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PubMed1986

[Comparative study of stability following the nailing of fractures of the femur shaft. An experimental study with cadaver bones].

Intramedullary osteosynthesis is preferred for shaft fractures of the long bones of the lower extremities because it generally results in early weight-bearing stability, allowing full function of the leg and rapid rehabilitation. Only transverse and short oblique fractures near the middle of the medullary cavity can be sufficiently stabilised using intramedullary nails alone. Additional stabilising aids such as cerclages or interlocking nails must be used for all other types of shaft fracture. There are no previous studies of stability that make a comparison between conventional intramedullary nails and interlocking bolts with or without cerclages. This paper set out to clarify therapeutically relevant questions in the light of a comparative study of stability. To this end a comparative experimental investigation was carried out using femora from human cadavers to determine whether or not either method of intramedullary osteosynthesis attained the stability of the intact bone. The study also set out to establish the relevance of the type of fracture, localisation and mode of fitting to the resulting stability of the osteosynthesis. A comparison was also made of the stability in the bone-implant complex of two different commercially available interlocking nails. Finally, the question was raised as to the clinical conclusions that can be drawn from the present investigation. The bone-implant complex represented by a fracture of the femur secured by an interlocking nail is a complex mechanical system. An optical measuring arrangement was developed so as to ensure that this system was not subject to any interference resulting from the method of measurement. This consisted of a laser light source which projected a beam of light parallel to the axis of the femur shaft across the fracture onto a mirror system attached to the bone. The rays of light reflected from the mirrors were recorded as dots of light on measuring screens. A load of up to 1000 N was gradually applied along the bearing axis, and the resulting changes in the position of the parts of the osteosynthesised fracture relative to one another indicated by the deviation of the beams of light. The axial tilt and rotation of the pieces of bone could be determined from the coordinates of the dots of light. The stability of nailed transverse femoral fractures (n = 6), short oblique fractures (n = 6), long oblique fractures (n = 6) and comminuted fractures (n = 6) was determined and compared with the deformation of intact femora. The stability of all types of osteosynthesis was several times less than that of the intact bones.(ABSTRACT TRUNCATED AT 400 WORDS)

Aged↗
PubMed1986

Failure of stabilization of the spine with methylmethacrylate. A retrospective analysis of twenty-four cases.

Twenty-four patients who had a major complication after attempted stabilization of the spine with methylmethacrylate were referred for treatment. The initial instability that necessitated stabilization was caused by a traumatic condition in fifteen of these patients and by a metastatic tumor in nine. The average length of time before failure of fixation was 208.3 days for the patients who had a traumatic condition and 193.7 days for those who had a neoplasm. In eleven patients a progressive neural deficit developed postoperatively, and in six others the recovery of neural function was possibly hindered by the cement. A deep wound infection developed in six patients, and in five of them treatment by removal of the methylmethacrylate and metal, followed by a prolonged period of cervical traction, was required. Loosening and failure of fixation was the most common complication--it occurred in twelve of the fifteen patients who had a traumatic lesion and in eight of the nine who had a neoplasm. Salvage operations that included removal of the cement and conventional bone-grafting procedures were performed in eleven of the twelve patients who had loosening associated with a traumatic lesion and in six of the eight who had loosening and a tumor. Stability was restored in every patient. Improved long-term results can be achieved by using grafts of iliac bone and triple-wire stabilization methods (a midline wiring between the spinous processes and two iliac-crest grafts, one on each side, wired to the posterior elements) instead of methacrylate in the primary treatment of traumatic injuries. The treatment of choice for instability caused by neoplastic destruction of two or more vertebral bodies includes a construct of methylmethacrylate anteriorly. However, if cement is used, early augmentation with posterior fusion of the spine and stabilization should be considered. As a rule, combined anterior and posterior stabilization is recommended for the reconstruction of a spine that is unstable due to neoplastic destruction. In general, whenever methylmethacrylate is used for spinal stabilization, it should be augmented with grafts of iliac bone to provide long-term stability.

