Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 ± 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ

The equilibrium dissociation of recombinant human IFN-γ was monitored as a function of pressure and sucrose concentration. The partial molar volume change for dissociation was −209 ± 13 ml/mol of dimer. The specific molar surface area change for dissociation was 12.7 ± 1.6 nm2/molecule of dimer. The first-order aggregation rate of recombinant human IFN-γ in 0.45 M guanidine hydrochloride was studied as a function of sucrose concentration and pressure. Aggregation proceeded through a transition-state species, N*. Sucrose reduced aggregation rate by shifting the equilibrium between native state (N) and N* toward the more compact N. Pressure increased aggregation rate through increased solvation of the protein, which exposes more surface area, thus shifting the equilibrium away from N toward N*. The changes in partial molar volume and specific molar surface area between the N* and N were −41 ± 9 ml/mol of dimer and 3.5 ± 0.2 nm2/molecule, respectively. Thus, the structural change required for the formation of the transition state for aggregation is small relative to the difference between N and the dissociated state. Changes in waters of hydration were estimated from both specific molar surface area and partial molar volume data. From partial molar volume data, estimates were 25 and 128 mol H2O/mol dimer for formation of the aggregation transition state and for dissociation, respectively. From surface area data, estimates were 27 and 98 mol H2O/mol dimer. Osmotic stress theory yielded values ≈4-fold larger for both transitions.

Purification and Characterization of a Galactose-Rich Basic Glycoprotein in Tobacco1

We found a galactose-rich basic glycoprotein (GBGP) in the cell walls of cultured tobacco (Nicotiana tabacum) cells. GBGP and extensin were isolated as the major components of basic, salt-extracted cell wall glycoproteins. GBGP and extensin were separated by gel filtration in 6 m guanidine hydrochloride as 49- and 90-kD peaks, respectively, and further purified with reverse-phase chromatography. The protein moiety of GBGP constitutes about one-half of the molecule (w/w) and contains lysine (16%), proline (12%), hydroxyproline (10%), tyrosine (4%), alanine (7%), leucine (6%), and cystine (1.4%). Galactose accounted for 72% of the sugar moiety, arabinose content was low (17%), and a significant amount of mannose (7%) was found. No immunological cross-reaction was detected between GBGP and extensin. The antibody against native GBGP with sugar chains reacted with other glycoproteins on the gel blots, whereas the antibodies against deglycosylated GBGP and native extensin were highly specific. Immunolocalization analysis in tobacco stems showed that GBGP is specific to parenchyma tissue and that extensin localizes in the epidermis. This tissue-specific and exclusive distribution suggests important functions of these basic glycoproteins.

Initiation sites of protein folding by NMR analysis.

Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable.

Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase.

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl] -4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33 angstroms resolution and refined to an Rfactor 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2.

Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels.

4-Hydroxylation of estradiol by human uterine myometrium and myoma microsomes: implications for the mechanism of uterine tumorigenesis.

