(1) A, dH...O(HB2)= 1.635(1) A, dN-H(HB1) = 1.046(1) A, dN-H(HB2) = 1.057(1) A). Furthermore, the charge density analysis reveals that HB2 has a larger covalent character than HB1, with considerable polarization of the density towards the acceptor atom. The Gatti and Bader source function (S) is introduced to the analysis of strong HBs. The source function is found to be a sensitive measure for the nature of a hydrogen bond, and comparison with low-barrier and single-well hydrogen bonding systems (e.g., benzoylacetone and nitromalonamide) shows that the low-barrier hydrogen bond (LBHB) state is characterized by an enormously increased hydrogen atom source contribution to the bond critical point in the HB. In this context, HB2 can be characterized as intermediate between localized HBs and delocalized LBHBs.

Binding Sites↗
PubMed2006

Oxidative-stress and IL-6 mediate the fibrogenic effects of [corrected] Kupffer cells on stellate cells.

The impact of Kupffer cells (KCs) on the hepatic stellate cell (HSC) fibrogenic response was examined in an in vitro coculture model of primary KCs and HSCs. Coculture with KCs induced a more activated phenotype and greater proliferation compared to HSC cultured alone. Similar results were obtained on Matrigel which maintains HSCs quiescent. The effect of KCs on HSC collagen I involved transcriptional regulation, as determined by nuclear in vitro transcription run-on assays, promoter studies, and Northern blot analysis, while stability of the COL1A1 and COL1A2 mRNA were similar. The minimal COL1A1 and COL1A2 promoter regions responsible for the KC effects were localized to the -515 and -378 base pair (bp) regions, respectively. Intracellular and extracellular collagen I protein, H2O2, and IL-6 increased in a time-dependent fashion, especially for HSCs in coculture. Catalase prevented these effects as well as the transactivation of both collagen promoters. The rate of collagen I protein synthesis and intracellular collagen I degradation remained similar but the t(1/2) of the secreted collagen I was lower for HSC in coculture. MMP13, a protease that degrades extracellular collagen I, decreased in the cocultures, while TIMP1, a MMP13 inhibitor, increased; and these effects were prevented by catalase, anti-IL-6, and siRNA-IL-6. Cocultured HSC showed elevated phosphorylation of p38 which when inhibited by catalase, anti-IL-6, and siRNA-IL-6 it blocked TIMP1 upregulation and collagen I accumulation. In conclusion, these results unveil a novel dual mechanism mediated by H2O2 and IL-6 by which KCs may modulate the fibrogenic response in HSCs.

Animals↗
PubMed1988

The use of dense alumina-alumina ceramic combination in total hip replacement.

The purpose of this article was to review the laboratory and clinical performances since 1970 of a total hip prosthesis using alumina-alumina combination. The chemical and physical properties of dense alumina ceramic were studied in relation to biocompatibility, mechanical strength, and surface properties. Through the examination of 35 retrieved implants, it was found that the long-term success of alumina-alumina total hip replacement depends on both the ceramic microstructure (small grain size with uniform distribution, minimum porosity, absence of inclusions) and implant geometry (sphericity deviation +/- 1 micron, radius tolerance between components 7-10 microns). Alumina component wear and fractures have disappeared with the use of high-performance materials and severe manufacturing quality control. Examination of human biopsies from well-fixed prostheses showed that alumina particles deposits increase with time with only a low-grade macrophagic reaction. When loosening occurred, an inflammatory reaction appeared; this reaction was less striking than with loose metal-polyethylene prostheses, however. The long-term behavior of cementless alumina cup fixation depends upon initial positioning and stability; survivorship analysis of the cemented ceramic cups showed an 88% survival probability after 8 years with a 1.6% average annual probability of revision. The percentage of surviving was 100% after 8 years in patients who were less than 50 years old. Aseptic loosenings occurring at the cup-cement interface were assumed to be related to stress protection secondary to the high rigidity of the ceramic leading to a weakening of the spongious bone supporting the cement mantle. Good bone stock quality as well as high-quality ceramic appear to be the prerequisites for durable fixation of alumina sockets.

Aluminum↗
PubMed1990

Cell cycle regulation of an exogenous human poly(ADP-ribose) polymerase cDNA introduced into murine cells.