Adult↗
PubMed1983

An approach to the assessment of membrane stability of cultured cells.

A simple method for assessing the combined stability of the plasma and lysosomal membranes of cultured cells is described. Monolayers of normal, human glial cells were incubated in situ in an isotonic, buffered sucrose solution (pH 5.0) containing the acid phosphatase (AP) enzyme substrate p-nitrophenyl phosphate (PNPP). The rate of appearance, in the solution, of the reaction product p-nitrophenol (PNP) was measured spectrophotometrically, curves then plotted, and fitted by computer. "Lag time" (LT) was calculated, and an index of membrane lability constructed, termed "fragility index" (FI). Transmission electron microscopy (TEM), "vital" staining of the cells with fluorescein diacetate (FDA) and Evans Blue (EB), and use of a Gomori-type cytochemical technique, indicate that the data reflects the combined stability of lysosomal and plasma membranes. The latter playing the more critical role. Cell cultures pre-incubated with various membrane labilizing or stabilizing agents were compared. Control, 0.3 M sucrose, and normal saline treated cells demonstrated similar stability. Distilled water decreased AP latency (increased fragility), and the magnitude of this effect was time dependent. Cells fixed in glutaraldehyde (GA) retained much of their osmotic reactivity, as confirmed by distilled water treatment. Oxygen derived free radicals caused pronounced fragility, while dexamethasone, a membrane stabilizing agent, decreased membrane fragility. Triton X-100 abolished latency completely, and total AP activity was very rapidly recovered outside the cells in the surrounding incubation medium. These results suggest this technique yields a measure of membrane stability which is sensitive enough to differentiate between known stabilizers and labilizers of membranes. Hence, this may prove an easy and useful aid for the assessment of how various substances and environments modulate the lysosomal and plasma membrane stability of cultured cells.

Acid Phosphatase↗
PubMed1998

Protein stability in the amorphous carbohydrate matrix: relevance to anhydrobiosis.

The formation of intracellular glass is proposed to be relevant to protein stabilization and survival of anhydrobiotic organisms in the dry state. The stability of proteins in the amorphous carbohydrate matrix and its relevance to seed survival have been investigated in the present study. Glucose-6-phosphate dehydrogenase (G6PDH) was preserved in the amorphous glucose/sucrose (1:10, w/w) matrix by freeze-drying. The stability of freeze-dried G6PDH was examined at temperatures above and below the glass transition temperature (Tg). The rate of G6PDH inactivation in the amorphous carbohydrate matrix deviated significantly from the Arrhenius kinetics, and conformed to the Williams-Landel-Ferry (WLF) relationship. The temperature dependence of G6PDH inactivation in two sets of samples with different Tg values was compared. Identical temperature dependence of G6PDH inactivation was observed after temperature normalization by (T-Tg). Seed survival of Vigna radiata Wilczek (mung bean) showed a similar WLF kinetics at storage temperatures T > or = Tg. In situ protein stability in mung bean embryonic axes was studied using differential scanning calorimetry (DSC). Thermal stability of seed proteins exhibited a strong dependence on the Tg of intracellular glass. These results indicate an important role of the glassy state in protein stabilization. Our data suggest an association between protein stability in intracellular glass and seed survival during storage.

Calorimetry, Differential Scanning↗
PubMed2002

Long term stability of rh-Cu/Zn-superoxide dismutase (SOD)-liposomes prepared by the cross-flow injection technique following International Conference on Harmonisation (ICH)-guidelines.