Estradiol is converted to catechol estrogens via 2- and 4-hydroxylation by cytochrome P450 enzymes. 4-Hydroxyestradiol elicits biological activities distinct from estradiol, most notably an oxidant stress response induced by free radicals generated by metabolic redox cycling reactions. In this study, we have examined 2- and 4-hydroxylation of estradiol by microsomes of human uterine myometrium and of associated myomata. In all eight cases studied, estradiol 4-hydroxylation by myoma has been substantially elevated relative to surrounding myometrial tissue (minimum, 2-fold; mean, 5-fold). Estradiol 2-hydroxylation in myomata occurs at much lower rates than 4-hydroxylation (ratio of 4-hydroxyestradiol/2-hydroxyestradiol, 7.9 +/- 1.4) and does not significantly differ from rates in surrounding myometrial tissue. Rates of myometrial 2-hydroxylation of estradiol were also not significantly different from values in patients without myomata. We have used various inhibitors to establish that 4-hydroxylation is catalyzed by a completely different cytochrome P450 than 2-hydroxylation. In myoma, alpha-naphthoflavone and a set of ethynyl polycyclic hydrocarbon inhibitors (5 microM) each inhibited 4-hydroxylation more efficiently (up to 90%) than 2-hydroxylation (up to 40%), indicating > 10-fold differences in Ki (<0.5 microM vs. > 5 microM). These activities were clearly distinguished from the selective 2-hydroxylation of estradiol in placenta by aromatase reported previously (low Km, inhibition by Fadrozole hydrochloride or ICI D1033). 4-Hydroxylation was also selectively inhibited relative to 2-hydroxylation by antibodies raised against cytochrome P450 IB1 (rat) (53 vs. 17%). These data indicate that specific 4-hydroxylation of estradiol in human uterine tissues is catalyzed by a form(s) of cytochrome P450 related to P450 IB1, which contribute(s) little to 2-hydroxylation. This enzyme(s) is therefore a marker for uterine myomata and may play a role in the etiology of the tumor.

Genetic and redox determinants of nitric oxide cytotoxicity in a Salmonella typhimurium model.

Paradoxically, nitric oxide (NO) has been found to exhibit cytotoxic, antiproliferative, or cytoprotective activity under different conditions. We have utilized Salmonella mutants deficient in antioxidant defenses or peptide transport to gain insights into NO actions. Comparison of three NO donor compounds reveals distinct and independent cellular responses associated with specific redox forms of NO. The peroxynitrite (OONO-) generator 3-morpholinosydnonimine hydrochloride mediates oxygen-dependent Salmonella killing, whereas S-nitrosoglutathione (GSNO) causes oxygen-independent cytostasis, and the NO. donor diethylenetriamine-nitric oxide adduct has no antibacterial activity. GSNO has the greatest activity for stationary cells, a characteristic relevant to latent or intracellular pathogens. Moreover, the cytostatic activity of GSNO may best correlate with antiproliferative or antimicrobial effects of NO, which are unassociated with overt cell injury. dpp mutants defective in active dipeptide transport are resistant to GSNO, implicating heterolytic NO+ transfer rather than homolytic NO. release in the mechanism of cytostasis. This transport system may provide a specific pathway for GSNO-mediated signaling in biological systems. The redox state and associated carrier molecules are critical determinants of NO activity.

Determination of the angle between the anticodon and aminoacyl acceptor stems of yeast phenylalanyl tRNA in solution.

A principal feature of the crystal structures of tRNAs is an L-shaped tertiary conformation in which the aminoacyl acceptor stem and the anticodon stem are approximately perpendicular. However, the anticodon-acceptor interstem angle has not been precisely quantified in solution for any tRNA. Such a determination would represent an important test of the predicted global conformation of tRNAs in solution. To this end, we have constructed a yeast tRNA(Phe) heteroduplex RNA molecule in which the anticodon and acceptor stems of the tRNA have each been extended by approximately 70 base pairs. A comparison of the rotational decay times of the heteroduplex molecule and a linear control yields an interstem angle of 89 +/- 4 degrees in 4 mM magnesium chloride/100 microM spermine hydrochloride, essentially identical to the corresponding angle observed in the crystal under similar buffer and temperature conditions. The current approach is applicable to the study of a wide variety of RNA molecules that possess elements of nonhelical structure.

Viral particles are required for infection in neurodegenerative Creutzfeldt-Jakob disease.

