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PubMedDate not supplied

Analysis of the thermal and pH stability of human respiratory syncytial virus.

Respiratory syncytial virus (RSV) was studied as a function of pH (3-8) and temperature (10-85 degrees C) by fluorescence, circular dichroism, and high-resolution second-derivative absorbance spectroscopies, as well as dynamic light scattering and optical density as a measurement of viral aggregation. The results indicate that the secondary, tertiary, and quaternary structures of RSV are both pH and temperature labile. Derivative ultraviolet absorbance and fluorescence spectroscopy (intrinsic and extrinsic) analyses suggest that the stability of tertiary structure of RSV proteins is maximized near neutral pH. In agreement with these results, the secondary structure of RSV polypeptides seems to be more stable at pH 7-8, as evaluated by circular dichroism spectroscopy. The integrity of the viral particles studied by turbidity and dynamic light scattering also revealed that RSV is more thermally stable near neutral pH and particularly prone to aggregation below pH 6. By combination of the spectroscopic data employing a multidimensional eigenvector phase space approach, an empirical phase diagram for RSV was constructed. The pharmaceutical utility of this approach and the optimal formulation conditions are discussed.

Circular Dichroism↗
PubMed1996

STAB-SD: a Shine-Dalgarno sequence in the 5' untranslated region is a determinant of mRNA stability.

Transcription of the Bacillus thuringiensis cryIIIA toxin gene is initiated at nucleotide position -558 (relative to the cryIIIA start codon). However, the major cryIIIA transcript is an mRNA with its 5' end at nucleotide position -129. Fusions to the lacZ reporter gene revealed that the cryIIIA 5' untranslated region downstream of nucleotide position -129 acts as a 5' mRNA stabilizer. Deletion and mutation analysis suggest that the determinant of stability is a Shine-Dalgarno (SD) sequence close to the 5' end of the stable transcript. This SD sequence, designated STAB-SD, does not direct translation initiation. However, mutations predicted to weaken the putative interaction between the SD sequence and the 3' end of 16S rRNA lead to reduced mRNA stability. The binding of a 30S subunit to STAB-SD may be required for stabilization of cryIIIA mRNA as a transcript with a 5' end at nucleotide position -129. Similar STAB-SD sequences are found in the 5' untranslated regions of other cryIII genes, and in the 5' untranslated regions of genes from Gram-positive bacteria other than B. thuringiensis and may therefore be a widespread determinant of mRNA stability.

Bacillus thuringiensis↗
PubMed1996

Characterization of crystalline formate dehydrogenase H from Escherichia coli. Stabilization, EPR spectroscopy, and preliminary crystallographic analysis.

The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenum-coordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 microg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations (>/=3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 A resolution and belong to a space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146.1 A and c = 82.7 A. There is one monomer of FDH per crystallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation. A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes. These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme.

Crystallography, X-Ray↗
PubMed2000

Neutron activation of NDDP, a liposomal platinum antitumor agent.

UNLABELLED: Cis-bis-neodecanoato-trans-R,R-1,2-diamminocyclohexane platinum (II) [NDDP] is a liposome-entrapped platinum compound currently, in phase II clinical trials, that has been shown to undergo intraliposomal activation. The objective of this study was to determine the feasibility of activating NDDP and using the induced radioactivity to monitor NDDP distribution and penetration. METHODS: Neutron activation analysis (NAA) was done on NDDP using the nuclear reactor at Texas A & M University, College Station, Texas. After a 3-hour irradiation, the NDDP samples were analyzed using an HPGE (high purity germanium) detector to determine the activation of the radioisotopic platinum. This was followed by HPLC-UV analysis to determine the stability of NDDP after exposure to the reactor's core. RESULTS: Platinum radioisotopes were produced along with potassium-40 and sodium-24. Irradiation did not result in any significant degradation of NDDP. CONCLUSIONS: (1) Irradiating fully synthesized NDDP is feasible for diagnostic use if a purification step is taken after the irradiation, and (2) radiation exposure is lessened by irradiating NDDP after synthesis rather than starting with high-specific-activity isotopes.

Antineoplastic Agents↗
PubMed1978

A comparative study of the activity of cefamandole and other cephalosporins and analysis of the beta-lactamase stability and synergy of cefamandole with aminoglycosides.

The antibacterial activity of cefamandole against 445 clinical isolates was investigated and compared with the activity of other known cephalosporins (cephalothin, cephaloridine, cephalexin, and cefazolin) and of two penicillins (ampicillin and carbenicillin). Cefamandole was the most active antibiotic against isolates of Citrobacter, Enterobacter, and Shigella, and its activity against Staphylococcus aureus, Bacteroides, and some members of the Enterobacteriaceae was comparable to that of the other antibiotics tested. The stability of cefamandole with respect to beta-lactamase was investigated and compared with that of cephalothin, cefazolin, and cephalexin. Cefamandole was stable with respect to the beta-lactamases of Enterobacter and some other members of the Enterobacteriaceae. No significant correlation was found between the antibacterial activity and the beta-lactamase stability of cefamandole, except with Enterobacter. The synergistic activity of cefamandole combined with gentamicin or amikacin was demonstrated by killing-curve techniques, isobolograms, and susceptibility data. Although 12%--46% of the isolates were synergistically inhibited by either combination, antagonism was not observed. No correlation between the hydrolysis of cefamandole by beta-lactamase and the synergistic activity of cefamandole combined with amikacin was demonstrated.

