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PubMed1997

Rhenium-188 labeled hydroxyapatite and rhenium-188 sulfur colloid. In vitro comparison of two agents for radiation synovectomy.

AIM: One therapeutic approach to rheumatoid arthritis and other inflammatory arthropathies besides surgical removal of inflamed synovium is radiation synovectomy using beta-emitting radionuclides to destroy the affected synovial tissue. Up to now the major problem associated with the use of labeled particles or colloids has been considerable leakage of radionuclides from the injected joint coupled with high radiation doses to liver and other non target organs. In this study we compared 188Re labeled hydroxyapatite particles and 188Re rhenium sulfur colloid for their potential use in radiation synovectomy. METHODS: To this end we varied the labeling conditions (concentrations, pH-value, heating procedural and analyzed the labeling yield, radiochemical purity, and in vitro stability of the resulting radiopharmaceutical. RESULTS: After optimizing labeling conditions we achieved a labeling yield of more than 80% for 188Re hydroxyapatite and more than 90% for the rhenium sulfur colloid. Both of the radiopharmaceuticals can be prepared under aseptic conditions using an autoclave for heating without loss of activity. In vitro stability studies using various challenge solutions (water, normal saline, diluted synovial fluid) showed that 188Re labeled hydroxyapatite particles lost about 80% of their activity within 5 d in synovial fluid. Rhenium sulfur colloid on the other hand proved to be very stable with a remaining activity of more than 93% after 5 d in diluted synovial fluid. CONCLUSION: These in vitro results suggest that 188Re labeled rhenium sulfur colloid expects to be more suitable for therapeutic use in radiation synovectomy than the labeled hydroxyapatite particles.

Arthritis, Rheumatoid↗
PubMed1980

[Investigations of stability with the fixateur externe during strain of bending. Vertical and eccentric strain during compression of the fracture line and while the distance of support is changed (author's transl)].

The Fixateur externe was investigated under vertical and eccentric load when the fracture line was compressed and the distance of support was changed. Three kinds of mounting were used: R (Fixateur externe as frame), Z (three dimensional Fixateur externe) and Z2S (three dimensional Fixateur externe with two screws according to Schanz). If the distance of outer support was increased, the stability became greatly reduced. A compression increased the stability around the fracture line and on the free end of the system. A compression force of 10 kp at the fracture line yielded the greatest increase in stability. If in case of the methods R and Z, the compression was increased beyond 70 kp, there resulted even an instability, which depended on the distance of support. The most stable method of mounting was the three-dimensional Fixateur externe with two screws according to Schanz. The stability was compared if there was either a defect or a compression at the line of the fracture: there resulted a strikingly increased instability, if the distance of support was increased. The compression made it possible to compensate partly for the loss of stability.

Biomechanical Phenomena↗
PubMed2005

Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients.

AIMS: To investigate the stability of Bifidobacterium animalis ssp. lactis VTT E-012010 (=Bb-12) during freeze-drying, storage and acid and bile exposure. The effect of harvesting time and composition and pH of the cryoprotectant on the survival was evaluated. The procedure was performed by using a milk-free culture medium and cryoprotectants to produce cells for nonmilk-based applications. METHODS AND RESULTS: Bifidobacterial cells were grown in fermenters in general edible medium for 15 or 22 h. The cell mass was freeze-dried either as non-neutralized or neutralized using sucrose, betaine or reconstituted skim milk (control) as cryoprotectants. For stability studies freeze-dried powders were stored at 37, 5 and -20 degrees C for 2-6 months. In addition, acid and bile tolerance of the powders was tested. Sucrose-formulated B. animalis ssp. lactis preparations had an excellent stability during storage at refrigerated and frozen temperatures for 5-6 months. They also had a good survival during storage at 37 degrees C for 2 months as well as during exposure to pH 3 and 1% bile acids. No difference was observed between 15 and 22 h grown cells or between non-neutralized and neutralized cells. Betaine proved to be a poor cryoprotectant compared with sucrose. CONCLUSIONS: Fermentation time and neutralization of cell concentrate before freeze-drying had no impact on the storage stability and bile and acid tolerance of freeze-dried bifidobacterial cells. The nonmilk-based production protocol using sucrose as a cryoprotectant yielded powdery preparations with excellent stability in adverse conditions (storage at elevated temperatures and during acid and bile exposure). SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that it is feasible to develop nonmilk-based production technologies for probiotic cultures. This provides new possibilities for the development of nondairy-based probiotic products.