Several models have been proposed for the infectious agents that cause human Creutzfeldt-Jakob disease (CJD) and sheep scrapie. Purified proteins and extracted nucleic acids are not infectious. To further identify the critical molecular components of the CJD agent, 120S infectious material with reduced prion protein (PrP) was treated with guanidine hydrochloride or SDS. Particulate and soluble components were then separated by centrifugation and molecularly characterized. Conditions that optimally solubilized residual PrP and/or nucleic acid-protein complexes were used to produce subfractions that were assayed for infectivity. All controls retained > 90% of the 120S titer (approximately 15% of that in total brain) but lost > 99.5% of their infectivity after heat-SDS treatment (unlike scrapie fractions enriched for PrP). Exposure to 1% SDS at 22 degrees C produced particulate nucleic acid-protein complexes that were almost devoid of host PrP. These sedimenting complexes were as infectious as the controls. In contrast, when such complexes were solubilized with 2.5 M guanidine hydrochloride, the infectious titer was reduced by > 99.5%. Sedimenting PrP aggregates with little nucleic acid and no detectable nucleic acid-binding proteins had negligible infectivity, as did soluble but multimeric forms of PrP. These data strongly implicate a classical viral structure, possibly with no intrinsic PrP, as the CJD infectious agent. CJD-specific protective nucleic acid-binding protein(s) have already been identified in 120S preparations, and preliminary subtraction studies have revealed several CJD-specific nucleic acids. Such viral candidates deserve more attention, as they may be of use in preventing iatrogenic CJD and in solving a fundamental mystery.

Efeitos da Imunocastração e de agonistas beta-adrenérgicos sobre a qualidade da carne de bovinos

Os agonistas beta-adrenérgicos (βAA) são conhecidos por aumentar a hipertrofia muscular e lipólise, neste caso uma maneira de se reduzir o efeito da lipólise seria a imunocastração. Dessa forma, o objetivo deste projeto foi avaliar o efeito dos βAA e da imunocastração sobre a qualidade da carne de bovinos Nelore. Foram utilizados noventa e seis bovinos Nelore, sendo que metade dos animais (n=48) receberam uma dose da vacina de imunocastração, e após 30 dias receberam a segunda dose. A outra metade dos animais (n=48) não recebeu nenhuma dose da vacina. Durante 70 dias os animais foram alimentados com uma dieta padrão composta de 24% volumoso e 76% de concentrado. Após 70 dias de confinamento os animais foram divididos em três grupos, dentro de bloco (peso inicial) e condição sexual e foram alimentados por 30 dias, com umas das seguintes dietas: CON - dieta padrão utilizada na fase anterior, sem a adição de βAA; ZIL - dieta padrão acrescida de 80 mg/dia Cloridrato de Zilpaterol; RAC - dieta padrão acrescida de 300 mg/dia Cloridrato de Ractopamina. Ao final desse período os animais foram abatidos e colhidas amostras do músculo Longissimus dorsi para as avaliações de qualidade de carne, lipídeos totais, perfil de ácidos graxos, análise sensorial do consumidor, perfil morfométrico muscular, expressão dos genes calpaína e calpastatina, comprimento de sarcômero. Para a maioria das características avaliadas não foram observadas interações entre os tratamentos. Ao avaliar o efeito da condição sexual, os animais imunocastrados apresentaram maiores intensidades de cor L, a e b, lipídios totais, ácidos oleico, palmítico e total de monoinsaturados e maior frequência para as fibras oxidativas (FO) e glicolíticas (FG) em relação aos não-castrados. Contudo, os animais não-castrados tiveram uma tendência a apresentarem uma carne mais macia na análise sensorial e obtiveram maior frequência das fibras oxidativasglicolíticas (FOG) em relação aos imunocastrados. Quanto ao efeito dos βAA, o grupo ZIL apresentaram uma carne menos macia na força de cisalhamento, maiores concentrações de ácidos heptadecanoico, linoleico, araquidonico ácido C20:3 N6C8C11C14, ômega 6, maior frequência para as FO e menor para FG em comparação ao grupo RAC e CON. No entanto, os animais do grupo CON e ZIL apresentaram maior área para as FO em comparação ao grupo RAC, enquanto que para as FOG, os animais do grupo CON tiveram maior área do que os animais do grupo RAC e ZIL. Na análise sensorial, os grupos RAC e ZIL receberam menores notas para os atributos textura e qualidade global em relação ao CON. Não foi observado efeito da condição sexual e dos βAA sobre a expressão dos genes e comprimento de sarcômero. Conclui-se que a condição sexual e a suplementação com os βAA podem alterar a qualidade da carne, perfil de ácidos graxos e morfométrico muscular, sem, contudo, alterar a expressão dos genes e do comprimento de sarcômero.