Acinetobacter↗
PubMed1993

Analysis of effects of tRNA:message stability on frameshift frequency at the Escherichia coli RF2 programmed frameshift site.

The codon that is in-frame prior to +1 frameshifting at the E.coli prfB (RF2 gene) frameshift site is randomized to create thirty-two variants. These alleles vary 1000-fold in frameshift-dependent expression in fusions to lacZ. Frameshifting is more frequent at sites where the in-frame codon ends in uridine, as if third position wobble pairs to message uridine facilitate slippage into the +1 frame. Consistent with other studies of programmed frameshift sites, efficient frameshifting depends on stable message:tRNA base pairs after rephasing. For complexes with mispairs, frameshift frequency depends on the nature, number, and position of mispairs. Central purine:purine mispairs are especially inhibitory. Relative stabilities of +1 rephased complexes are estimated from published data on the stabilities of tRNA:tRNA complexes. Stability correlates with frameshifting over its entire range, which suggests that stability is an important determinant of the probability of translation of the rephased complex.

Alleles↗
PubMed1975

Analysis of rifampin disk diffusion and stability in 7H10 agar.

Rifampin was incorporated into Middlebrook 7H10 medium either by adding an aliquot of the antibiotic into melted agar (final concentration 1.0 and 3.0 mug/ml) or by submerging a 5 or 15 mug of rifampin paper disk into 5 ml of melted agar contained in one quadrant of a Felson "X" plate. At intervals, plugs of agar were removed from the stored plates and assayed. Plates stored at 5 C for 28 days showed no loss of potency; at 37 C, the half-life of rifampin was 9 days. Stability of rifampin at these concentrations in 7H10 medium was independent of the method used for incorporation. Using the disk method, uniform rifampin concentrations of 0.75 mug/ml on day 5 for the 5-mug disk and 2.7 mug/ml on day 6 for the 15-mug disk were observed. Results indicated that the rifampin concentrations within the agar dilution and disk diffusion plates were equivalent at these times.

Agar↗
PubMed2006

Anterior cervical fixation: analysis of load-sharing and stability with use of static and dynamic plates.

BACKGROUND: Anterior plates provide stability following decompression and fusion of the cervical spine. Various plate designs have emerged, and they include static plates with fixed-angle screws, rotationally dynamic plates that allow the screws to toggle in the plate, and translationally dynamic plates that allow the screws to both toggle and translate vertically. The goal of this study was to document the effects of plate design following a single-level corpectomy and placement of a full-length strut graft and the effects following 10% subsidence of the graft. METHODS: A total of twenty-one cadaveric cervical spines (C2-T1) were randomized into three treatment groups and were tested for initial range of motion. A C5 corpectomy was performed, reconstruction was done with a full-length interbody spacer containing a load-cell, and an anterior cervical plate was applied. Load-sharing data were recorded with incremental axial loads. The range of motion was measured with +/- 2.5 Nm of torque in flexion-extension, lateral bending, and axial rotation. Then, the total length of the interbody spacer was reduced by 10% to simulate subsidence, and load-sharing and the range of motion were retested. RESULTS: With the full-length interbody spacer, there were no significant differences in the abilities of the constructs to share load or limit motion. Following shortening of the interbody spacer, the static plate construct lost nearly 70% of its load-sharing capability, while neither of the dynamic plate constructs lost load-sharing capabilities. Also, the static plate construct allowed significantly more motion in flexion-extension following simulated subsidence than did either of the dynamic plate constructs (p < 0.05). CONCLUSIONS: Although all of the tested anterior cervical plating systems provide similar load-sharing and stiffness following initial placement of the interbody spacer, the static plate system lost its ability to share load and limit motion following simulated subsidence of the interbody spacer. Both dynamic plate systems maintained load-sharing and stiffness despite simulated subsidence. CLINICAL RELEVANCE: This study provides an improved understanding of the immediate performance of anterior cervical fusion surgery with plate fixation.

Aged↗
PubMed1998

[Theoretical analysis of factors affecting heat exchange stability of human body with environment].

Life could not be normal without the heat produced by metabolism of human body being transmitted into environment. This paper discussed the ways of heat exchange of human body with the environment, and analyzed their effects on the stability of heat exchange theoretically. In addition, factors that affects the stability of heat exchange were studied. The results indicate that the environmental temperature is the most important factor.

