Synergistic Effect of 5-Nitro-2′-deoxyuridine with Ganciclovir Against Human Cytomegalovirus In Vitro

Abstract

In this paper we describe a practical synthesis of 5-nitro-2′-deoxyuridine (4) and 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-nitrouracil (11). These compounds were then evaluated for their ability to inhibit the growth of human cytomegalovirus (HCMV, strain AD169) in MRC-5 cells using a plaque reduction assay. Compound 11 was unable to inhibit the growth of HCMV at the highest concentration tested (100 μg/mL). However, compound 4 (5-NO₂-dU) exhibited marginal activity against HCMV in vitro in a dose-dependent manner with a 50% inhibitory concentrations (IC₅₀) of 1 to 5 μg/mL. Combinations of 5-NO₂-dU with ganciclovir synergistically inhibited HCMV induced cell killing in culture.     

Effect of Ser-129 phosphorylation on interaction of α-synuclein with synaptic and cellular membranes

In the healthy brain, less than 5% of α-synuclein (α-syn) is phosphorylated at serine 129 (Ser(P)-129). However, within Parkinson disease (PD) Lewy bodies, 89% of α-syn is Ser(P)-129. The effects of Ser(P)-129 modification on α-syn distribution and solubility are poorly understood. As α-syn normally exists in both membrane-bound and cytosolic compartments, we examined the binding and dissociation of Ser(P)-129 α-syn and analyzed the effects of manipulating Ser(P)-129 levels on α-syn membrane interactions using synaptosomal membranes and neural precursor cells from α-syn-deficient mice or transgenic mice expressing human α-syn. We first evaluated the recovery of the Ser(P)-129 epitope following either α-syn membrane binding or dissociation. We demonstrate a rapid turnover of Ser(P)-129 during both binding to and dissociation from synaptic membranes. Although the membrane binding of WT α-syn was insensitive to modulation of Ser(P)-129 levels by multiple strategies (the use of phosphomimic S129D and nonphosphorylated S129A α-syn mutants; by enzymatic dephosphorylation of Ser(P)-129 or proteasome inhibitor-induced elevation in Ser(P)-129; or by inhibition or stable overexpression of PLK2), PD mutant Ser(P)-129 α-syn showed a preferential membrane association compared with WT Ser(P)-129 α-syn. Collectively, these data suggest that phosphorylation at Ser-129 is dynamic and that the subcellular distribution of α-syn bearing PD-linked mutations, A30P or A53T, is influenced by the phosphorylation state of Ser-129.     

Effect of α-actinin on actin viscosity

As determined by analytical ultracentrifugation, purified α-actinin does not form stable complexes with G-actin, myosin, tropomyosin, or the tropomyosintroponin complex. However, α-actinin forms a stable complex with F-actin polymerized either in 100 mM KC1 or in 2mM MgCl₂ without KCl. Viscosity studies confirm that α-actinin interacts as strongly with Mg²⁺-polymerized actin as it does with KCl-polymerized actin.     

Synergistic Action of Recombinant α-Amylase and Glucoamylase on the Hydrolysis of Starch Granules

Barley α-amylase 1 mutant (AMY) and Lentinula edodes glucoamylase (GLA) were cloned and expressed in Saccharomyces cerevisiae. The purified recombinant AMY hydrolyzed corn and wheat starch granules, respectively, at rates 1.7 and 2.5 times that of GLA under the same reaction conditions. AMY and GLA synergistically enhanced the rate of hydrolysis by ∼3× for corn and wheat starch granules, compared to the sum of the individual activities. The exo-endo synergism did not change by varying the ratio of the two enzymes when the total concentration was kept constant. A yield of 4% conversion was obtained after 25 min 37°C incubation (1 unit total enzyme, 15 mg raw starch granules, pH 5.3). The temperature stability of the enzyme mixtures was ≤50°C, but the initial rate of hydrolysis continued to increase with higher temperatures. Ca⁺⁺ enhanced the stability of the free enzymes at 50°C incubation. Inhibition was observed with the addition of 10 mM Fe⁺⁺ or Cu⁺⁺, while Mg⁺⁺ and EDTA had lesser effect. Reference to a company and/or products is only for purposes of information and does not imply approval of recommendation of the product to the exclusion of others that may also be suitable. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