The current market position of liposomes as drug carriers is still being discussed with regard to large scale production, product characterisation and the stability of the dispersions. In this study, long term stability of liposomal suspensions with encapsulated rh-Cu/Zn-superoxide dismutase was tested according to the International Conference on Harmonisation (ICH) recommendations. The guidelines of the ICH provide general requirements for stability testing for registration and export in particular. The Institute of Applied Microbiology has examined a process to produce large amounts of pharmaceutical-grade liposomes for the treatment of inflammatory diseases by topical application. For the evaluation of its long-term storage stability, liposomal stability and protein stability were tested under appropriate conditions. Therefore, size alterations of the vesicles, protein release and protein activity were evaluated. During the observation period, neither significant alterations of the liposomes nor any protein degradation could be detected. In the light of these findings our liposomal formulations seem to provide chemical, physical and biological stability according to the definitions of the ICH. Appropriate lipid compounds and environmental factors, in combination with an optimised process and adequate storage conditions, facilitate the production of liposome dispersions suitable as drug carriers on the market.

Drug Stability↗
PubMed1999

The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease.

The effects of the peptide inhibitor acetyl pepstatin on the structural stability of the HIV-1 protease have been measured by high sensitivity calorimetric techniques. At 25 degrees C and pH 3.6, acetyl pepstatin binds to HIV-1 protease with an affinity of 1.6 x 10(7 )M-1 and an enthalpy of 7.3 +/- 0.5 kcal/mol, indicating that binding is not favored enthalpically and that the favorable Gibbs energy originates from a large positive entropy. Since the binding of acetyl pepstatin is associated with a negative change in heat capacity (-450 cal/K*mol) the association reaction becomes enthalpically favored at temperatures higher than 40 degrees C. The presence of the inhibitor stabilizes the dimeric structure of the protease in a fashion that can be quantitatively described by a set of thermodynamic linkage equations. The combination of titration and differential scanning calorimetry provides an accurate way of determining binding constants for high affinity inhibitors that cannot be determined by titration calorimetry alone. A structure-based thermodynamic analysis of the binding process indicates that the stabilization effect is not distributed uniformly throughout the protease molecule. The binding of the inhibitor selectively stabilizes those conformational states in which the binding site is formed, triggering a redistribution of the state probabilities in the ensemble of conformations populated under native conditions. As a result, the stability constants for individual residues do not exhibit the same change in magnitude upon inhibitor binding. Residues in certain areas of the protein are affected significantly whereas residues in other areas are not affected at all. In particular, inhibitor binding has a significant effect on those regions that define the binding site, especially the flap region which becomes structurally stable as a result of the additional binding free energy. The induced stabilization propagates to regions not in direct contact with the inhibitor, particularly to the strand between residues Pro9 and Ala22 and the helix between Arg87 and Gly94. On the other hand, the stability of the strand between Asp60 and Leu76 is not significantly affected by inhibitor binding. The structural distribution of binding effects define cooperative pathways within the protease molecule. Proteins 1999;36:147-156.

Algorithms↗
PubMed2007

Effect of siRNA nuclease stability on the in vitro and in vivo kinetics of siRNA-mediated gene silencing.

Small interfering RNA (siRNA) molecules achieve sequence-specific gene silencing through the RNA interference (RNAi) mechanism. Here, live-cell and live-animal bioluminescent imaging (BLI) is used to directly compare luciferase knockdown by unmodified and nuclease-stabilized siRNAs in rapidly (HeLa) and slowly (CCD-1074Sk) dividing cells to reveal the impact of cell division and siRNA nuclease stability on the kinetics of siRNA-mediated gene silencing. Luciferase knockdown using unmodified siRNAs lasts approximately 1 week in HeLa cells and up to 1 month in CCD-1074Sk cells. There is a slight increase in the duration of luciferase knockdown by nuclease-stabilized siRNAs relative to unmodified siRNAs after cationic lipid transfection, but this difference is not observed after electroporation. In BALB/cJ mice, a fourfold increase in maximum luciferase knockdown is observed after hydrodynamic injection (HDI) of nuclease-stabilized siRNAs relative to unmodified siRNAs, yet the overall kinetics of the recovery after knockdown are nearly identical. By using a mathematical model of siRNA-mediated gene silencing, the trends observed in the experimental data can be duplicated by changing model parameters that affect the stability of the siRNAs before they reach the cytosolic compartment. Based on these findings, we hypothesize that the stabilization advantages of nuclease-stabilized siRNAs originate primarily from effects prior to and during internalization before the siRNAs can interact with the intracellular RNAi machinery.