We have evaluated the regulation of expression of the poly(ADP-ribose) polymerase gene during cell growth and replication. In a synchronized population of HeLa cells or in serum-stimulated WI-38 cells, steady-state levels of the polymerase mRNA were highest at late S and S-G2 phases and negligible in early S phase. Transcription did not solely account for the significant increase in the mRNA levels observed in late S phase by Northern analysis. The stability of the mRNA was dependent upon the percent proliferating cells in the culture. Accordingly, polymerase mRNA from cells in early exponential phase was significantly more stable than from cells in stationary phase of asynchronous growth. To clarify these observations, we utilized a novel heterologous expression system that involved murine 3T3 cells transfected with a human poly(ADP-ribose) polymerase cDNA under the control of a non-cell cycle-specific promoter. Cells were synchronized, and a comparison was made of the endogenous (murine) and exogenous (human) polymerase mRNA levels. Both the endogenous and the exogenous mRNA were specifically stabilized by the same mechanisms and only during late S phase; therefore, we concluded that mRNA pools for the polymerase are regulated at the post-transcriptional level. The heterologous expression system confirmed that the post-transcriptional regulation system in the mouse cells can recognize and faithfully regulate the human cDNA in response to the murine cell cycle signals. More importantly, the presence of extra copies (human) of the polymerase gene did not provide an increased amount of the total polymerase mRNA or protein and, in fact, the sum of the endogenous and exogenous mRNA in the transfected cells was approximately the same as the level of endogenous transcript in the control cells. This suggested that there might be a limit to the amount of polymerase protein accumulating in the cellular pool and thus levels of poly(ADP-ribose) polymerase may be autoregulated.

Animals↗
PubMed1997

Analysis of the structure and stability of omega loop A replacements in yeast iso-1-cytochrome c.

Omega (omega)-loop A, residues 18-32 in wild-type yeast iso-1-cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase. Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low-temperature spectroscopy and cannot grow on nonfermentable media. Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native-like cytochrome c conformation, but are less stable than the wild-type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone. A single-site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.

Amino Acid Sequence↗
PubMed1989

Electrostatic effects in a large enzyme complex: subunit interactions and electrostatic potential field of the icosahedral beta 60 capsid of heavy riboflavin synthase.

The role of the electrostatic interactions in the stability of the icosahedral beta 60 capsid of heavy riboflavin synthase from Bacillus subtilis has been investigated using an approach based on the theory of Kirkwood and Tanford. The pH dependence of the electrostatic subunit interactions agrees well with experimental data. The electrostatic subunit interaction energy has a pronounced minimum at pH 8.2 for both the ligated and ligand-free capsid. The latter is characterized by a reduction of the magnitude and the pH range of the electrostatic attraction. It is found that only 8 charged groups, which form one cluster and two ion pairs, provide a significant contribution to the capsid stability. The analysis has shown that the aggregation/disaggregation equilibrium seems to be regulated by electrostatic interactions between beta-subunits forming dimers, which connect the relatively stable pentamers in the beta-60 capsid. The release of the ligand causes a reduction of the electrostatic attraction of the dimers, which may induce disaggregation of the capsid. The electrostatic potential field due to the titratable groups and alpha-helix macrodipoles has been calculated on the basis of the Coulomb relation. Two different values of the dielectric constant have been used for the protein and the surrounding solvent, respectively. The electrostatic potential shows a radially polar distribution with a positive pole at the inner capsid wall and a negative pole outside the capsid. An interesting feature of the electrostatic field is the formation of positive potential "channels" that coincide with the channels constituted by the pentameric and trimeric beta-subunit aggregates. It is supposed that the electrostatic potential field plays a role in enzyme-substrate recognition.

Bacillus subtilis↗
PubMed2005

Acetonitrile/water gas-phase reaction products observed by use of atmospheric pressure chemical ionization: adducts formed with sulfonamides.

Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the two most common mass spectrometric ionization methods used in the pharmaceutical industry. However, APCI analysis can sometimes lead to ambiguity in compound characterization and quantitation due to gas-phase reactions occurring between acetonitrile and water in the plasma, and between these plasma-generated compounds and the analyte. During the analysis of various sultams and sulfonamides we observed signals corresponding to m/z [M+44](+) and [M+60](+). Various solvent conditions and collisionally activated dissociation MS(n) experiments revealed that under the high-energy plasma conditions of APCI, the acetonitrile/water solvent mixture reacts undergoing acid-catalyzed hydrolysis producing acetamide, 59 Da. Further, the highly reactive 43 Da species ethanimine is also produced. These two compounds, normally not observed in APCI analysis, are stabilized by the sulfonamide and appear as adduct species in the mass spectra. The sulfone oxygens and the lone pair of electrons on the amide nitrogen play a role in stabilizing this adduct.