Bifidobacterium↗
PubMed1997

Computerized cephalometric evaluation of orthognathic surgical precision and stability in relation to maxillary superior repositioning combined with mandibular advancement or setback.

PURPOSE: A computerized, cephalometric, orthognathic surgical program (TIOPS) was applied in orthognathic surgical simulation, treatment planning, and postoperatively to assess precision and stability of bimaxillary orthognathic surgery. PATIENTS AND METHODS: Forty consecutive patients with dentofacial deformities requiring bimaxillary orthognathic surgery with maxillary superior repositioning combined with mandibular advancement or setback were included. All patients were managed with rigid internal fixation (RIF) of the maxilla and mandible and without maxillomandibular fixation (MMF). Preoperative cephalograms were analyzed and treatment plans produced by computerized surgical simulation. Planned, 5-week postoperative and 1-year postoperative maxillary and mandibular cephalometric-positions were compared. RESULTS: In the mandibular advancement group, the anterior maxilla was placed too far superiorly, with an inaccuracy of 0.4 mm. The posterior maxilla and the anterior mandible were placed in the planned positions. The lower posterior part of the mandibular ramus was placed too far anteriorly, with an inaccuracy of 2.0 mm. However, the mandibular condyles were accurately placed. In the setback group, the anterior maxilla was placed too far superiorly and posteriorly, with a vertical and sagittal inaccuracy of 1.0 mm and 0.7 mm, respectively. The posterior part of the maxilla was placed in a posterior position with an inaccuracy of 1.9 mm. The anterior mandible was placed too far anteriorly with an inaccuracy of 0.9 mm. The lower posterior part of the mandibular ramus was placed in a posterior position with an inaccuracy of 0.9 mm. However, the mandibular condyles were accurately placed. The statistical analysis of the 1-year stability data showed that the maxilla had moved 0.3 mm posteriorly in the advancement group and the lower incisors had moved 0.8 mm superiorly. No other significant positional maxillary or mandibular changes were found. In the setback group, the maxilla had moved 0.5 mm posteriorly, the anterior mandible 0.5 mm anteriorly, and the lower incisors 0.7 mm superiorly. No significant positional changes were seen in the mandibular ramus. CONCLUSION: The TIOPS computerized, cephalometric, orthognathic program is useful in orthognathic surgical simulation, planning, and prediction, and in postoperative evaluation of surgical precision and stability. The simulated treatment plan can be transferred to model surgery and finally to the orthognathic surgical procedures. The results show that this technique yields acceptable postoperative precision and stability.

Adolescent↗
PubMed1993

Liposome-encapsulated hemoglobin using film hydration processing to form artificial red blood cells.

Process parameters using a Microfluidizer M110 to produce liposome-encapsulated hemoglobin (LEH) were further studied to examine their effect on hemoglobin (Hb) encapsulation efficiency (yield), steady shear viscosity, mechanical stability, and oxygen delivery. Liposome formulation loading ratios of up to 300 mumol of lipid per ml of Hb solution were evaluated; a maximum yield was obtained at 300 mumol/ml. Liposomes containing encapsulated Hb concentrations as high as 15.5 g/100 ml were prepared. LEH particle size distribution, determined from negatively stained whole mount preparations using transmission electron microscopy, resulted in average vesicle sizes for optimal batches of about 155 nm. Steady shear viscosity of LEH (up to 40% by volume) in an isotonic-isooncotic solution of PBS containing either albumin or dextran were evaluated for shear rates to 2000 s-1. Values obtained were generally higher than those of whole blood at all shear rates tested. Little leakage of Hb from liposomes stored in isotonic PBS was observed as a function of storage time and shear rate. Administration of LEH supported life in rats whose hematocrit had been reduced via isovolemic exchange transfusion to levels well below 5%, which was incompatible with survival when exchange transfusion was performed with the isotonic-isooncotic PBS solution.