Effect of aluminum on the binding properties of α-chymotrypsin

 The effect of aluminum ions on the binding properties of α-chymotrypsin has been studied. The results show that aluminum does not affect the catalytic rate constant k _cat, but it acts as an enzyme activator favoring the binding of the substrate to the catalytic site (i.e. decreasing K _m). Furthermore, aluminum binding to α-chymotrypsin displays about a threefold decrease in its affinity for the macromolecular inhibitor bovine pancreatic trypsin inhibitor (BPTI). Altogether, the different effect of aluminum on the binding of synthetic substrates (e.g. N-α-benzoyl-l-tyrosine ethyl ester, BTEE) and macromolecular inhibitors (e.g. BPTI) to α-chymotrypsin suggests the occurrence of an aluminum-linked conformational change in the enzyme molecule which brings about a marked structural change at the primary and secondary recognition sites for substrates and inhibitors. The modulative effect exerted by aluminum on the enzyme hydrolytic activity has been investigated also as a function of pH. The ion-linked effect appears to be dependent on the pH in a complex fashion, which suggests that aluminum binding is controlled by the protonation of at least two classes of residues on the enzyme molecule. Received: 5 December 1996 / Accepted: 11 March 1997     

Effect of polyethylene glycol on Bacillus subtilis α-amylase purification with raw and modified starch

Polyethylene glycol was found to enhance adsorption of Bacillus subtilis -amylase on starch in optimum concentration 10 % (w/w). Degree of adsorption at 12°C was increased from 83 to 98 % and from 30 to 81 % for cross-linked and raw starch, resp. Higher sorption capacity and easy desorption of -amylase without temperature or pH change was reached at 22 °C. Yield of -amylase 95 % and purification factor 8.3 were achieved on the cross-linked starch column. The method is suitable for -amylase isolation from PEG phase after its microbial production in aqueous two-phase systems.     

Effect of α-tocopherol tissue levels on beef quality

To evaluate meat quality of beef with different α-tocopherol tissue levels, 55 feedlot steers were fed a barley-based finisher diet with four vitamin E supplementation levels (0, 350, 700 and 1400 IU DL-α-tocopheryl acetate/animal per day) for 120 days. Although the increase in oxidation levels overtime was much smaller (P < 0.001) in the high-medium and high groups, α-tocopherol tissue levels did not affect (P > 0.05) pH, proximate analysis, drip and cooking losses, and shear force of steaks. No effect of α-tocopherol tissue levels was found in retail evaluation of steaks after a short ageing time of 6 days, but with 21 days of ageing, a delay in formation of metmyoglobin (P = 0.008) was observed in steaks with higher tissue levels of α-tocopherol. Similar results were found for ground beef (25% fat) prepared from 6-day aged meat. Thus, higher α-tocopherol tissue levels protect ground beef and long-aged steaks from discolouration and lipid oxidation.     

Effect of pH and Glucose on the Stability of α-lactalbumin

The objective of this study is to understand the influence of pH and effect of cosolvent (glucose) on the stabilization of bovine α-lactalbumin by using ultrasonic techniques. Values of density, ultrasonic velocity and viscosity were measured for bovine α-lactalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12) solutions mixed with and without the cosolvent at 30 °C. These measurements were used to calculate few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, relaxation time, relative association constant, the partial apparent specific volume and the partial apparent specific adiabatic compressibility for the said systems. The obtained results revealed a strong comparison between the effects of acidic and alkaline pH values on protein denaturation, i.e., the acidic pH are instantaneous and are of less magnitude whereas alkaline pH are slower but sharper. Further the present study supports the fact that the presence of glucose stabilizes α-lactalbumin against denaturation due to pH variation, which may be due to the strengthening of non-covalent interactions and the steric exclusion effect.     