Animals↗
PubMed2004

mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure.

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.

Antigens, Surface↗
PubMed1997

Regulation of CCR2 chemokine receptor mRNA stability.

During inflammatory and immunological responses, leukocytes respond to external stimuli by altering the stability of cytokine and cytokine receptor messages. Change in message stability is an effective mechanism for rapidly regulating steady state levels of mRNA. Cytokine messages containing A-U-rich elements located in the 3' untranslated region (ARE) are the best studied examples of this process. AREs have been shown to act as targeting motifs for degradation of cytokine and transcription factor messages. We have recently observed that the interleukin-8 (IL-8) receptor messages, IL-8RA and B (CXCR1 and CXCR2), also undergo changes in stability in response to the inflammatory stimulator lipopolysaccharide (LPS). To determine whether regulation of message stability is a common mechanism for modulation of chemokine receptor mRNA we explored whether the stability of the CC chemokine receptor message for CCR2 (monocyte chemotactic protein-1 receptor) is also regulated by LPS. We found that LPS induces a rapid loss of steady state levels of CCR2 message through message degradation. Furthermore, LPS stimulated the decay of Poly(A) CCR2 mRNA faster than total CCR2 RNA, indicating that deadenylation is the first step in LPS-induced CCR2 RNA degradation. We conclude from these experiments that LPS stimulates the rapid degradation of CCR2 messages through a two-step process, deadenylation followed by degradation of the message body. In contrast to the results obtained for CCR2 mRNA, macrophage inflammatory protein-1alpha messages, which contain an ARE motif, were stabilized by LPS stimulation, indicating that chemokine and chemokine receptor mRNA stability are regulated by different and opposing mechanisms.

Adenine↗
PubMed2002

Differential scanning calorimetric studies of the thermal stability of plasmid DNA complexed with cationic lipids and polymers.

The thermal stabilities of supercoiled (SC) and linear/open circular (LIN/OC) forms of plasmid DNA when complexed with cationic lipids or cationic polymers used for cellular transfection were assessed using differential scanning calorimetry. Differences in the stability of SC DNA produced by the cationic lipids DOTAP (1,2-dioleoyltrimethyl ammoniumpropane chloride), DSTAP (1,2-distearyltrimethyl ammoniumpropane chloride), and DDAB (dimethyldioctadecylammonium bromide) upon complexation suggest possible effects of headgroup structure on the stability of SC DNA and minimal effects of lipid acyl chain saturation/unsaturation. Complexation of DNA with the cationic polymers polyethylenimine (PEI) or poly-L-lysine (PLL) (but not poly-L-arginine) resulted in a decreased stability of SC DNA when the DNA was in charge excess, although all polymers stabilized SC DNA when the polymer was in charge excess. The effects of these cationic polymers on the stability of SC DNA can be explained by changes produced in the tertiary structure of SC DNA upon binding and may reflect the importance of the topological constraint of supercoiling upon the stability of the resulting complexes.

Calorimetry, Differential Scanning↗
PubMed2004

Influenza vaccine powder formulation development: spray-freeze-drying and stability evaluation.

