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PubMed1990

Detection of methylated asparagine and glutamine residues in polypeptides.

A residue of gamma-N-methylasparagine (gamma-NMA) is found at position beta-72 of many phycobiliproteins. delta-N-Methylglutamine is present in some bacterial ribosomal proteins. gamma-NMA was synthesized by reacting the omega-methyl ester of aspartate with methylamine and delta-N-methylglutamine by reaction of pyroglutamate with methylamine. These derivatives and the omega-methyl esters of aspartate and glutamate were characterized by melting point, by thin-layer chromatography, by amino acid analysis, by NMR spectroscopy, and after conversion to the phenylthiohydantoin (PTH) derivative. The gamma-NMA residues in peptides from allophycocyanin, C-phycocyanin, and B-phycoerythrin were stable under the conditions of automated sequential gas-liquid phase Edman degradation. On HPLC, PTH-gamma-NMA co-eluted with PTH-serine and was accompanied by a minor component eluting just prior to dimethylphenylthiourea. Similar results were obtained on manual derivatization of synthetic gamma-NMA to prepare the PTH derivative. The PTH-delta-N-methylglutamine standard eluted near the position of dimethylphenylthiourea under the usual conditions employed for the identification of PTH-amino acid derivatives in automated protein sequencing.

Amino Acid Sequence↗
PubMed1989

Mussel glue protein has an open conformation.

Both native glue protein from marine mussels and a synthetic nonhydroxylated analog were analyzed by far-uv CD under a variety of conditions. Analysis of the CD spectra using various models strongly suggest a primarily random coil structure for both forms of the protein, a fact also supported by the absence of spectral change for the glue protein upon dilution into 6 M guanidine hydrochloride. The nonhydroxylated analog, which consists of 20 repeats of the peptide sequence Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys, was further characterized by enzyme modification using mushroom tyrosinase. Enzymatic hydroxylation of tyrosines was found to be best fit by a model containing two rate constants, 5.6 (+/- 0.6) X 10(-3) and 7.2 (+/- 0.3) X 10(-2) min-1. At equilibrium, HPLC analysis of digests showed nearly 100% conversion of Tyr-9 and only 15 to 35% conversion of Tyr-5. The Chou and Fasman rules for predicting structure were applied to the repeat sequence listed above. The rules predict the absence of alpha helix and beta pleated sheets in the structure of this peptide. On the other hand, beta turns are predicted to be present with Tyr-5 being in the region of highest probability. These data suggest that the protein in solution has only a small amount of secondary structure.

Amino Acid Sequence↗
PubMed1976

Sphingolipid base metabolism. Sphinganine-1-phosphate lyase: identification of ethanolamine 1-phosphate as product.

Ethanolamine 1-phosphate has been characterized as a product of the action of rat liver microsomal sphinganine 1-phosphate lyase on erythro-sphinganine 1-phosphate. The product was characterized by various forms of chromatography, gas-liquid chromatography-mass spectral analysis of appropriate derivatives, and by conversion to ethanolamine. The results of detailed studies of the mass spectral fragmentation of the tetra-trimethylsilyl derivative of ethanolamine 1-phosphate are also reported.

Animals↗
PubMed1984

Mutagenic activity of possible metabolites of 4-nitrobiphenyl ether.

A series of possible metabolites--4-nitrosobiphenyl ether (4-NO), 4-hydroxylaminobiphenyl ether (4-NHOH), 4-aminobiphenyl ether (4-NH2), 4-hydroxyacetylaminobiphenyl ether (4-N(OH)Ac), 4-acetoxyacetylaminobiphenyl ether (4-N(OAc)Ac)involved in the toxic effects of 4-nitrobiphenyl ether (4-NO2) was synthesized and tested for mutagenic activity toward Salmonella typhimurium TA100 strain in the presence and the absence of liver homogenates of guinea pig treated with Kaneclor-500. 4-NO2, 4-NO and 4-NHOH showed direct-acting mutagenicity. 4-NO and 4-NHOH showed high mutagenic activity, while the mutagenic activity of 4-NO2 was very weak compared to 4-NO and 4-NHOH. 4-NO showed antimicrobial action at high concentrations. The other three compounds tested induced no mutation. Upon addition of NAD(P)H, the mutagenic activities of 4-NO and 4-NHOH were slightly enhanced, but no enhancement was observed by addition of NAD(P)+. Metabolic activation with guinea pig liver homogenates enhanced the mutagenic activities of 4-NO2 and 4-NO, and converted 4-NH2, 4-N(OH)Ac and 4-N(OAc)Ac to the product(s) responsible for the mutagenic activity. Addition of bis(p-nitrophenyl)phosphate, a deacetylase inhibitor, inhibited the mutagenic activities of 4-N(OH)Ac and 4-N(OAc)Ac by about 70% in the presence of NADPH and about 77% in the absence of NADPH. High performance liquid chromatography (HPLC) analysis of non-enzymatic conversion-products of 4-NHOH and 4-BO with and without NADPH indicated that 4-NHOH disappeared after 30 min of incubation and was converted completely to 4-NO without NADPH, while with NADPH, 4-NHOH disappeared very slowly and was detected even after 4 h of incubation. In the case of 4-NO, no decrease of 4-NO was observed without NADPH, while with NADPH 4-NO decreased quickly and a significant amount of 4-NHOH appeared. The mechanism of the NAD(P)H-dependent increase in mutagenicity is also discussed.