Body Temperature Regulation↗
PubMed2003

Proteomic analysis of intestinal epithelial cells expressing stabilized beta-catenin.

Aberrant accumulation of beta-catenin protein because of mutation of either the beta-catenin or adenomatous polyposis coli gene plays an essential role in the development of colorectal carcinoma. We established previously a stable clone of the rat small intestinal epithelial cell line IEC6, which is capable of inducing stabilized beta-catenin protein lacking NH(2)-terminal glycogen synthase kinase-3beta phosphorylation site under a strict control of the tetracycline-regulatory system. This clone, IEC6-TetOFF-beta-catenin DeltaN89, shows in vitro polypoid growth on the removal of doxycycline and seems to be an appropriate model for analyzing the molecular mechanisms of early intestinal carcinogenesis. Of >2000 protein spots displayed by newly developed two-dimensional difference gel electrophoresis, 22 were found to be up- or down-regulated on the induction of stabilized beta-catenin. The majority of these proteins fell into two categories: (a) redox-status regulatory proteins and (b) cytoskeleton-associated proteins. Representatively, a key redox-status regulatory protein, manganese superoxide dismutase, up-regulated in IEC6 cells expressing stabilized beta-catenin protein, was overexpressed in adenoma and adenocarcinoma cells of familial adenomatous polyposis patients in parallel with the accumulation of beta-catenin. These results suggest that aberrant accumulation of beta-catenin might contribute to colorectal carcinogenesis by affecting redox status in the mitochondria of intestinal epithelial cells.

Adenocarcinoma↗
PubMedDate not supplied

Heart preservation: analysis of cardioprotective infusate characteristics. Membrane stabilization, calcium antagonism, and protease inhibition on myocardial viability: a biochemical, ultrastructural, functional study.

Thirty-three canine hearts were isolated after initial cardioplegia and preserved for 6 hours in 4 degrees C saline solution with intermittent infusion of cardioprotective solution every hour. Reperfusion was observed for 2 hours under normothermic cross-circulation. Hearts were divided into five groups depending on the agent(s) added to the K(+)-Mg2+ cardioplegic solution (K(+)-Mg(2+)-CP) infused. Control hearts (n = 6) received K(+)-Mg(2+)-CP solution alone; group I (n = 7) received lidocaine, 200 mg/L, added to the K(+)-Mg(2+)-CP solution; group II (n = 7) received betamethasone (250 mg/L) added to the formula for group I; group III (n = 6) received diltiazem (200 micrograms/L) added to the formula for group II; group IV (n = 7) received aprotinin (150 KIU/L) added to the formula of group III. Coronary sinus MB fraction of creatine kinase level was significantly decreased at 60 and 120 minutes of reperfusion in group II, as was mitochondrial aspartate aminotransferase level at 2 hours of reperfusion. Lysosomal enzyme release decreased in group IV. Myocardial adenosine triphosphate levels and total adenine nucleotides showed no significant difference among the groups at the end of reperfusion; however, myocardial adenosine diphosphate and adenosine monophosphate levels during reperfusion increased significantly in group I, and myocardial adenosine diphosphate and adenosine monophosphate levels at the end of reperfusion in groups I and IV were significantly higher than those of the control. Calcium overload, which was lowest in group II, was not completely prevented during reperfusion in any group. Left ventricular end-systolic pressure volume relationship in group II showed the "best" functional recovery. In addition, the ultrastructure of the left ventricular myocardium was well preserved in all groups. These results suggest that membrane stabilization with lidocaine and betamethasone affords beneficial effects on myocardial biochemical and functional viability. Diltiazem appears to be less effective in preventing calcium overload during ischemia-reperfusion, and protease inhibition with aprotinin (150 KIU/ml) seems to be highly effective in suppressing lysosomal enzyme activation-release and maintaining myocardial adenosine diphosphate and adenosine monophosphate levels.

Adenine Nucleotides↗
PubMed1991

[Analysis of factors affecting the thermal stability of alpha 2-macroglobulin in solution].

The factors affecting the thermal stability of alpha 2-macroglobulin (alpha 2M) in aqueous solution have been analyzed with an experiment of orthogonal design. The optimum conditions for heat treatment of alpha 2M have been established. Under these conditions, the trypsin-binding activity of alpha 2M can be fully retained after heat treatment at 60 degrees C for 10 hours.

Drug Stability↗
PubMed1984

Improved recovery and stability of ethanol in automated headspace analysis.

Solutions are reported here for two problems in automated headspace gas chromatographic analysis for ethanol. The first is the catalytic oxidation of ethanol by oxyhemoglobin, a process that is limited only by the amount of oxygen in the sealed vessel. This reaction is prevented by the addition of sodium dithionite. The other problem, the lack of a suitable salting-out agent, led to the selection of ammonium sulfate, which improved recovery of ethanol as well as enhancing method precision. A procedure incorporating these improvements is described.

Chromatography, Gas↗