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PubMed2004

Application of principles of evidence-based medicine to occlusal treatment for temporomandibular disorders: are there lessons to be learned?

Critical evaluation of treatment methods has become an important part of health care and will certainly have a major influence on decisions about acceptable treatment methods in the future. Evidence-based medicine (EBM) means the systematic, explicit, and judicious implementation of the best evidence in patient care. The most reliable sources of evidence are high-quality systematic reviews and randomized controlled trials (RCTs). A systematic EBM approach could be particularly useful in the treatment of temporomandibular disorders (TMD), where controversial and conflicting ideas about management are common. In this field, concerns about the lack of evidence are often expressed. This article aims to elucidate and discuss the application of EBM to the treatment of TMD, using the most controversial treatments (i.e., occlusal treatments) as an example. By applying the principles of EBM to TMD treatments, we wish to highlight some of the important issues that form the basis for high-quality care in this field. A systematic review of occlusal treatments (occlusal splints and occlusal adjustment) updated to January 2003 revealed 16 RCTs of occlusal splints and 4 of occlusal adjustment. The overall quality of the trials was fairly low. Recently, however, some high-quality RCTs of occlusal splints have been published. The most obvious methodologic shortcomings in published trials included problems in defining the patient population, inadequacies in performing randomization and blinding, problems in defining the therapies or appropriate control treatments, short follow-ups, and problems in monitoring patient compliance. Occlusal splint studies yielded equivocal results. Even in the most studied area, stabilization splints for myofascial face pain, the results do not justify definite conclusions about the efficacy of splint therapy. Their clinical effectiveness to relieve pain also seems modest when compared with pain treatment methods in general. None of the occlusal adjustment studies provided evidence supporting the use of this treatment method. The clinical implications of the findings and future perspectives are discussed.

Dentistry↗
PubMed2006

[Fusion tags technology and their applications].

Fusion tags are originally developed to facilitate the purification of recombinant protein from crude extracts. In recent years, the discovery of different tags and the development of fusion strategy make the function of fusion tags diversified. However, there was no a cure-all fusion tag for different applications. We here give an overview of fusion tag technology and the different applications of fusion tags, including the purification, detection and oriented immobilization of recombinant protein, the visualization of bioevent in vivo, the enhancement of the yield of protein, the improvement of the solubility and stability of the expressed protein.

Recombinant Proteins↗
PubMed1986

The mechanism of the search for homology promoted by recA protein. Facilitated diffusion within nucleoprotein networks.

recA protein promotes the homologous pairing of single strands with duplex DNA by polymerizing on the single strands to make presynaptic nucleoprotein filaments which are polyvalent with respect to duplex DNA and which consequently form large networks or coaggregates when duplex DNA is added. Previous work has shown that efficient homologous pairing occurs within these networks. In the experiments described here, we observed that the length of the duplex DNA determined the stability of coaggregates, their steady state level, and the yield of joint molecules. Correspondingly, heterologous duplex DNA when preincubated with presynaptic filaments excluded subsequently added homologous duplex DNA from coaggregates and inhibited homologous pairing; the extents of exclusion and inhibition were determined by the length of the heterologous duplex DNA. On the other hand, long heterologous duplex DNA when added together with short homologous duplex DNA was capable of stimulating the absorption of the homologous molecules into coaggregates and increasing the rate of homologous pairing. In reactions involving short duplex molecules, polyamines exerted comparable effects on coaggregation and homologous pairing. We conclude that coaggregates are instrumental in homologous pairing, that they constitute distinct domains that are responsible for the processive or first order character of the pairing reaction, and that they act by concentrating DNA and facilitating diffusion.

DNA Restriction Enzymes↗
PubMedDate not supplied

[The use of immobilized enzymes for the synthesis of nucleosides and nucleotides including the ones labeled with radioactive isotopes].

Nucleoside phosphorylases from E. coli immobilized in polyacrylamide gel were used for production of nucleosides from nitrous bases during the transglucosylase reaction. Phosphorylation of nucleosides resulted in the formation of 5'-nucleotides was performed in the presence of carrot nucleoside phosphotransferase immobilized in polyacrylamide gel. Using 14C-labelled nitrous bases as starting substrates, labelled nucleosides and nucleotides can be obtained with the 75-80% yield that have radioactive purity of 95-99%. The stability of the immobilized enzymes was being studied under the conditions of the reactions using them in the batch and plug flow reactors.