Interaction of α2-macroglobulin with L-asparaginase

Summary Obvious protection of the catalytic activity of Esch. coli L-asparaginase by ₂-macroglobulin (₂M) was observed under conditions otherwise propitious to the dissociation of the tetrameric molecule into inactive subunits, i.e. very diluted enzyme solutions or the presence of either SDS or urea. The degree of protection depended on enzyme and ₂M concentrations respectively, and on the preincubation time of the ₂M-enzyme mixture prior to substrate addition. The formation of a catalytically active complex between ₂M and L-asparaginase was confirmed by gel filtration on a Sephadex-G column and by polyacrylamide gel electrophoresis. The fact that the migration distance of the active complex corresponded to the migration of ₂M and the absence in that case of a migration band corresponding to the intact molecule suggest that complexing of the enzyme with ₂M prevented its dissociation into subunits and thus its inactivation. Addition of ₂M to the already dissociated enzyme molecule did not restore its catalytic activity.Alpha₂-macroglobulin was shown to have an inhibiting effect on the proteolytic activity of almost all proteases and no effect on their esterolytic activity. Furthermore, it prevents the inhibition of esterolytic activity by some natural compounds^1–5. The effect of ₂M on other types of catalytic activity has not been investigated enough to afford a generalization of the possible role of this macroglobulin in the control of enzyme activity in the body.This paper reports the results of an in vitro study of the effect of ₂M on the catalytic activity of an important amidase, i.e. L-asparaginase (L-asparagine amidohydrolase 3.5.1.1), which in recent years has been used in the treatment of acute lymphocytic leukemia in children^6,7.Abbreviations ₂M ₂-macroglobulin - E enzyme - SDS sodium dodecylsulfate Part of the results were reported at the 10th International Congress of Biochemistry, Hamburg 1976, Abst. p. 377.     

Therapeutic Effect of α-Emitting 224Ra-Labeled Calcium Carbonate Microparticles in Mice with Intraperitoneal Ovarian Cancer

BACKGROUND: Ovarian cancer patients with chemotherapy-resistant residual microscopic disease in the peritoneal cavity have a considerable need for new treatment options. Alpha-emitting radionuclides injected intraperitoneally may be an attractive therapeutic option in this situation as they are highly cytotoxic, while their short range in tissues can spare surrounding radiosensitive organs in the abdomen. Herein we evaluate the therapeutic efficacy of a novel α-emitting compound specifically designed for intracavitary radiation therapy. METHODS: The α-emitter ²²⁴Ra was absorbed on calcium carbonate microparticles. Immunodeficient, athymic nude mice with human ovarian cancer cells growing intraperitoneally were treated with different activity levels of ²²⁴Ra-microparticles. Tumor growth, survival, and tolerance of the treatment were assessed. Two tumor models based on the cell lines, ES-2 and SKOV3-luc, with different growth patterns were studied. RESULTS: In both models, intraperitoneal treatment with ²²⁴Ra-microparticles gave significant antitumor effect with either considerably reduced tumor volume or a survival benefit. An advantageous discovery was that only a few kilobecquerels per mouse were needed to yield therapeutic effects. The treatment was well tolerated up to a dose of 1000 kBq/kg with no signs of acute or subacute toxicity observed. CONCLUSIONS: Intraperitoneal α-therapy with ²²⁴Ra-microparticles demonstrated a significant potential for treatment of peritoneal micrometastases in ovarian carcinoma.     

Effect of Trehalose on Oligomeric State and Anti-Aggregation Activity of αB-Crystallin

Biochemistry (Moscow) - αB-Crystallin (αB-Cr), one of the main crystalline lens proteins, along with other crystallins maintains lens transparency suppressing protein aggregation and thus...     

