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Anchor fixation and other modifications of endoscopic bladder neck suspension.

The long-term efficacy of surgical treatment of stress urinary incontinence can be improved by modifications that reduce the possibility of suspending suture detachment. Fifty-three women with stress urinary incontinence underwent consecutive endoscopic bladder neck suspensions with new modifications developed in an effort to decrease suspending suture detachment. Those modifications included: (1) technique of needle passage to capture a maximum volume of urethropelvic fascia lateral to the bladder neck and urethra while avoiding injury to the bladder, (2) pubic bone fixation of the suspending suture using a small anchor developed for orthopedic use, and (3) a simple technique to limit tension of the suspending sutures. Procedures were outpatient in 60 percent of patients (93% of the last 27 patients). Seventy percent of patients did not require intermittent catheterization beyond the day when their indwelling catheter was removed. The postoperative success rate (absence of stress urinary incontinence) at one month was 100 percent. There were 4 failures on follow-up up to fifteen months. Urgency incontinence decreased from 59 percent preoperatively to 15 percent postoperatively. The complication of osteitis pubis was not noted. Patient rating of satisfaction postoperatively was high. These modifications constitute a safe alternative to procedures that effectively suspend the bladder neck. An assessment of any change in long-term efficacy as a result of these modifications will require continued follow-up.

Adult↗

A functional requirement for modification of the wobble nucleotide in tha anticodon of a T4 suppressor tRNA.

Temperature-sensitive mutants of E. coli have been isolated which restrict the growth of strains of bacteriophage T4 which are dependent upon the function of a T4-coded amber or ochre suppressor transfer RNA. One such mutant restricts the growth of certain ochre but not amber suppressor-requiring phage. Analysis of the T4 tRNAs synthesized in this host revealed that many nucleotide modifications are significantly reduced. The modifications most strongly affected are located in the anticodon regions of the tRNA'S. The T4 ochre suppressor tRNAs normally contain a modified U residue in the wobble position of the anticodon; it has been possible to correlate tha absence of this specific modification in the mutant host with the restriction of suppressor activity. Furthermore, the extent of this restriction varies dramatically with the site of the nonsense codon, indicating that the modification requirement is strongly influenced by the local context of the mRNA. An analysis of spontaneous revertants of the E. coli ts mutant indicates that temperature sensitivity, restriction of phage suppressor function, and undermodification of tRNA are the consequences of a single genetic lesion. The isolation of a class of partial revertants to temperature insensitivity which have simultaneously become sensitive to streptomycin suggests that the translational requirement for the anticodon modification can be partially overcome by a change in the structure of the ribosome.

Anticodon↗

Effect of chemical modification on the cryoprecipitation of monoclonal human cryoglobulin M.

The effect of the chemical modification of lysine, histidine, arginine, tyrosine, tryptophan residues and carboxylic groups on the cryoproperties of monoclonal human cryoglobulin M has been studied. The modification of 35-40 lysine residues and that of 42-45 arginine residues in the molecule of cryo-IgM has been shown to result in practically complete inhibition of the cryoprecipitation. The same effect is observed on the modification of 60 histidine residues per molecule and on modification of 50 or 51 carboxylic groups. At the same time the modification of practically all the reagent-exposed tryptophan (10 residues per molecule) and tyrosine residues (55 residues per molecule) does not lead to any noticeable decrease in the cryoprecipitation. The conformations of the modified and native proteins are identical according to the circular dichroism data.

Anisoles↗

Pseudophosphorylation of the smooth muscle 20 000 dalton myosin light chain. An artifact due to protein modification.

The use of isoelectric focusing as a technique for quantifying the stoichiometry of phosphorylation of the 20 kDa smooth muscle myosin light chain (LC20) was found to overestimate true levels of phosphorylation under certain conditions due to the occurrence of LC20 charge modification. Modification of unphosphorylated LC20 produced a band of 'pseudophosphorylated' LC20 which co-focused with phosphorylated LC20. LC20 modification was found to occur when samples were subjected to electrophoresis under nonreducing conditions in the presence of ammonium persulfate. The overestimation of LC20 phosphorylation due to pseudophosphorylation was examined for both purified myosin and extracts from contracting smooth muscle and found to be greatest at low levels of LC20 phosphorylation. A simple theoretical model was developed which accurately predicted the effects of charge modification on the measured level of phosphorylation. LC20 modification was shown to be completely eliminated by the inclusion of dithiothreitol in extraction buffers and the pre-electrophoresis of sodium thioglycolate into gels.

Ammonium Sulfate↗

Effect of chemical modifications on the K99 and K88ab fibrillar adhesins of Escherichia coli.