Animals↗
PubMed1999

Intrabody construction and expression. I. The critical role of VL domain stability.

We have constructed a panel of hyperstable immunoglobulin VL domains by a rational approach of consensus sequence engineering and combining stabilizing point mutations. These prototype domains unfold fully reversibly, even when the conserved structural disulfide bridge is reduced. This has allowed us to probe the factors that limit the expression yield of soluble immunoglobulin domains in the reducing environment of the cytoplasm (intrabodies). The most important factor is thermodynamic stability, and there is an excellent quantitative correlation between stability and yield. Surprisingly, an unprocessed N-terminal methionine residue can severely compromise VL stability, but this problem can be overcome by changing the amino acid following the initiator methionine residue. Transcription from the strong T7 promoter does not increase the amount of soluble material over that obtained from the tetA promoter, but large amounts of inclusions bodies can be obtained. Elevated temperature shifts protein from a productive folding pathway to aggregation. The structural disulfide bridge does not form in the cytoplasm, but the two consensus cysteine residues can be quantitatively oxidized in vitro. In summary, stability engineering provides a plannable route to the high-yield cytoplasmic expression of functional intrabody domains.

Amino Acid Sequence↗
PubMed1995

Ligand-induced changes in the conformational stability of bovine trypsinogen and their implications for the protein function.

Bovine trypsinogen was used as a model protein for studying changes in the conformational stability induced by pH or binding of the calcium ion. Spectrophotometrically monitored thermal unfolding of trypsinogen and beta-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to beta-trypsin, trypsinogen exhibits lower enthalpy of denaturation delta Hden, higher denaturational heat capacity change delta Cp,den, but very similar temperature of denaturation Tden. pH-dependence of the conformational stability of the ligand-free trypsinogen, measured also by GdnCl-induced unfolding, is bell shaped with the maximum free energy of unfolding delta Gden = 10.9 kcal/mole at pH 5.5 (4.5 pH units below its isoelectric point). At pH 8.3 the conformational stability of the zymogen drops to delta Gden = 3.2 kcal/mole, but increases by delta delta Gden = 6.1 kcal/mole in the presence of Ca2+. This significant stabilization of the zymogen by the calcium ion is also pH-dependent. To assess the effect of Ca2+ on the trypsinogen molecule, the spectrophotometric titrations and NOESY spectra were carried out. Based on the structural analysis, the long range effects between Ca2+-->Ile73-->Trp141 and the interdomain His40-Asp194 ion pair are proposed to be partially responsible for trypsinogen stabilization. Additionally, the steady-state parameters for hydrolysis of the oligopeptide amide substrate catalysed by free trypsinogen, its complexes with Ca2+ and the IleVal dipeptide and by beta-trypsin were measured. It appears that in the pH range 5.5 to 8.3 the stability and the catalytic activity/ligand binding properties are fully separated. Whereas the deprotonation of His57 accounts for the increase of kcat/km parameter, deprotonation of His40 is involved in the huge decrease of the conformational stability. Similarly, a large stabilization by the calcium ion is not accompanied by changes in enzymatic activity. Presented data are encouraging for an enzyme design directed toward improved stability.

Animals↗
PubMed1995

[The contribution and usefulness of routine intraoperative transesophageal echocardiography in cardiac surgery. An analysis of 130 consecutive cases].