Animals↗
PubMed1990

Formylation of tetrahydrofolate by formyl phosphate.

Formyl phosphate is the putative intermediate in the formylation of tetrahydrofolate (THF) catalyzed by N10-formylTHF synthetase. In this study the non-enzymic reaction between formyl phosphate and THF was examined at 5 degrees C. 1H-NMR, HPLC and kinetic analysis of the proton-catalyzed conversion of the product to N5,10-methenylTHF were used to identify the product. In contrast to the enzyme reaction, which produces N10-formylTHF, N5-formylTHF was the only formylated THF derivative formed. The reaction was conducted at pH values of 3, 5, and 7, with the highest yield being obtained at pH 5 (64-85%, based on THF). The enzyme, therefore, changes the regioselectivity of this reaction by increasing the reactivity of the 10-nitrogen and either decreasing the reactivity of the 5-nitrogen or limiting its accessibility to formyl phosphate. 2-Mercaptoethanol, present in the reaction mixture to protect THF from O2, was also formylated by formyl phosphate, at the oxygen position.

Chemical Phenomena↗
PubMedDate not supplied

Structural relationships between the isoenzymes of human placental alkaline phosphatase: a serum factor converts M-PLAP to A- and B-PLAP.

A protein factor has been found in serum which converts the M form of placental alkaline phosphatase (PLAP) to the A and B forms. The identity of the conversion products has been confirmed by analysis of their dimers and polypeptides. Proteolysis is not implicated in this phenomenon. This report establishes microvillous M-PLAP as the precursor of the A and B forms.

Alkaline Phosphatase↗
PubMed1991

Sequence analysis of HLA-Bw53, a common West African allele, suggests an origin by gene conversion of HLA-B35.

In the West African population of the Gambia the class I antigen HLA-Bw53 is found at high frequency. We used the polymerase chain reaction to amplify cDNA from an individual homozygous for this allele and determined the nucleotide sequence of the polymorphic alpha 1 and alpha 2 domains. The HLA-Bw53 sequence is identical to HLA-B35 except for a short sequence at the 3' end of exon 2 (encoding the alpha 1 domain) which specifies a Bw4 rather than a Bw6 motif. This suggests an origin for HLA-Bw53 involving a gene conversion of HLA-B35 by an allele containing this Bw4 sequence. The alpha 2 domain shared by HLA-Bw53, -B35, and -Bw58 is particularly common in sub-Saharan Africans.

Adult↗
PubMed1995

Comparison of experimental and theoretical parameters of the Moolgavkar-Venzon-Knudson incidence function for the stages of initiation and promotion in rat hepatocarcinogenesis.

Mathematical descriptions of complex biological phenomena, such as cancer, require an experimental format that faithfully recapitulates the biological process. In addition, the biological process must dictate the parameters in the mathematical formula. Evidence from the epidemiology of several human cancers and from experimental carcinogenesis in several organ systems indicates that cancer is a multistage process. The initiation-promotion-progression format of experimental carcinogenesis mimics the development of cancer in humans and other animals. In rats, the altered hepatic focus model of hepatocarcinogenesis has been well characterized and, coupled with the method of quantitative stereology, permits accurate determination of the number and the volume fraction of such altered foci per liver. The placental isozyme of glutathione S-transferase (PGST) is reportedly the best single marker of preneoplasia in the rat liver. Recently, single hepatocytes expressing PGST have been proposed as putatively initiated cells. Quantitation of individual hepatic cells and altered hepatic foci expressing PGST in the livers of rats subjected to an initiation-promotion protocol permits determination of the congruence of the Moolgavkar-Venzon-Knudson (MVK) model with experimental data. The best fit of the MVK model for the preneoplastic stages of hepatocarcinogenesis assumes that all hepatocytes are susceptible and that single hepatocytes expressing PGST are the initiated cell population for the focal lesions that express PGST. Further refinement of the initiation-promotion-progression model to permit accurate quantitation of early malignant conversion should allow a more complete analysis of the congruence of the MVK model for human cancer risk determination. In addition, the MVK model may be extended to other model systems and to human cancers in which early preneoplasia can be quantitated. Furthermore, the use of a more biologically based risk-assessment protocol, such as the MVK model rather than the stochastic one-hit model presently used, would permit incorporation of the present knowledge on the pathogenesis of cancer. To apply experimental data to a mathematical model that reflects the biological processes underlying human cancer development will require integration of the cell kinetics and experimental data to a mathematical model that reflects the biological processes underlying human cancer development including the pharmacokinetic and pharmacodynamic properties of the treatment chemicals.