The role of specific amino acid residues of the K88ab and K99 fibrillar adhesins in the binding to erythrocytes and antibodies has been studied by chemical modification. It appeared that: (1) The integrity of the single disulfide bridge in the K99 subunits is essential for the binding of the fibrillae to the glycolipid receptors, but not for the recognition and binding of specific anti-K99 antibodies. (2) Modification of one lysine residue per subunit with 4-chloro-3,5-dinitrobenzoate results in the loss of the adhesive capacity of K99 fibrillae. Lysine residue are not important for the adhesive activity of K88ab fibrillae. Three or five lysine residues per subunit, respectively, can be modified without an effect on the immunological properties of the K99 and K88ab fibrillae. (3) Limited reaction of K99 and K88ab fibrillae with 2,3-butanedione destroys the adhesive activity of both fibrillae. This inactivation corresponds with the loss of one (K99) or two (K88ab) arginine residues per subunit. Ultimately, in K99 three, and in K88ab four, arginine residues per subunit can be modified without affecting the binding of specific antibodies. (4) Modification of five out of the nine carboxyl groups contained in the K99 subunit suppresses the recognition of specific anti-K99 antibodies, but carboxylates are not important for the adhesive activity of K99 fibrillae. Modification of two additional carboxylates in K99 results in an insoluble product. (5) Tyrosine residues are most probably not present in the adhesive or antigenic sites of K99 fibrillae. Modification of six out of the ten tyrosine residues in the K88ab subunit results in a decrease in adhesive activity but has no effect on the reaction with anti-K88ab antibodies.

Adhesins, Escherichia coli↗

Chemical modification of lysine and arginine residues in the myosin regulatory light chain inhibits phosphorylation.

The contribution of lysine and arginine residues to the substrate specificity of the myosin light-chain kinase has been studied using chemically modified myosin light chains. Succinylation or maleylation of the myosin light chains caused complete inhibition of their phosphorylation. Modification of 50% of the lysine residues resulted in 90% inhibition of phosphorylation and this was accompanied by a 25-fold increase in the apparent Km. In contrast, phosphorylation of the myosin light chains by the cAMP-dependent protein kinase was relatively insensitive to lysine modification, with only a 15% reduction in phosphorylation following succinylation of 50% of the lysine residues. Treatment with either cyclohexane-1,2-dione or camphorquinone-10-sulfonic acid resulted in between 90 and 98% inhibition of myosin light-chain phosphorylation. These reagents caused modification of both lysine and arginine residues, and accordingly only part of the inhibition can be attributed to arginine modification. Modification of all of the cysteine and methionine residues caused only a 40% inhibition of phosphorylation. The results of this study support the concept that lysine and arginine residues act as essential specificity determinants for the myosin light-chain kinase in protein substrates.

Animals↗

Chemical modification of a beta-glucosidase from Schizophyllum commune: evidence for essential carboxyl groups.

The beta-glucosidase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed Con A-Sepharose. The participation of carboxyl groups in the mechanism of action of the enzyme was delineated through kinetic and chemical modification studies. The rates of beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside were determined at 27 degrees C and 70 mM ionic strength over the pH range 3.0-8.0. The pH profile gave apparent pK values of 3.3 and 6.9 for the enzyme-substrate complex and 3.3 and 6.6 for the free enzyme. The enzyme is inactivated by Woodward's K reagent and various water-soluble carbodiimides; chemical reagents selective for carboxyl groups. Of these reagents, 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide in the absence of added nucleophile was the most effective and a kinetic analysis of the modification indicated that one molecule of carbodiimide is required to bind to the beta-glucosidase for inactivation. Employing a tritiated derivative of the carbodiimide, 44 carboxyl groups in the enzyme were found to be labelled while the competitive inhibitor deoxynojirimycin protected three residues from modification. Treatment of the enzyme with tetranitromethane resulted in the modification of five tyrosine residues with approx. 28% diminution of enzymic activity. Titration of denatured enzyme with dithiobis(2-nitro-benzoic acid) indicated the absence of free thiol groups. Reaction of the enzyme with diethyl pyrocarbonate resulted in the modification of four histidine residues with the retention of 78% of the original enzymatic activity. The divalent transition metals Cu2+ and Hg2+ were found to be potent inhibitors of the enzyme, binding in an apparent irreversible manner.

1-Deoxynojirimycin↗

Change of substrate specificity by chemical modification of lysine residues of porcine pancreatic alpha-amylase.