Enzyme Stability↗
PubMed1988

Clearance and distribution of recombinant murine gamma-interferon in mice.

Recombinant murine gamma-interferon (rIFN-gamma) was radiolabeled by a novel procedure which does not require the use of preiodinated Bolton-Hunter reagent (specific activities of 0.5-3.0 microCi/micrograms). Gel filtration chromatography of the radiolabeled preparation yielded two peaks. The early eluting peak contained disulfide stabilized aggregates with minimal interferon antiviral activity. The second peak contained activity that was consistently greater than or equal to that of the nonradiolabeled rIFN-gamma. Two bands with apparent molecular weights of 17,000 and 34,000 were observed when the second peak was analyzed by SDS gel electrophoresis. Fractions comprising each of the two chromatography peaks were pooled separately and subjected to gel filtration again on identical columns 24 h after completion of the first column run. The elution volumes of each peak remained unchanged suggesting that the two forms are not in rapid equilibrium. The plasma clearance rates of [125I]rIFN-gamma before and after purification by chromatography were initially rapid but multiphasic. The slower phases of clearance did not result from stable association of the rIFN-gamma with plasma proteins. In organ distribution studies, the liver and spleen sequestered significant amounts of [125I]rIFN-gamma; however, the highest concentration of rIFN-gamma was recovered in the kidneys. A functional nephrectomy procedure was used to further study the role of the kidneys in rIFN-gamma clearance. Eliminating the kidneys significantly increased the amount of rIFN-gamma retained in the circulation, particularly at later times when the vascular [125I]rIFN-gamma levels were approximately threefold higher than in nonnephrectomized mice.

Animals↗
PubMed1985

[Analysis by reflection. A new method of observing human chromosomes].

When metaphase preparations of human cells are stained with Giemsa and submitted to epi-illumination with white light a brilliant yellow-green reflexion from the chromosomes is produced, quite comparable to fluorescence after acridine orange staining. Beside a remarkable stability of the image during repeated observations, this method yields excellent resolution.

Chromosomes, Human↗
PubMedDate not supplied

[Congenital kyphosis with neurological disorders].

The paper outlines the results of examination and surgical treatment of a large group of patients with congenital sternolumbar kyphoses complicated by development of neurological symptoms. The results of radioopaque investigations showed there was a relationship of the magnitude of intracanal changes to the size of kyphotic deformity. The comparative study of the outcomes of surgical management of patients with congenital kyphoses demonstrated the high efficiency of interventions, whose complex included central spondylosis. This operation decreases spinal deformity, yields decompression of intracanal nervous structures and ensures further stability of the vertebral column.

Adolescent↗
PubMed1994

Post-translational requirements for functional factor V and factor VIII secretion in mammalian cells.

Coagulation factors V and VIII are homologous glycosylated plasma proteins that provide essential functions for hemostasis. Factor V is secreted as a single chain polypeptide, whereas factor VIII is processed intracellularly to yield a metal-ion-associated heterodimer that is stabilized through interaction with von Willebrand factor. In transfected mammalian cells, factor V is more efficiently secreted than factor VIII. To provide insight into the different secretion efficiencies, we compared the post-translational processing requirements for factor V and factor VIII expressed in mammalian cells. In contrast to factor VIII, factor V was not detected in association with the immunoglobulin-binding protein (BiP), a chaperonin protein of the endoplasmic reticulum (ER). Depletion of intracellular ATP levels by treatment of cells with low concentrations of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), protonophore that uncouples oxidative phosphorylation, inhibited secretion of factor VIII but had no effect on the secretion of factor V. Inhibition of N-linked oligosaccharide addition by treatment with tunicamycin prevented secretion of both factor V and factor VIII, whereas treatment with an inhibitor of complex oligosaccharide addition, deoxymannojirimycin, did not affect secretion, although the specific activities of both factor V and factor VIII were slightly increased. Thus, complex oligosaccharide addition was not required for secretion or functional activity of either factor V or factor VIII. Depletion of intralumenal calcium with the ionophore A23187 did not affect secretion of either factor V or factor VIII. In the presence of A23187, the secreted factor V was fully functional, whereas the factor VIII heavy and light chains were not associated and the secreted molecule was inactive. In addition, A23187 treatment inhibited addition of serine/threonine (O)-linked oligosaccharides to factor V and factor VIII. The differences between factor V and factor VIII were further evaluated by characterization of a single chain mutant factor VIII. The single chain factor VIII was secreted with an efficiency similar to wild-type factor VIII and also required von Willebrand factor for stabilization. In addition, the activity of single chain factor VIII was also inhibited by A23187 treatment, suggesting a unique metal-ion requirement within the secretory pathway for functional factor VIII folding. The differences identified in BiP association, ATP requirements, and metal-ion dependence for effective functional secretion of these two molecules may underlie mechanisms accounting for their different secretion efficiencies.