Animals↗
PubMed2004

Talking about taste: using a discursive psychological approach to examine challenges to food evaluations.

This study is concerned with developing the interdisciplinary nature of food research, and with examining eating practices as they occur in everyday situations. The aim is to demonstrate how discursive approaches may contribute to eating research using a specific analytical example. A discursive psychological approach is used to examine mealtime conversations from 10 families with the analysis focusing on how food evaluations are challenged in interaction-for example, asking someone to justify what they think is 'wrong' with the food. Data are presented with 7 examples of the 30 challenges that were found within the data corpus. The analysis demonstrates how people may be held accountable for their expressed taste preferences when being challenged, and how this contributes to our understanding of eating as primarily an individual and embodied experience. It is argued that a specific and detailed analysis of eating interactions provides an alternative way of conceptualising food evaluations as discursive rather than mentalistic concepts. A discursive approach also opens up practical ways in which the social and familial aspects of eating may be examined as they occur as part of food practices.

Adult↗
PubMedDate not supplied

Testing of properties of digital modules used in gamma-ray spectrometry.

The properties of digital modules (analog-to-digital converters (ADC), digital signal processor (DSP)) in spectrometric chain are the decisive factors which affect the quality of spectrometric measurements. Several properties, integral and differential linearity, channel profiles and throughput, of various ADC and DSP models by CANBERRA were tested.

Analog-Digital Conversion↗
PubMed2005

Cloning, expression, and differentiation-dependent regulation of SMAF1 in adipogenesis.

With the aim of identifying novel molecular pathways in the adipocyte, we conducted differential screening of DNA filter arrays with probes from 3T3-L1 preadipocytes and adipocytes, and discovered a novel 0.7kb transcript we term small adipocyte factor 1 (SMAF1). SMAF1 encodes a wholly novel 10kDa protein. Transfection and localization studies of a SMAF1-EGFP fusion construct indicate nuclear localization, suggestive of a possible regulatory role. Northern blot analysis of various murine tissues indicates adipose tissue-restricted expression, and fractionation of adipose tissue reveals that SMAF1 is expressed soley in adipocytes and not in the stromal-vascular cell population. Northern blot analysis of brown and white adipogenic conversion reveals that expression of SMAF1 closely parallels emergence of an adipocyte phenotype and that TNFalpha-mediated dedifferentiation of 3T3-L1 adipocytes results in a rapid decline of SMAF1 transcript. These data indicate that SMAF1 is closely tied to the adipocyte phenotype and predict a novel and possibly regulatory role for this gene in adipocyte function.

3T3-L1 Cells↗
PubMed2006

Microcystin analysis in human sera and liver from human fatalities in Caruaru, Brazil 1996.

In 1996, an extensive exposure of Brazilian hemodialysis patients at a dialysis center, using a municipal water supply water contaminated with cyanotoxins, provided the first evidence for acute lethal human poisoning from the cyclic peptide hepatotoxins called microcystins. During this outbreak, 100 of 131 patients developed acute liver failure and 52 of these victims were confirmed to have been exposed to lethal levels of microcystins. Detection and quantitation of microcystins in these biological samples posed some analytical challenges since there were no well-established and routine analytic methods to measure total microcystins in tissue or sera samples. At the time of the 1996 exposure we used analytic methods that combined the use of enzyme linked immunosorbant assay (ELISA), analytical high performance liquid chromatography (HPLC), electrospray ionization ion-trap mass spectroscopy (ES-ITMS) and matrix assisted laser desorption ionization-time of flight spectroscopy (MALDI-TOF). In the intervening years these methods have been improved and others developed that allow a more quantitative and critical analysis of microcystin contaminated tissue and sera. For these reasons, and to see how storage with time might effect the detection and stability of microcystins in these matrices, we reanalyzed selected liver tissues and sera from the Caruaru victims in Brazil. We developed and validated a procedure to measure total microcystins in Caruaru human sera and liver tissue using a combination of ELISA, liquid chromatography and liquid chromatography-mass spectrometry (LC/MS), GC/MS and MS/MS techniques. GC/MS and LC/MS were followed by MS/MS to obtain a fingerprint fragment spectra for the microcystins. The validity of the extraction procedure for free microcystins was confirmed by recovery experiments with blood sera spiked with microcystin-LR. We removed proteins with the Microcon Centrifugal Filter prior to LC/MS and ELISA analysis. A solid phase extraction (SPE) procedure was used for analysis of protein bound microcystins by conversion of ADDA to erythro-2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) combined with GC/MS. We found that the GC/MS method yielded a higher concentration of microcystin than that obtained by ELISA and LC/MS. We hypothesize that this difference is due to better GC/MS detection of the covalently bound form of microcystins in human liver tissue. We also concluded that microcystins are very stable when stored under these conditions for periods of almost 10 years.