Lysine residues of porcine pancreatic alpha-amylase (PPA) were modified with trinitrobenzenesulfonate (TNBS). 6 out of 21 lysine residues were modified per PPA molecule. Amylase activity (hydrolysis of the alpha-1,4-D-glucoside bond) was decreased to about 50% of the native enzyme, as judged from the kcat value at pH 6.9 after the modification, whereas maltosidase activity (hydrolysis of p-nitrophenyl-alpha-D-maltoside producing p-nitrophenol and maltose) was increased to about 250%. The increase in maltosidase activity of the modified PPA was due to the increase in kcat, but not to the decrease in Km. Modification of PPA with five kinds of acid anhydrides also caused the same effect as TNBS, including the number of modified lysine residues. The degree of increase in maltosidase activity was fairly proportional to the volume of the incorporated modification reagent. A modification protection study in the presence of maltotriitol (G3OH), which protected two out of six modifiable lysine residues against modification, suggested that a lysine residue at the substrate-binding site contributes to the change of substrate specificity.

Anhydrides↗

Covalent modifications of chromosomal proteins during aging.

Covalent modifications of proteins introduce negative or positive charges into the molecules and thereby cause alterations in the ionic interactions of protein-protein or DNA-protein complexes. Whereas modifications of histones largely affect the organization of chromatin, those of non-histone proteins are believed to be involved in the expression of genes. These modifications during aging have been reviewed here. The available data suggest that the extent of covalent modifications of histones and non-histone chromosomal (NHC) proteins change during aging and such modifications may have an important role in the differential expression of genes at different phases of life span of an organism.

Acetylation↗

Covalent protein modifications and gene expression changes in rodent liver following administration of methapyrilene: a study using two-dimensional electrophoresis.

The effect of methapyrilene (MP), a mitochondrial proliferator and presumed nongenotoxic carcinogen, has been examined in rodent liver by means of high-resolution two-dimensional electrophoretic analysis of total proteins. Using this approach, we have discovered protein modifications in rat liver resulting from 1 week MP treatment that suggest the involvement of a reactive drug metabolite. The restriction of these molecular charge modifications to mitochondrial proteins indicates that such a reactive metabolite must be generated and confined within the mitochondrion. Quantitative changes in numerous nonmitochondrial proteins are also observed. Following a 4-week recovery period, almost all the 1-week treatment changes are reversed, reestablishing a protein pattern close to that of the controls. At the end of a 10-week exposure, the mitochondrial protein modifications are increased and are accompanied by a variety of quantitative protein changes indicative of a large shift in gene expression and/or cell type composition. When a 4-week untreated recovery period follows the 10-week treatment, small quantitative changes persist. In the mouse, where MP appears not to induce mitochondrial proliferation or tumorigenesis, 1 week treatment nevertheless produces mitochondrial protein changes in vivo consistent with attack by a reactive metabolite, but at a level substantially lower than that seen in the rat. Features of the mitochondrial protein modification indicate that it is covalent, does not involve cysteine or tryptophan, and results from binding of a negatively charged adduct. The possibility that the putative reactive metabolite could also attack mitochondrial (but not nuclear) DNA suggests that MP could be genotoxic in an unconventional way. Detection of protein modification by two-dimensional gel analysis appears to offer a general method for the detection and characterization of processes generating reactive metabolites.

Animals↗

Effects of digital grey-scale modification on the diagnosis of small approximal carious lesions.

The aim of this study was to evaluate the accuracy of approximal caries diagnosis from digitized radiographs and digitally modified radiographic images, compared with conventional radiography. Twenty bitewing radiographs were digitized and from the digitized radiographs mirror-images were produced, resulting in 40 digital images. The caries progress at three approximal surfaces was graded by 12 dentists from conventional radiographs, digital unmodified images and digitally modified images. The image modifications were derived from the cumulative probability of grey values in the original digital image using one of five probability distributions: uniform, exponential, Rayleigh, hyperbolic cube root and hyperbolic logarithmic. Interobserver agreement was substantial. The unmodified digital images produced sensitivities comparable to radiographs but their specificities were lower. When the diagnostic task was to discriminate between 'caries' and 'no caries', the exponential, hyperbolic cube root and hyperbolic logarithmic modifications and radiographs performed equally well. With the decision cut off between 'dentinal caries' and 'no dentinal caries', the sensitivity of the hyperbolic logarithmic type of modification was statistically significantly superior to that of radiographs, but this modification was also associated with a statistically significant reduction of specificity. It is concluded that in particular the hyperbolic logarithmic modification can be an alternative to conventional radiography in incipient approximal caries diagnosis and restorative decision making.

Color↗

Application of high-performance liquid chromatography for recognition of covalent nucleic acid modification with anticancer drugs.