Adenosine Triphosphate↗
PubMed1994

Initiation of DNA replication by simian virus 40 T antigen is inhibited by the p107 protein.

The p107 protein is related to the Rb protein by a 60-kDa region of homology called the pocket domain that binds cellular proteins such as E2F and cyclins A and E as well as the viral oncoproteins E1A, E7, and SV40 T antigen. The p107 and Rb proteins have both been implicated as negative regulators of cell growth. We have examined the effect of the unphosphorylated pocket domain of p107 on specific stages of the T antigen-mediated replication of SV40 DNA in vitro. The pocket domain inhibited replication by preventing the assembly of T antigen at the SV40 core origin DNA containing binding site II. However, both proteins formed a ternary complex with DNA containing T antigen-binding site I. The pocket domain of p107 did not inhibit the oligomerization of T antigen in the absence of DNA, and the p107 derivative is bound to the intermediates of this reaction. In the unwinding assay, once T antigen was preassembled as hexamers at the core origin, the pocket domain bound to and stabilized the complex, resulting in an increase in the yield of unwound product. Preformed T antigen hexamers complexed with the pocket domain bind to a synthetic replication fork, and this complex supported unwinding. Consistent with this, the p107 pocket domain had no effect on the helicase activity of T antigen in an assay using a partial duplex substrate. However, a complex containing p107 and T antigen assembled at the core origin did not support SV40 DNA replication in HeLa cell crude extracts or in the monopolymerase reaction. This inhibition is due to the inability of the complex to bind to DNA polymerase alpha, which is required for the initiation of DNA synthesis. The data suggest pathways by which the pocket domain of p107 can negatively regulate T antigen-mediated replication in vitro. The binding of T antigen to p107 is discussed with respect to its role in mitigating negative cell growth control, resulting in viral mediated transformation.

Animals↗
PubMed1996

[Curved lamellar keratotomy for correction of astigmatism. Experimental and initial clinical results].

BACKGROUND: The first experiments for surgical correction of higher astigmatism were reported more than 100 years ago. A lot of different procedures were strongly recommended at the beginning but then abandoned later on because they could not fulfill the expectations regarding the postoperative results and the complications. On the other hand, lamellar preparation of the cataract incision has been considered a major advance in ophthalmology. The main advantage of this incision is that it yields stable postoperative refraction as well as high mechanical stability very early (postoperatively). These findings prompted us to combine the advantages mentioned above with those of the arcuate transverse incision. In this report we present our experimental and clinical results with arcuate lamellar keratotomy. MATERIALS AND METHODS: Experiments were carried out on 22 cadaver bulbi. The optical zones ranged from 6 to 8 mm and the length of the arcuate incisions was between 2 and 7 mm. The clinical data presented here were obtained from 20 patients with a 4-week follow-up. These 20 patients had undergone cataract surgery previously with an induced astigmatism ranging from 2.5 to 5 D. Patients were treated with an optical zone of 7 mm or 8 mm. The length of the arcuate incision was 3 mm. All incisions were paired. RESULTS: Our experiments (cadaver bulbi) showed an approximately linear decrease of the effect with increasing width of the optical zone and increasing are length. Our clinical results demonstrate that the astigmatism induced by our procedure (including potential overcorrection) was 3.41 +/- 1.33 D on the the first day postoperatively. All astigmatism was measured with the Zeiss keratometer. After 1 and 4 weeks the results were 3.98 +/- 1.35 and 3.71 +/- 1.29 D, respectively. The induced astigmatism also depended on the width of the optical zone. In the group with a 7 mm optical zone the induced astigmatism was 4.5 +/- 1.56 D after 4 weeks. This effect was remarkably higher than in the 8 mm group with an average of 3.35 +/- 0.94 D of induced astigmatism. There were no significant differences between visual acuity under glare conditions and the number of endothelial cells preoperatively and at 4 weeks follow-up, nor were there variations in refraction, depending on the time of day. CONCLUSIONS: Due to the relatively high standard deviation of the induced astigmatism we must keep trying to make the results of our procedure more predictable.