Bacterial Toxins↗
PubMed1985

On the mechanism of muscular contraction.

A thermodynamic analysis is presented for the energy conversion by muscle contraction. During the cyclic processes the major change in energy of the myosin-actin system is due to bond formation between myosin heads and actin. To account for the high efficiency of a working muscle the work done is connected directly to the formation of myosin-actin bond. It is suggested that successively stronger bonds are formed by a stepwise movement of myosin heads over an interval between two troponin molecules on the actin filament. At the end of the interval, where the bond has maximum strength, energy is supplied to break the bond. Here the work is not primarily connected to the 45 degrees rotation of myosin heads as is commonly done. A way of separating the different kinds of energy losses is presented.

Actomyosin↗
PubMed1994

Effect of high dose vitamin C on urinary oxalate levels.

The effect of high dose ascorbate on urinary oxalate levels in healthy adults was investigated using a modified ion chromatography method. Subjects ingested 1, 5 and 10 gm. supplemental ascorbate per day for 5 days, separated by 5 days of no supplementation. Urine ascorbate levels demonstrated variable increases with ascorbate supplementation. Ascorbate added directly to urine in vitro resulted in statistically significant but modest increases in measured oxalate. Addition of 5.68 mmol./l. ascorbate increased measured urinary oxalate by 36 mumol./l., implying conversion of ascorbate to oxalate during analysis. Measurement of 24-hour urinary oxalate levels with 5 and 10 gm. ascorbate per day showed similar, modest increases, which could be entirely accounted for by oxalate production during analytical procedures. Thus, no genuine increase in urinary oxalate was demonstrable despite a greatly increased ascorbate intake.

Adult↗
PubMed2003

Properties of elastomeric calcium phosphate cement-chitosan composites.

OBJECTIVE: Self-hardening calcium phosphate cements (CPC) have been shown to be efficacious in a number of clinical applications. For some applications it is desirable to have CPC in a non-rigid resorbable elastomeric matrix. In the present study, chitosan was evaluated as the matrix for preparing CPC-chitosan composites. METHODS: Cement specimens were prepared by mixing CPC powder (an equimolar mixture of tetracalcium phosphate and dicalcium phosphate anhydrous) with a chitosan solution at a powder/liquid ratio of 2-2.5. The setting time was measured by a Gilmore needle method. A standard three-point flexural test was used to fracture the specimens at a crosshead speed of 0.5 mm/min. Powder X-ray diffraction analysis was used to determine the conversion of the CPC to hydroxyapatite. RESULTS: The CPC-chitosan composites were more stable in water than conventional CPC. They did not disintegrate even when placed in water immediately after mixing. The CPC-chitosan paste hardened within 10 min in all cases. The 1d mean flexural modulus (GPa) for the control CPC was 5.3 (0.3) (mean (standard deviation); n=5), and that for CPC-chitosan composites were between 2.7 (0.3) and 4.7 (0.3). The 1d mean flexural strength (MPa) for the control was 16.6 (1.9), and that for the CPC-chitosan ranged from 4.5 (0.5) and 12.0 (1.0) (n=5). Chitosan did not interfere the conversion of CPC components to hydroxyapatite. SIGNIFICANCE: This study demonstrates that CPC-chitosan composites are stable in a wet environment and have acceptable mechanical strengths for clinical applications.

Biocompatible Materials↗
PubMed2002

Synthesis and photopolymerization of N,N'-dimethyl,-N,N'-di(methacryloxy ethyl)-1,6-hexanediamine as a polymerizable amine coinitiator for dental restorations.