PURPOSE: To assess the value of routine intraoperative transesophageal echocardiography (TEE) in unselected patients (P) undergoing cardiac surgery. DESIGN: Routine intraoperative TEE in unselected patients undergoing cardiac surgery and evaluation of its usefulness and contribute to a successful surgery. PATIENTS: In-hospital patients from cardiology, cardiac surgery and intensive care departments of a general hospital classified as a reference medical centre in cardiovascular pathology. MATERIAL AND METHODS: From February 1994 to May 1994 an intraoperative TEE was routinely performed in patients undergoing cardiac surgery. A total of 130 TEE studies were made corresponding to 128 patients, 69 males and 61 females, with a mean age of 49.4 +/- 12.1 years. Indications for surgery were as following: a) Coronary artery bypass grafting (C.A.B.G.)-39; b) Valvular surgery-76; c) C.A.B.G. and valvular surgery-5; d) Thoracic aorta pathology associated or not to aortic valve surgery and/or coronary arteries re-implantation-6; e) Other-4. TEE performed using either a monoplane, biplane or multiplane probe and the images acquisition was made before thoracotomy and cardiopulmonary bypass (CPB) and after CPB with hemodynamic stabilization. RESULTS: Pre CPB imaging yielded unsuspected findings in 11 P (8.5%) that changed the planned surgery in 7 cases (5.4%). Post CPB echo study, which was performed after hemodynamic stabilization, modified the surgical strategy in 13 cases (10.9%) avoiding 1 predetermined surgical procedure (tricuspid ring annuloplasty) and a new surgery in another case, leading to a 3 non-predetermined surgical procedures (by showing significative tricuspid regurgitation), to a further surgery in the same valve in 6 patients (mitral paravalvular leak with significative regurgitation in 1P, another mitral prosthesis dysfunction in 1P, residual mitral valve insufficiency after mitral valve repair in 3 P and aortic prosthesis dysfunction in 1 P), to a intraaortic counterpulsation balloon device in 1 P and to new CPB due to an insufficient extraction of calcified pericardium in one case of constrictive pericarditis. In 23 cases (17.7%) TEE has modified the anesthetic procedure by demonstrating signs of hemodynamic changes before Swan-Ganz catheter, thus prompting changes in the administrations of fluids and either in inotropic or vasodilator agents. In total, intraoperative TEE has changed the surgical and/or anesthetic plan in 43 cases (33.0%). There was no difficulties or complications related to the procedure. CONCLUSION: These data indicate that intraoperative TEE is useful in formulating the surgical plan and assessing immediate operative results as well as a guide to anesthetic procedures. Its high rentabillity in modifying the surgical and/or anesthetic plans lead us to believe that it must be used as a routine procedure in patients undergoing cardiac surgery.

Adult↗
PubMed1992

Oxygen yield and thermoluminescence characteristics of a cyanobacterium lacking the manganese-stabilizing protein of photosystem II.

Previous experiments have shown that a Synechocystis sp. PCC 6803 mutant (delta psbO) lacking the extrinsic manganese-stabilizing protein (MSP) exhibits impaired, but significant levels of H2O-splitting activity [Burnap, R., & Sherman, L.A. (1991) Biochemistry 30, 440-446]. [14C]DCMU-binding experiments now show that the number and affinity of DCMU-binding sites (normalized to chlorophyll) are equivalent in delta psbO and the wild type, suggesting equal concentrations of assembled reaction centers. A similar conclusion is reached on the basis of measurements of PSII electron transport (DPC-supported DCPIP reduction) by mutant and wild-type thylakoids. The pattern of flash O2 yield by delta psbO cells measured with a bare platinum electrode exhibits a period four oscillation (with a maximum on the third flash), indicating that the H2O-splitting enzyme in delta psbO retains the basic mechanistic features found in normal cells. However, the amplitude of these signals is smaller and more highly damped than those obtained from wild-type cells, suggesting the absence of MSP results in a higher miss probability and/or a reduction in the number of centers competent in oxygen evolution. Analysis of the rise kinetics of the ampermeric signal on the bare platinum electrode indicates that the S3-[S4]-S0 transition is retarded by at least a factor of 5 in the mutant. Thermoluminescence emission peak temperatures indicate that the S2QA-, S2QB-, and S3QB-charge pairs are significantly more stable with respect to recombination in the mutant. The intensities of the thermoluminescence emissions are also significantly reduced in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed2000

The stability of house dust mite allergens in glycerinated extracts.