Silver-catalysed multicomponent 1,3-dipolar cycloaddition of 2-oxoaldehydes-derived azomethine ylides

The silver-catalysed multicomponent reaction between ethyl glyoxylate, 2,2-dimethoxyacetaldehyde, or phenylglyoxal as aldehyde components with a α-amino ester hydrochloride and a dipolarophile in the presence of triethylamine is described. This domino process takes place at room temperature by in situ liberation of the α-amino ester followed by the formation of the imino ester, which is the precursor of a metalloazomethine ylide. The cycloaddition of this species and the corresponding dipolarophile affords polysubstituted proline derivatives. Ethyl glyoxylate reacts with glycinate, alaninate, phenylalaninate and phenylglycinate at room temperature in the presence of representative dipolarophiles affording endo-2,5-cis-cycloadducts in good yields and high diastereoselection. In addition, 2,2-dimethoxyacetaldehyde is evaluated with the same amino esters and dipolarophiles, under the same mild conditions, generating the corresponding endo-2,5-cis-cycloadducts with higher diastereoselections than the obtained in the same reactions using ethyl glyoxylate. In the case of phenylglyoxal the corresponding 5-benzoyl-endo-2,5-cis cycloadducts are obtained in short reaction times and similar diasteroselection.

Federal and State laws pertaining to methadone /

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Doxorubicin pharmacokinetics following a single-dose infusion to sulphur-crested cockatoos (Cacatua galerita)

Objective To determine the pharmacokinetics of doxorubicin in sulphur-crested cockatoos, so that its use in clinical studies in birds can be considered. Design A pharmacokinetic study of doxorubicin, following a single intravenous (IV) infusion over 20 min, was performed in four healthy sulphur-crested cockatoos (Cacatua galerita). Procedure Birds were anaesthetised and both jugular veins were cannulated, one for doxorubicin infusion and the other for blood collection. Doxorubicin hydrochloride (2 mg/kg) in normal saline was infused IV over 20 min at a constant rate. Serial blood samples were collected for 96 h after initiation of the infusion. Plasma doxorubicin concentrations were assayed using an HPLC method involving ethyl acetate extraction, reverse-phase chromatography and fluorescence detection. The limit of quantification was 20 ng/mL. Established non-parametric methods were used for the analysis of plasma doxorubicin data. Results During the infusion the mean +/- SD for the C-max of doxorubicin was 4037 +/- 2577 ng/mL. Plasma concentrations declined biexponentially immediately after the infusion was ceased. There was considerable intersubject variability in all pharmacokinetic variables. The terminal (beta-phase) half-life was 41.4 +/- 18.5 min, the systemic clearance (Cl) was 45.7 +/- 18.0 mL/min/kg, the mean residence time (MRT) was 4.8 +/- 1.4 min, and the volume of distribution at steady state (V-SS) was 238 131 mL/kg. The extrapolated area under the curve (AUC(0-infinity)) was 950 +/- 677 ng/mL.h. The reduced metabolite, doxorubicinol, was detected in the plasma of all four parrots but could be quantified in only one bird with the profile suggesting formation rate-limited pharmacokinetics of doxorubicinol. Conclusions and clinical relevance Doxorubicin infusion in sulphur-crested cockatoos produced mild, transient inappetence. The volume of distribution per kilogram and terminal half-life were considerably smaller, but the clearance per kilogram was similar to or larger than reported in the dog, rat and humans. Traces of doxorubicinol, a metabolite of doxorubicin, were detected in the plasma.

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.