Acetonitriles↗
PubMed1996

Expression and functional characterization of a single chain Fv antibody directed against secretions involved in plant nematode infection process.

Expression in plants of antibodies directed against proteins essential for pathogenesis could provide an alternative approach to engineer new resistance traits into crops. Salivary secretions of the root-knot nematode Meloidogyne incognita are known to play a key role during this nematode infection process. From a hybridoma cell line producing an IgM monoclonal antibody specific to these secretions, we have constructed a synthetic gene that encodes an antigen-binding single-chain Fv protein (scFv). The scFv gene was created by polymerase chain reaction amplification of variable domain encoding regions from the IgM antibody. The cloned scFv was initially expressed in Escherichia coli as a 33-kDa protein which could be purified to near homogeneity by immobilized metal affinity chromatography. The produced scFv is fully functional since it shows the same specificity towards a crude extract of M. incognita infective larvae as the corresponding parental IgM. Transient expression assays with tobacco leaf protoplasts using different targeting signals resulted in a high intracellular accumulation of scFv, especially when fused to the tetrapeptide KDEL retention signal. Activity analysis and stability characterization of this scFv in tobacco protoplast represent the first step before plant transformation in order to test a new form of resistance to root-knot nematode in plants.

Amino Acid Sequence↗
PubMed2000

Synthetic melanin suppresses production of proinflammatory cytokines.

An overproduction of proinflammatory cytokines mediates the damaging sequelae of inflammation in pathologic conditions such as rheumatoid arthritis, graft-vs-host reaction, cachexia, and sepsis syndrome. We examined the cytokine regulatory activity of synthetic melanin, exemplified by biosynthetic l-glycine-l-tyrosine-based polymer (ME-1) and chemosynthetic dihydroxyphenylalanine-based polymer (MC-1). At nontoxic concentrations, both compounds effectively (>/=60%) and reversibly suppressed the production of tumor necrosis factor (TNF), even when applied after stimulation of human peripheral blood monocytes with lipopolysaccharide (LPS). The inhibitory activity of melanin was selective with regard to cytokine response but not inducer- or cell-type-specific. In addition to TNF, melanin inhibited production of interleukin (IL)-1beta, IL-6, and IL-10 but not granulocyte-macrophage colony-stimulating factor by the LPS-stimulated monocytes. Melanin was equally effective in inhibiting production of TNF by monocytes stimulated with the purified protein derivative of Mycobacterium tuberculosis and production of IL-6 by IL-1alpha-stimulated human fibroblasts and endothelial cells. Northern blot analysis, mRNA stability determination, immunoprecipitation studies on metabolically labeled intracellular TNF, and pulse chase experiments revealed that melanin reduced efficiency of mRNA translation. The finding that melanin arrests ongoing cytokine synthesis suggests that this compound may be useful as an adjunct therapy for conditions showing involvement of proinflammatory cytokines.

Cell Survival↗
PubMed1997

Multiple sequence threading: an analysis of alignment quality and stability.

Methods that compare a protein sequence directly to a structure can be divided into those that construct a molecular model (threading methods) and those that perform a sequence alignment with the structure encoded as a sequence of structural states (one-dimensional/three-dimensional (1D/3D) matching). The former take into account the internal packing of the molecule but the latter do not. On the other hand, it is simple to include multiple sequence data in a 1D/3D comparison but difficult in a threading method. Here, a protein sequence/structure alignment method is described that uses a combination of matching predicted and observed residue exposure, predicted and observed secondary structure (1D/3D) together with pairwise packing interactions in the core (threading). Using a variety of distantly related and analogous protein structures, the multiple sequence threading (MST) method was compared to a single sequence threading (SST) method (that uses complex potentials of mean-force) and also to a multiple sequence alignment (MSA) program. It was found that the MST method produced alignments that were better than the best that could be obtained with either the SST or MSA method. The method was found to be stable to error in both secondary structure prediction and predicted exposure and also under variation of the key parameters (fully described in an Appendix). The contribution of the pairwise term was found to be small but without it, the correct alignments were less stable and structurally unreasonable deletions were observed when matching against larger structures. Using the parameters derived for alignment, the method was able to recognise related folds in the structure databank with a specificity comparable to other methods.

Algorithms↗