BACKGROUND: Mite allergen vaccines are important diagnostic and immunotherapeutic reagents. Previous studies on mite allergen stability under different storage conditions have yielded contradictory results. OBJECTIVE: We sought to compare, over a 12-month period, the stability of mite allergens reconstituted in 50% glycerol and stored at different temperatures and to examine the role of protease inhibitors in enhancing allergen stability. METHODS: Lyophilized allergen extracts were reconstituted in 50% glycerol, with and without protease inhibitors, and stored at -70 degrees C, -20 degrees C, 4 degrees C, or 37 degrees C for 12 months. At 6 and 12 months, the extracts were compared with freshly dissolved extracts by competition ELISA with pooled allergic sera, 2-site ELISA with mite-specific mAbs, and immunoblot analyses. RESULTS: The overall potencies of the stored extracts measured by competition ELISA were stable at -20 degrees C and 4 degrees C. As determined by means of the immunoblot and 2-site ELISA, Der f 1 levels decreased at 4 degrees C. Levels of Der f 2, Der p 1, and Der p 2 decreased in at least one of the allergen-specific assays. Storage at 37 degrees C led to overall loss of potency and allergen content, whereas storage at -70 degrees C was associated with a moderate loss of potency that increased with multiple freeze-thaw cycles. Protease inhibitors had no effect on allergen stability. CONCLUSION: Although overall potency of the extracts, as measured by competition ELISA, was preserved at -20 degrees C and 4 degrees C, allergen-specific assays indicated loss of allergens. These findings suggest that the competition ELISA is insensitive to decreases in the concentrations of individual allergens.

Animals↗
PubMed2004

Muscle activity, internal loads, and stability of the human spine in standing postures: combined model and in vivo studies.

STUDY DESIGN: The load in active and passive spinal components as well as the stability margin in standing postures +/- load in hands are studied using both computational model and in vivo studies. OBJECTIVE: To investigate muscle activity, spinal loads, and system stability in standing postures. SUMMARY OF BACKGROUND DATA: Study of the human trunk yields a redundant system, the satisfactory solution of which remains yet to be done. Existing biomechanical models are often oversimplified or attempt to solve the problem by equilibrium of loads at only one cross section along the spine. METHODS: In vivo measurements are performed to obtain kinematics (by skin markers) as input data into model and EMG activity (by surface electrodes) for validation of predictions. A thoracolumbar model, while accounting for nonlinear ligamentous properties and trunk musculature, solved the redundant active-passive system by a novel kinematics-based approach that used both the posture and gravity/external loads as input data. In both studies, neutral standing posture was considered with weights up to 380 N held in hands with arms extended close to the body either in front or on sides. RESULTS: Predicted muscle forces were in satisfactory agreement with measured EMG activities. The activity in extensor muscles significantly increased with the load magnitude when held in front, a trend that disappeared as loads were held on sides. Abdominal muscles remained relatively silent. Large compression forces of approximately 2000 N were computed in lower lumbar levels when 380 N was held in front. Coactivity in abdominal muscles markedly increased internal loads and stability margin. CONCLUSION: A tradeoff exists between lower loads in passive tissues (i.e., tissue risk of failure) and higher stability margins as both increase with greater muscle coactivation. Greater muscle activity observed under load held in front did not necessarily yield larger stability margin as the position of load appeared to play an important role as well. The strength of the proposed model is in realistic consideration of both passive-active structures under postures and gravity/external loads, yielding results that satisfy kinematics, equilibrium, and stability requirements in all directions along the spine.

Adult↗
PubMed1999

Stability and cytotoxicity of Fab-ricin A immunotoxins prepared with water soluble long chain heterobifunctional crosslinking agents.