Effect of phenobarbital on the oligosaccharide structure of rat α-acid glycoprotein

We have recently shown that the administration of phenobarbital to rats leads t an increased serum α₁-acid glycoprotein content with alterations in the relative proportion of the sugar moiety. Therefore, α₁-acid glycoprotein was purified from normal (α₁-acid glycoprotein_N) and phenobarbital-treated rats (α₁-acid glycoprotein_PB). Glycans were separated by AX-10 chromatography and analysed by gas chromatography. It appears that, compared to α₁-acid glycoprotein_N, α₁-acid glycoprotein_PB had a higher carbohydrate content (31.7% compared to 26%) and a non-negligible amount of neutral oligosaccharide (12.2% compared to 1.3%). No tetrasialyl oligosaccharides in α₁-acid glycoprotein_PB were detected, whereas their relative proportion in α₁-acid glycoprotein_N was 27%.     

Effect of Trifluoroethanol on the Structural and Functional Properties of α-Crystallin

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.     

Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with α-synuclein at the single molecule level

Background

Abnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain.

Results

Cohorts of mice were pulse labeled with ¹³?C₆-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid ?? (A??). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and A?? clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of A?? deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of A??, suggesting that ABCA1 does not regulate A?? metabolism in the brain.

Conclusions

Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that this technique will be applicable to the study of protein dynamics in the pathogenesis of several neurodegenerative diseases, and could aid in the evaluation of novel therapeutic agents.     

Effect of Anoxia on α-Amylase Induction in Maize Caryopsis

α -amylase as well as other enzymes involved in starch degradation under anoxia. Carbohydrates resulting from starch breakdown allow maize caryopses to avoid sugar starvation. Most interestingly, this correlates well with the ability of maize caryopses to sustain relatively prolonged anaerobiosis, in agreement with the hypothesis linking carbohydrate availability to anoxia tolerance. Received 25 September 1999/ Accepted in revised form 24 February 2000     

Effect of additives on the thermostability ofBacillus stearothermophilus α-amylase

The effect of additives on the thermostability ofBacillus stearothermophilus -amylase was determined. Polyols, dimethyl formamide, and dimethyl sulfoxide all increased the half life of the enzyme approximately 2-fold when tested at a 10% (w/v) addition. These results suggest that the enzyme's structure is stabilized against thermal denaturation through ionic interactions. Addition of dextran or polyvinyl alcohol (hydrophilic polymers which increase the viscosity of the solution) had a slight positive effect on enzyme stability while addition of polyethylene glycol or polyvinylpyrrolidone (hydrophobic polymers which increase the viscosity of the solution) resulted in a 2-fold decrease in enzyme half life.     

Effect of Oxidation of αA- and αB-Crystallins on their Structure, Oligomerization and Chaperone Function

The purpose of this study was to investigate the effect of metal-catalyzed oxidation by H₂O₂ on the structure, oligomerization, and chaperone function of αA- and αB-crystallins. Recombinant αA-and αB-crystallins were prepared by expressing them in E. coli and purifying by size-exclusion chromatography. They were incubated with 1.5 mM H₂O₂ and 0.1 mM FeCl₃ at 37 ^∘C for 24 hrs and the reaction was stopped by adding catalase. Structural changes due to oxidation were ascertained by circular dichroism (CD) measurements and chaperone activity was assayed with alcohol dehydrogenase (ADH) and insulin as target proteins. The oligomeric nature of the oxidized proteins was assessed by molecular sieve HPLC. The secondary structure of the oxidized αA- and αB-crystallins has been substantially altered due to significant increase in random coils, in addition to decrease in β-sheet or α-helix contents. The tertiary structure also showed significant changes indicative of different mode of folding of the secondary structural elements. Chaperone function was significantly compromised as supported by nearly 50% loss in chaperone activity. Oxidation also resulted in the formation of higher molecular weight (HMW) proteins as well as lower molecular weight (LMW) proteins. Thus, oxidation leads to disintegration of the oligomeric structure of αA- and αB-crystallins. Chaperone activity of the HMW fraction is normal whereas the LMW fraction lacks any chaperone activity. So, it appears that the formation of the LMW proteins is the primary cause of the chaperone activity loss due to oxidation.