N,N'-dimethyl,-N,N'-di(methacryloxy ethyl)-1,6-hexanediamine (NDMH) was synthesized for the purpose of replacing both triethylene glycol dimethacrylate (TEGDMA) and the non-polymerizable amine which is added as a coinitiator in dental resin mixtures, 2,2-bis[4(2-hydroxy-3-methacryloxypropoxy)phenyl] propane (bis-GMA), camphorquinone (CQ) and ethyl-4-dimethylaminobenzoate (EDAB) were used as monomer, photoinitiator and coinitiator, respectively, in these model dental resin systems. Mixtures of bis-GMA/TEGDMA/CQ/EDAB and bis-GMA/TEGDMA/CQ/NDMH were found to reach final conversions of about 45%, slightly higher than his-GMA/NDMH/CQ (40%) under comparable visible light irradiation conditions. In addition, samples cured to these conversions were tested with dynamic mechanical analysis. The bis-GMA/TEGDMA/CQ/EDAB, his-GMA/TEGDMA/CQ/NDMH and bis-GMA/NDMH/CQ mixtures were found to have approximately the same glass transition temperature and modulus. Finally, the water sorption and solubility of bis-GMA/NDMH/CQ were higher than those of the bis-GMA/TEGDMA/CQ/EDAB, and bis-GMA/TEGDMA/CQ/NDMH. However, the values were still within the range of the ISO 9000's standards. These results suggest that NDMH is a viable alternative to conventional photocuring dental resins, serving both as a diluent and coinitiator, since there are no large differences in physical and mechanical properties when using NDMH to replace the amine coinitiator and TEGDMA diluent. The key advantage to this system is that the dimethacrylate NDMH can copolymerize with bis-GMA and TEGDMA, limiting the amount of extractable amine.

Amines↗
PubMed2000

Application of simultaneous excitation/detection to generate real-time excitation profiles in fourier transform ion cyclotron resonance mass spectrometry

Simultaneous excitation/detection (SED), which permits observation of ion motion during an excitation event, is used to generate real-time Fourier transform ion cyclotron resonance (FTICR) excitation profiles that track the radial extent of ion motion in a trapped-ion cell. The conventional FTICR excitation profile is collected in a series of individual experiments in which peak magnitude is monitored as excitation voltage is increased. In contrast, SED permits the single-scan detection of ion cyclotron motion within the trapped-ion cell and consequently yields the data that produces a real-time excitation profile. Data analysis techniques are presented that facilitate conversion of a time domain SED profile into an excitation profile. An order of magnitude decrease in the amount of time is required to acquire an excitation profile, while the precision of the measurement is improved. To demonstrate the utility of the technique, it is applied to the study of axial and radial ion loss mechanisms for argon, benzene, and acetophenone ions under different conditions. SED excitation profiles are also used to illustrate the facility of quadrupolar excitation for minimizing radial ion loss.

Journal Article↗
PubMed2001

Experimental and theoretical vibrational study of isatin, its 5-(NO2, F, Cl, Br, I, CH3) analogues and the isatinato anion.

Effects of 5-R substitution (R = NO2, F, Cl, Br, I, CH3) and N-deprotonation on the 4000-400 cm(-1) region of the low temperature FT IR spectrum and the molecular structure of solid isatin are investigated. Harmonic IR spectra and molecular geometries of the 5-R isatins (except for Br and I analogues) are calculated at the HF/6-31G(d, p) level and compared with the experimental solid-state data. In general, substitution has small effect on the molecular structure and the IR spectrum of isatin. The v(CO) triplet in the IR spectra of isatin and its 5-substituted analogues is resulted by vibrational splitting of the out-of-phase CO stretching, v(op)[(CO)2]. While the frequency of the v(op)[(CO)2] mode is relatively less affected by 5-substitution and mainly depends on the substituent mass, the frequency of the in-phase stretching, v(ip)[(CO)2], is strongly sensitive to both mass and electronic properties of the substituent. Substitution at C5 has relatively greater influence on the electron density and the force constant of the amide than on the ketone carbonyl group. Strong electron-donors shorten and stabilize the unusually long alpha-dicarbonyl CC bond, while electron-accepting groups tend to stretch this bond further. N-Deprotonation brings to elongation of the five membered-ring along the N-C(CO(ketone)) vector and expansion of the bonds within the alpha-dicarbonyl part. Theoretical v(CO) frequency of isatin is lowered for about 180 cm(-1) upon conversion into isatinato ion. Harmonic vibrational analysis reveals that only the highest-frequency v(CO) mode of the isolated isatinato anion can be considered good group vibration for empirical assignments in spectra of solid isatinates. Owing to the solid-state influences on the v[(CO)2] modes, no reliable spectra structure correlations could be established from the present experimental spectroscopic data.

Chemical Phenomena↗