The effects of the hindered and non-hindered water soluble long-chain disulfide bonds on the stability and cytotoxicity of the ricin A chain (RTA) immunotoxin were examined. The RTA immunotoxins were prepared with the Fab fragments of anti-common acute lymphoblastic leukemia antigen (CALLA) monoclonal antibody (Fab-RTA) using sulfosuccinimidyl-6-[(-methyl-(-(2-pyridyldithio)toluamido]hexanoate (S-LC-SMPT) and sulfosuccinimidyl-6-[3-(2-pyridyldithio)-propionamido]hexanoate (S-LC-SPDP). The prepared Fab-RTA immunotoxins were evaluated for their conjugation yield, immunoreactivity, thermal and disulfide bond stability and cytotoxicity. The conjugation yield of the Fab-RTA immunotoxin from the water soluble long chain crosslinking agents, S-LC-SMPT and S-LC-SPDP, were comparable. Both Fab-RTA immunotoxins exhibited a similar immunoreactivity and thermal stability in aqueous solution. However, S-LC-SMPT -mediated Fab-RTA, sterically hindered, showed an enhanced disulfide bond stability in vitro over S-LC-SPDP mediated one. In the cytotoxicity against antigenic cell Daudi, the S-LC-SMPT -mediated RTA immunotoxin maintained a comparable cytotoxicity, compared with S-LC-SPDP mediated Fab-RTA immunotoxin.

Animals↗
PubMed2002

Optimization of reaction conditions and stabilization of phenylalanine ammonia lyase-containing Rhodotorula glutinis cells during bioconversion of trans-cinnamic acid to L-phenylalanine.

Studies were performed to elucidate the optimal reaction conditions (pH, temperature, ammonia concentration and biocatalyst loading) for bioconversion of trans-cinnamic acid (t-CA) to L-phenylalanine (L-Phe) by L-phenylalanine ammonia lyase (PAL) containing Rhodotorula glutinis cells. All treatments with permeabilizing agents stimulated L-Phe production and also enhanced instability of the catalyst, except Triton X-100 which gave a superior (56%) increase in conversion as compared to the control and a significant stabilization of PAL enzyme. Inclusion of several activity modifiers and stabilizer additives in reaction mixtures were shown to enhance the yield of L-Phe and maintained PAL stability over several successive incubations during the bioconversion process. Maximum stabilization of PAL and enhancement of L-Phe production was achieved with addition of 20% polyhydric alcohol (glycerol). The production of L-Phe continued to the fifth cycle and the total yield increased 2.3 times compared to the yield produced by the control (without glycerol addition) during the repeated batch process. Reducing agents such as 2-mercaptoethanol and thioglycolic acid were added to the bioconversion mixture in order to reduce the effects of oxygen on PAL catalyst life. Production of L-Phe by addition of 400 mgL(-1) of thioglycolic acid was maximized over the control by 55%. When both 20% glycerol and 400 mgL(-1) thioglycolic acid were simultaneously present in the reaction mixture, reuseability and stability of biocatalyst (PAL) were extended to eight consecutive cycles and conversion rate and overall productivity of L-Phe were higher than that of the control. These results may lead to improvements in the production of the essential amino acid L-Phe.

Ammonia↗
PubMed1988

Experimental diabetes in lactating sheep: effects of alloxan on plasma insulin, glucose, glucose kinetics and milk characteristics.

After intravenous administration of alloxan (50 mg kg-1 liveweight) to lactating ewes, there were triphasic changes in plasma glucose and insulin. Almost immediately, plasma insulin decreased and hyperglycaemia occurred, then, between c. 5-12 h, insulin increased and ewes became hypoglycaemic. Thereafter, insulin decreased and glucose increased from c. 20 h after alloxan and the diabetic state was established. Changes in glucose production and utilization correlated with changes in plasma glucose. Exogenous insulin was administered from 30 h after alloxan, and it took some 2 weeks to stabilize ewes. During this period, when mild hyperglycaemia persisted, milk yields and feed intakes were decreased but milk fat content was elevated. Once ewes were stabilized, plasma glucose, milk yield, feed intake and milk fat content returned to levels prior to alloxan. These observations are consistent with insulin playing a role in the aetiology of the 'low milk fat syndrome' in the ruminant. It appears that the alloxan-treated, insulin-stabilized ewe would be a useful model for studying the role of insulin during lactation, but it is necessary to allow time for animals to overcome effects of administration of alloxan.

Alloxan↗
PubMed1984

Two methods compared for measuring lipase activity in plasma after heparin administration.