The purpose of this study was to develop a spray-freeze-drying (SFD) process for preparing an influenza vaccine dry powder formulation suitable for epidermal powder immunization. After preformulation of two types of flu vaccines, their dry-powder formulations were prepared by SFD. Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Chemical and biochemical stability of vaccine antigens was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single radial immunodiffusion assay, and in vivo immunogenicity in a mouse model. We demonstrated that SFD could produce high-density particles-a critical parameter for effective skin penetration. From the stability perspective, the stress posed by SFD was mild because the antigen in the dry powder retained its stability, potency, and immunogenicity. Among several formulations screened, we noted that formulation composition has a significant role in the powder's long-term physical and biochemical stability. One formulation, in particular, containing sub-unit vaccine (45 microg of antigen in 1 mg of powder) with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability, including acceptable biochemical stability after being exposed to a highly humid environment. After all, we have not only demonstrated the suitability of SFD to prepare powders for epidermal powder immunization but also developed a systematic formulation development strategy that allowed the optimization of an influenza vaccine dry powder formulation. More important, this study led to the selection of a formulation system that had been successfully tested in a human clinical study.

Aerosols↗
PubMed2004

Retrospective statistical analysis of lyophilized protein formulations of progenipoietin using PLS: determination of the critical parameters for long-term storage stability.

Although certain criteria have become recognized as being essential for a stable lyophilized formulation, the relative importance of different stability criteria has not been demonstrated quantitatively. This study uses multivariate statistical methods to determine the relative importance of certain formulation variables that affect long-term storage stability of a therapeutic protein. Using the projection to latent structures (PLS) method, a retrospective analysis was conducted of 18 formulations of progenipoietin (ProGP), a potential protein therapeutic agent. The relative importance of composition, pH, maintenance of protein structure (as determined by infrared (IR) spectroscopy), and thermochemical properties of the glassy state (as measured by differential scanning calorimetry (DSC)) were evaluated. Various stability endpoints were assessed and validated models constructed for each using the PLS method. Retention of parent protein and the appearance of degradation products could be adequately modeled using PLS. The models demonstrate the importance of retention of native structure in the solid state and controlling the pH. The relative importance of T(g) in affecting storage stability was low, as all of the samples had T(g) values above the highest storage temperature (40 degrees C). However, other indicators of molecular mobility in the solid state, such as change in DeltaC(p) upon annealing, appear to be important, even for storage below T(g). For the first time, the relative importance of certain properties in controlling long-term storage stability could be assessed quantitatively. In general, the most important parameters appear to be pH and retention of native structure in the solid state. However, for some stability endpoints, the composition (concentration of protein or various excipients), as well as some DSC parameters, were found to be significant in predicting long-term stability.

Calorimetry, Differential Scanning↗
PubMed2006

Stability studies of testosterone and epitestosterone glucuronides in urine.

The stability of testosterone glucuronide (TG), epitestosterone glucuronide (EG) and the T/E ratio in urine has been studied. Samples were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Urine samples were submitted to a solid-liquid cleanup followed by extraction of unconjugated testosterone (T) and epitestosterone (E) with tert-butyl methyl ether (free fraction). The remaining aqueous phase was hydrolyzed with beta-glucuronidase and extracted at alkaline pH with n-pentane. Analytes were analyzed by GC/MS as their enol-trimethylsilyl (TMS) derivatives. The urine for stability testing was obtained from an excretion study after the administration of T to healthy volunteers. The homogeneity of the sample was verified before starting the stability study. The stability of TG and EG was evaluated at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 degrees C and -20 degrees C was determined at different time intervals for 22 months. For short-term stability testing, analyte concentration was evaluated in urine stored at 37 degrees C for 3 and 7 days. The effect of repeated freezing (at -20 degrees C) and thawing (at room temperature) was studied for up to three cycles. Data obtained in this work demonstrated the stability of TG, EG and the T/E ratio in sterilized urine samples stored at 4 and -20 degrees C for 22 months and after going through repeated freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 37 degrees C due to the partial cleavage of the glucuronide conjugates; however, the T/E ratio was not affected. These results show the feasibility of preparing reference materials containing TG and EG to be used for quality control purposes.

Doping in Sports↗
PubMed2004

Plasmid copy number and plasmid stability.