Aged↗
PubMed1976

[Measurement of emulsion stability].

For the routine assessment of the stability of water-in-oil emulsions, the authors suggest to determine the beginning of de-emulsification or to measure the stability constant at 38 degrees C., since these methods yield fairly reproducible results and require but little time and laboratory outfit. More accurate results will be obtained only by conductance measurements.

Chemical Phenomena↗
PubMed2001

Spatio-temporal pattern formation on spherical surfaces: numerical simulation and application to solid tumour growth.

In this paper we examine spatio-temporal pattern formation in reaction-diffusion systems on the surface of the unit sphere in 3D. We first generalise the usual linear stability analysis for a two-chemical system to this geometrical context. Noting the limitations of this approach (in terms of rigorous prediction of spatially heterogeneous steady-states) leads us to develop, as an alternative, a novel numerical method which can be applied to systems of any dimension with any reaction kinetics. This numerical method is based on the method of lines with spherical harmonics and uses fast Fourier transforms to expedite the computation of the reaction kinetics. Numerical experiments show that this method efficiently computes the evolution of spatial patterns and yields numerical results which coincide with those predicted by linear stability analysis when the latter is known. Using these tools, we then investigate the rĵle that pre-pattern (Turing) theory may play in the growth and development of solid tumours. The theoretical steady-state distributions of two chemicals (one a growth activating factor, the other a growth inhibitory factor) are compared with the experimentally and clinically observed spatial heterogeneity of cancer cells in small, solid spherical tumours such as multicell spheroids and carcinomas. Moreover, we suggest a number of chemicals which are known to be produced by tumour cells (autocrine growth factors), and are also known to interact with one another, as possible growth promoting and growth inhibiting factors respectively. In order to connect more concretely the numerical method to this application, we compute spatially heterogeneous patterns on the surface of a growing spherical tumour, modelled as a moving-boundary problem. The numerical results strongly support the theoretical expectations in this case. Finally in an appendix we give a brief analysis of the numerical method.

Animals↗
PubMed2001

In vitro stability of alpha-helical peptide nucleic acids (alphaPNAs).

Alpha-helical peptide nucleic acids (alphaPNAs) are synthetic molecules that merge the alpha-helix secondary structure of peptides with the codified Watson-Crick base pairing capability of nucleic acids. It is now demonstrated that alphaPNAs made up of either L- or D-amino acids are resistant to degradation by the proteases present in human serum. The increased stability of alphaPNAs towards proteases may be attributable to the presence of unnatural nucleoamino acid residues [-NHCH(CH(2)OCH(2)B)CO-, where B=thymine or cytosine] since the replacement of these amino acids by serine yields a control peptide that does break down in human serum. The stability of alphaPNAs towards proteases makes them attractive candidates for further development as antisense agents.

Amino Acid Sequence↗
PubMed1980

[Steroid transformation with immobilized microorganisms. III. Degradation of the side chain of cholesterol derivatives with immobilized Mycobaterium phlei and M. smegmatis cells].

The cholesterol derivatives 3,3-ethylendioxycholest-5-en I and 4-cholesten-3-(O-carboxymethyl)oxime II were transformed into the corresponding androstenedione derivatives by selective cleavage of the sterol side chain with immobilized preparations of Mycobacterium phlei IMET SG 1026 and M. smegmatis IMET H 124. The influence of three immobilization methods on fermentation activity and stability was investigated. The immobilized cells of M. phlei IMET SG 1026 were found to be suitable to transform the structure modified sterol II in a periodic process over a long period into the androstenedione in a yield of 50--70%. Immobilized cells of M. phlei have higher transformation stability in comparison with free intact cells.