We compared two methods for the direct selective measurement of hepatic lipase and lipoprotein lipase activities in human plasma after intravenous administration of heparin. Except for the emulsifier (gum arabic vs lecithin), the two assay media for hepatic lipase are essentially similar. Results for hepatic lipase by these two assays correlate well (r = 0.99). The assays for lipoprotein lipase in the two procedures differ in the way that hepatic lipase activity is eliminated (immunological inhibition vs a specific substrate emulsion), and also with regard to the emulsifier. The substrate emulsion stabilized by gum arabic (immunological assay) consistently yielded about three times higher enzymic activity than the specific substrate stabilized by lecithin. Experiments in which purified enzymes were used demonstrated that this systematic difference can be accounted for by the different emulsifiers. The satisfactory correlation (r = 0.92) between the two lipoprotein lipase assays, however, demonstrates that they measure the same enzymic activity.

Adult↗
PubMed2005

Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase.

Mitochondrial creatine kinase (MtCK) plays a central role in energy homeostasis within cells that display high and variable rates of ATP turnover. Vertebrate MtCKs exist primarily as octamers but readily dissociate into constituent dimers under a variety of circumstances. MtCK is an ancient protein that is also found in invertebrates including sponges, the most primitive of all multi-cellular animals. We have cloned, expressed, and purified one of these invertebrate MtCKs from a marine polychaete worm, Chaetopterus variopedatus (CVMtCK). Size exclusion chromatography and dynamic light scattering (DLS) were used to characterize oligomeric state in comparison with that of octameric chicken sarcomeric isoform (SarMtCK). At protein concentrations >1 mg/ml, CVMtCK was predominantly octameric (>90%). When diluted to 0.1 mg/ml, CVMtCK dissociated into dimers much more rapidly than SarMtCK when observed under identical conditions. The rate of dissociation for both MtCKs increased as temperature rose from 2 to 28 degrees C, and in CVMtCK, fell at higher incubation temperatures. The fraction of octameric CVMtCK at equilibrium increased with temperature and then fell. Temperature transition studies showed that octamers and dimers were rapidly interconvertible on a similar time scale. Importantly, when CVMtCK was converted to the transition state analog complex (TSAC), both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. These results clearly show distinct differences in octamer stability between CVMtCK and SarMtCK, which could impact function under physiological circumstances. Furthermore, the large yield of recombinant protein and high stability of CVMtCK in the TSAC suggest that this protein might be a good target for crystallization efforts.

Adenosine Diphosphate↗
PubMed2005

Crystal structure of the interferon-induced ubiquitin-like protein ISG15.

The biological effects of the ISG15 protein arise in part from its conjugation to cellular targets as a primary response to interferon-alpha/beta induction and other markers of viral or parasitic infection. Recombinant full-length ISG15 has been produced for the first time in high yield by mutating Cys78 to stabilize the protein and by cloning in a C-terminal arginine cap to protect the C terminus against proteolytic inactivation. The cap is subsequently removed with carboxypeptidase B to yield mature biologically active ISG15 capable of stoichiometric ATP-dependent thiolester formation with its human UbE1L activating enzyme. The three-dimensional structure of recombinant ISG15C78S was determined at 2.4-A resolution. The ISG15 structure comprises two beta-grasp folds having main chain root mean square deviation (r.m.s.d.) values from ubiquitin of 1.7 A (N-terminal) and 1.0 A (C-terminal). The beta-grasp domains pack across two conserved 3(10) helices to bury 627 A2 that accounts for 7% of the total solvent-accessible surface area. The distribution of ISG15 surface charge forms a ridge of negative charge extending nearly the full-length of the molecule. Additionally, the N-terminal domain contains an apolar region comprising almost half its solvent accessible surface. The C-terminal domain of ISG15 was superimposed on the structure of Nedd8 (r.m.s.d. = 0.84 A) bound to its AppBp1-Uba3 activating enzyme to model ISG15 binding to UbE1L. The docking model predicts several key side-chain interactions that presumably define the specificity between the ubiquitin and ISG15 ligation pathways to maintain functional integrity of their signaling.

Amino Acid Substitution↗
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↗