Many expression systems in research and industry use plasmids as vectors for the production of recombinant proteins or non-proteinous recombinant substances. Plasmids have an essential impact on productivity. Related factors are plasmid copy number, structural plasmid stability and segregational plasmid stability. Plasmid copy number determines the gene dosage accessible for expression and many plasmids lead generally to a high productivity. To analyze an expression system the quantification of plasmid copy number is very helpful. Therefore, different methods for the determination of plasmid copy number are described. Structural plasmid stability exists, when all generated plasmids have the correct base sequence. The analysis of structural instabilities is not trivial and some methods are reported. When all daughter cells get at least one plasmid during cell division, the culture is segregational stable. The development of plasmid free cells can lead to a significant loss in productivity. Different methods for lab scale and industrial scale help to avoid segregational instability. Since plasmids are used as pharmaceuticals, additional aspects of stability have to be taken into account. These include stability during downstream processing, stability after application and stability during storage and shipping.

Animals↗
PubMed2005

The effect of stabilizers and denaturants on the cold denaturation temperatures of proteins and implications for freeze-drying.

PURPOSE: The aim of the study is to investigate the effects of stabilizers and denaturants on the thermal and cold denaturation temperatures of selected proteins in systems of interest to freeze-drying. METHODS: Beta-lactoglobulin and phosphoglycerate kinase (PGK) were chosen as model proteins. Protein thermal and cold denaturation temperatures were determined by both conventional and modulated differential scanning calorimetry and verified by tryptophan emission spectroscopy in selected systems. RESULTS: The cold denaturation of beta-lactoglobulin was reversible, whereas the thermal denaturation was only reversible at high scanning rate (10 degrees C/min). The cold denaturation temperatures of beta-lactoglobulin decreased with an increase in protein concentration (self-stabilization). The cold denaturation temperature increased with increases in pH (from pH 2 to 7) with about 4.6 degrees C increase per unit pH change. All stabilizers studied (i.e., sucrose, trehalose and glycerol) increased the thermal denaturation temperature of the proteins studied and decreased the cold denaturation temperature. The effect of sucrose in decreasing the PGK cold denaturation temperature [40 degrees C per molar concentration increase (40 degrees C/M)] was of the same magnitude as for beta-lactoglobulin (36 degrees C/M). The effect of stabilizers on cold denaturation temperatures is much greater than the effect on thermal denaturation temperatures. With sucrose, the beta-lactoglobulin thermal denaturation temperature increases only about 5 degrees C from 0 to 2.7 M, whereas the decrease in cold denaturation temperature was more than 35 degrees C even at sucrose concentrations as low as 0.9 M. Denaturants (urea and guanidine hydrochloride) increased the cold denaturation temperatures of proteins and thereby destabilized protein; the magnitudes were 9 degrees C/M (urea on Tcd of beta-lactoglobulin) and 65 degrees C/M (guanidine hydrochloride on PGK) compared with literature data of 16 degrees C/M (guanidine hydrochloride on beta-lactoglobulin). The cold denaturation temperatures of beta-lactoglobulin and PGK extrapolated to zero concentration of denaturants were -14 and -26 degrees C, respectively. CONCLUSIONS: The protein cold denaturation temperature was pH-, protein concentration-, and additive-dependent. Stabilizers, such as sugars and/or polyols, can stabilize both protein thermal and cold denaturation, whereas the denaturants destabilize protein cold denaturation. The stabilization effect on protein cold denaturation is much larger than on thermal denaturation, a result of great importance in protein freeze-drying.

Calorimetry, Differential Scanning↗
PubMed1991

The role of internal packing interactions in determining the structure and stability of a protein.