Adsorption↗
PubMedDate not supplied

99mTc-labeling and in vitro and in vivo evaluation of HYNIC- and (Nalpha-His)acetic acid-modified [D-Glu1]-minigastrin.

Gastrin/CCK-2 receptors are overexpressed in a number of tumors such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). Recently [D-Glu1]-minigastrin (MG) has been radiolabeled with 131I, 111In, and 90Y and evaluated in patients. This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling agent and (Nalpha-His)Ac as tridentate ligand for 99mTc(CO3) labeling. Labeling was perfomed at high specific activities using Tricine and EDDA as coligands for HYNIC-MG and [99mTc(OH2)3(CO)3]+ for (Nalpha-His)Ac-MG. Stability experiments were carried out by reversed phase HPLC analysis in PBS, serum, histidine, and cysteine solutions, as well as rat liver and kidney homogenates. Receptor binding and internalization experiments were performed using CCK-2 receptor positive AR42J rat pancreatic tumor cells. Biodistribution experiments were carried out in nude mice carrying AR42J tumors by injection of 99mTc-labeled peptide with or without coinjection of 50 microg of minigastrin I human (MGh). HYNIC-MG and (Nalpha-His)Ac-MG could be radiolabeled at high specific activities (>1 Ci/micromol). For HYNIC-MG, high labeling yields (>95%) were achieved using Tricine and EDDA as coligands. Stability experiments of all 99mTc-labeled conjugates revealed a high stability of the label in PBS and serum as well as toward challenge with histidine and cysteine. Incubation in kidney homogenates resulted in a rapid degradation of all conjugates with <10% intact peptide after 60 min at 37 degrees C, with no considerable differences between the radiolabeled conjugates; a somewhat lower degradation rate was seen in liver homogenates. Protein binding varied considerably with lowest levels for 99mTc-EDDA/HYNIC-MG. Competition experiments of unlabeled conjugates on AR42J membranes versus [125I-Tyr12]-gastrin I showed high CCK-2 receptor affinity for all conjugates under study. Internalization behavior was very rapid for all radiolabeled conjugates in the order of 99mTc-(Nalpha-His)Ac-MG > 99mTc-EDDA/HYNIC-MG > 99mTc-Tricine/HYNIC-MG. In tumor-bearing nude mice the highest tumor-uptake was observed with 99mTc-EDDA/HYNIC-MG (8.1%ID/g) followed by 99mTc-Tricine/HYNIC-MG (2.2%ID/g) and 99mTc-(Nalpha-His)Ac-MG (1.2%ID/g) which correlated with kidney uptake (101.0%ID/g, 53.8%ID/g, 1.8%ID/g respectively). In this series of compounds 99mTc-EDDA/HYNIC-MG with its very high tumor/organ ratios except for kidneys seems to be the most promising agent to target CCK-2 receptors. Despite promising properties concerning receptor binding, internalization, and in vitro stability, 99mTc-(Nalpha-His)Ac-MG showed low tumor uptake in vivo.

Acetates↗
PubMed1994

Identification of N-acetylcysteine as a new substrate for rat liver microsomal glutathione transferase. A study of thiol ligands.