Cassette mutagenesis has been used to investigate how internal packing interactions help to specify a protein's three-dimensional structure and stability. Three interacting residues in the hydrophobic core of the N-terminal domain of lambda repressor were randomized combinatorially. The randomization was restricted to the five amino acids Val, Leu, Ile, Met and Phe, thereby generating a sterically diverse set of core sequences composed solely of hydrophobic residues. We have isolated 78 of the 125 possible sequences generated by this randomization. Approximately 70% of the isolated sequences show some level of biological activity, and thus still carry sufficient information to encode the basic structure of lambda repressor. An assay based on the temperature dependence of activity in vivo has been used to estimate the relative activities and thermal stabilities of the set of mutants. In addition, nine mutants have been purified and their stabilities and DNA binding activities characterized in vitro. Of the 56 active sequences, only two, in addition to the wild-type, maintain the wild-type level of stability and activity. All three of these proteins satisfy stringent requirements for specifically shaped residues at each position. All of the remaining active sequences have reduced stabilities and/or reduced DNA binding affinities. These and previous results suggest that there are two levels of structural information encoded in core residues. At the first level, the basic structural information appears to reside largely in the hydrophobic character of these residues. The majority of sequences that simply maintain hydrophobicity at core positions are able to adopt the overall lambda repressor fold and maintain moderate stability. At the second, more detailed level, specific steric features of these residues and their packing interactions clearly act as important determinants of the protein's precise structure and stability. These results imply that many of the basic structural features of a protein could be predicted from relatively simple, degenerate sequence patterns.

DNA, Bacterial↗
PubMed1990

Comparison of the effect of various chemical stabilizers and lyophilization cycles on the thermostability of a Vero cell-adapted rinderpest vaccine.

The thermostability of a rinderpest vaccine produced on Vero cells was evaluated using a variety of chemical stabilizers and lyophilization protocols. Three stabilizer preparations and three lyophilization schedules were examined using accelerated stability testing at 37 degrees C. The vaccine preparation exhibiting the greatest stability at 37 degrees C was tested at three additional temperatures, 42, 45 and 56 degrees C, and an Arrhenius plot was constructed from the data. The stability of the reconstituted vaccine produced with the two most efficacious stabilizers was examined using three different diluent preparations. The stabilization method and high Vero cell virus batch titers resulted in a lyophilized vaccine which maintained the minimum required dose of log10 2.5 TCID50 tissue culture infectious dose for more than 20 weeks at 37 degrees C.

Analysis of Variance↗
PubMed2004

Molecular recognition and stability of 99mTc-UBI 29-41 based on experimental and semiempirical results.

99mTc-UBI 29-41 is an antimicrobial peptide fragment that directly radiolabeled with 99mTc shows high in vitro and in vivo stability, rapid background clearance, minimal accumulation in non-target tissues and rapid detection of infection sites. Molecular mechanics (MM) calculation has been an essential tool in explaining experimental results associated with molecular recognition and stability. This work is an attempt to explain the 99mTc-UBI 29-41 specificity for bacteria and to understand from a structural point of view, the experimental results indicative of a molecular recognition and stability not well favored for two other cationic peptides (99mTc-Tat-1-Scr and 99mTc-Tat-2-Scr ) used as control. Structures of 99mTc-UBI, 99mTc-Tat-1-Scr, 99mTc-Tat-2-Scr and of the corresponding free cationic peptides were built and the optimized structures, in the best stable configurations, were calculated by a MM procedure. In order to correlate the calculated and experimental results, in vitro stability tests with cysteine challenge and stability to dilution in human serum and in saline solution, were performed for the three labeled cationic peptides. The three complexes can be represented by the general formula [Tc(V)(O)(H2O)2(Lysn=1,2-Argn=0,1-peptide)]10+,11+. The potential energies were 104.5, 95.6 and 90.8 kcal/mol for 99mTc-Tat-1-Scr, 99mTc-Tat-2-Scr and 99mTc-UBI 29-41, respectively. Experimental and calculated results were in good agreement. It is thus possible to predict and explain that in similar solution media 99mTc-Tat-2-Scr would be more stable than 99mTc-Tat-1-Scr and why 99mTc-UBI shows the highest stability. In conclusion, the in vitro specific binding to bacteria and the accumulation at infection sites in humans of 99mTc-labeled UBI could be the result of its high thermodynamic stability, selectivity and stereospecificity.

Bacterial Infections↗