N-Acetyl-L-cysteine serves as an efficient substrate for the rat liver microsomal glutathione transferase with 1-chloro-2,4-dinitrobenzene as second substrate (8.8 +/- 0.37 mumol/min mg). The activity is actually higher than that obtained with glutathione (2-4 mumol/min mg). In examining the activity of liver subcellular fractions, no activity with N-acetyl-L-Cys could be detected in dialyzed or N-ethylmaleimide-treated (in order to remove endogenous glutathione) cytosol. The activity in rat liver microsomes was 0.11 +/- 0.007 mumol/min mg, which is accounted for by the content of microsomal glutathione transferase. Thus, N-acetyl-L-Cys can be used as a specific substrate for determining the conjugating activity of microsomal glutathione transferase. N-Acetyl-L-Cys was also shown to function as a substrate for the enzyme when other second substrates than 1-chloro-2,4-dinitrobenzene (with varying electrophilicity) are used. The pH dependence of microsomal glutathione transferase was studied. The kcat/Km(1-chloro-2,4-dinitrobenzene) was dependent on pH with an apparent pKa of 6, > or = 9, and > or = 8 with saturating glutathione, gamma-L-Glu-L-Cys, and N-acetyl-L- cysteine, respectively. Apparently the enzyme has the ability to lower the pKa of glutathione by 3 orders of magnitude. The kcat/Km(thiol) did not vary appreciably with pH (except for N-acetyl-L-cysteine), indicating that no rate-determining deprotonation occurs on the enzyme itself between pH 5.5 and 9. The abilities of histidine-, lysine-, and arginine-selective reagents to inactivate the enzyme when N-acetyl-L-cysteine and gamma-L-Glu-L-Cys were used as substrates were investigated. The activity toward N-acetyl-L-cysteine was decreased considerably less after treatment with the arginine-selective reagent phenylglyoxal, as compared to the activity toward GSH and gamma-L-Glu-L-Cys. This indicates that an arginine makes contact with gamma-L-Glu residue in GSH. With the other reagent/substrate combinations tested the enzyme was inactivated almost completely. The ability of microsomal glutathione transferase to stabilize the Meisenheimer complex formation between 1,3,5-trinitrobenzene and various glutathione analogues, including non-substrate thiols, has been examined. It is shown that, in general, substrates exhibited higher formation constants (approaching 50 mM-1) than non-substrates (4.5 +/- 1.7 mM-1, n = 7), whereas simpler thiols did not yield enzyme-bound complexes. The fact that the enzyme can stabilize Meisenheimer complexes from non-substrate thiol analogues of glutathione offers new possibilities for examining the substrate interactions of glutathione transferases.

Acetylcysteine↗
PubMed2005

An investigation of prebiotic purine synthesis from the hydrolysis of HCN polymers.

The polymerization of concentrated NH4CN solutions has been studied at various temperatures and ammonia concentrations. The products of the oligomerization of ammonium cyanide include adenine and guanine, as well as trace amounts of 2,6-diaminopurine. Our results indicate that the adenine yield is not strongly dependent on temperature. Guanine is produced in lower yield. The original studies by Oró and Kimball (1961) showed that the 6 N HCl hydrolysis of the NH4CN polymerization supernatant greatly increased the adenine yield. However, this hydrolysis also decomposes adenine and other purines. Therefore, we have measured the yields from an NH4CN polymerization as a function of hydrolysis time, and found that shorter hydrolytic periods give higher yields of adenine. We have also investigated the hydrolysis of the supernatant at pH 8, which is a more reasonable model of primitive oceanic conditions, and found that the adenine yield is comparable to that obtained with acid hydrolysis (approximately 0.1%). The yield of adenine does not decline at longer hydrolysis times because of the greater stability of adenine at pH 8. The insoluble black polymer formed from NH4CN has been analyzed by both acid and neutral hydrolysis. In both cases adenine yields of approximately 0.05% were obtained. This suggests that the polymer may have been as important a prebiotic source of purines as the usually analyzed supernatant.

Adenine↗
PubMed2006

Second generation, orally active, antimalarial, artemisinin-derived trioxane dimers with high stability, efficacy, and anticancer activity.

In only two steps and in 63% overall yield, naturally occurring 1,2,4-trioxane artemisinin (1) was converted into C-10-carba trioxane conjugated diene dimer 4. This new dimer was then transformed easily in one additional 4 + 2-cycloaddition step into phthalate dimer 5, and further modification led to bis-benzyl alcohol dimer 7 and its phosphorylated analogues 8 and 9. Bis-benzyl alcohol dimer 7 is the most antimalarially active in vitro, 10 times more potent than artemisinin (1). Bis-benzyl alcohol dimer 7 is approximately 1.5 times more orally efficacious in rodents than the antimalarial drug sodium artesunate and is about 37 times more efficacious than sodium artesunate via subcutaneous administration. Both dimers 5 and 7 are thermally stable neat even at 60 degrees C for 24 h. Phthalate dimer 5 is very highly growth inhibitory but not cytotoxic toward several human cancer cell lines; both dimers 5 and 7 very efficiently and selectively kill human cervical cancer cells in vitro in a dose-dependent manner with no cytotoxic effects on normal cervical cells.

Administration, Oral↗