Covalent modification of DNA by antineoplastic agents represents a potent biochemical lesion which can play a major role in drug mechanism of action. The ability to measure levels of DNA covalent modifications in target cells in vivo may, therefore, be seen as the ultimate form of therapeutic drug monitoring. Additionally, elucidation of the structure of critical DNA adducts and definition of their role in tumour cell cytotoxicity will provide more selective targets for rational drug design of new cancer chemotherapeutic agents. High-performance liquid chromatography has contributed significantly to all these areas. In vivo levels of nucleic acid covalent modifications are in the range of 1 in 10(5)-10(8) nucleotides precluding the use of conventional high-performance liquid chromatographic detection methods. Several classes of natural product anticancer drugs have been shown to bond covalently to nucleic acids under optimal laboratory conditions. These have proved more accessible to high-performance liquid chromatographic analysis because of their lipophilicity and strong UV chromophores. However, the majority of experimental evidence to date suggests that with the exception of mitomycin C and morpholino-anthracyclines these compounds do not exert their primary mechanism of action through nucleic acid covalent modification. DNA adducts of alkylating and platinating agents are more difficult to detect by high-performance liquid chromatography and can be chemically unstable. These compounds interact with DNA on the basis of chemical kinetics. Thus, the principle sites of attachment tend to be with the most nucleophilic base (guanine) at its most reactive centre (N-7 position). Limited in vivo high-performance liquid chromatographic studies with all classes of anticancer drugs indicate a much more complex pattern of adductation than would have been anticipated from in vitro studies alone. Some of these differences are probably due to methodological artefacts but these studies stress the need for sensitive detection methods and reliable sample preparation (nucleic acid extraction and digestion techniques) when attempting to determine nucleic acid covalent modifications by anticancer drugs.

Animals↗

Elimination of slow pathway conduction: an accurate indicator of clinical success after radiofrequency atrioventricular node modification.

OBJECTIVES: The purpose of this study was to determine the optimal end point of radiofrequency atrioventricular (AV) node modification using anatomically guided slow pathway approaches in patients with AV node reentrant tachycardia. BACKGROUND: The optimal end point for AV node modification using radiofrequency energy is uncertain, although elimination of inducible AV node reentrant tachycardia has been used. METHODS: We followed up 51 consecutive patients (40 women, 11 men, mean age +/- SD 41 +/- 16 years) with symptomatic AV node reentrant tachycardia for 12 +/- 6 months (range 4 to 24) after radiofrequency AV node modification using an anatomically guided slow pathway approach. Inducible AV node reentrant tachycardia was eliminated in all patients, whereas residual slow pathway conduction persisted in 12 patients (24%) after ablation. One study was complicated by complete AV block and two patients were lost to follow-up (one with and one without residual slow pathway conduction). RESULTS: Clinical recurrence of AV node reentrant tachycardia was documented in seven patients (14%) 3 days to 3 months (median 1 month) after ablation. The recurrence rate was significantly higher in patients with than in those without residual slow pathway conduction (6 [55%] of 11 vs. 1 [3%] of 37, p < 0.01). The recurrence rate was not different between patients with only residual slow pathway conduction and those with residual slow pathway conduction and inducible single echo cycles (three [60%] of five in both groups, p = NS). The number of radiofrequency energy applications was not significantly different between those without and those with recurrence (20 +/- 17 vs. 16 +/- 9, p = NS). Junctional tachycardia during application of radiofrequency energy tended to be more frequently observed in those with a successful outcome (77% vs. 57%, p > 0.05). Of the 22 patients who underwent modification before 1992, residual slow pathway conduction was present in 9 (41%) of 22 patients. Atrioventricular node reentrant tachycardia recurred in five (56%) of these nine patients. A greater effort made in 1992 to eliminate slow pathway conduction in 29 patients resulted in residual slow pathway conduction in only 3 (11%) with recurrence in 2 (4%). CONCLUSIONS: Complete elimination of slow pathway conduction is feasible in the majority of patients. Elimination of slow pathway conduction is highly predictive of long-term success after AV node modification using an anatomically guided approach.

Adult↗

Cell cycle modifications of breast cancers during neoadjuvant chemotherapy: a flow cytometry study on fine needle aspirates.

Breast cancer cells from 92 patients were obtained by repeated fine needle sampling and analysed by flow cytometry for cell cycle modifications during neoadjuvant chemotherapy. Modifications of the histograms were observed for 47 of the 71 informative cases (66%), the most frequent concerning S-phase (increase or decrease) and G2M accumulation. These modifications correlated well with the efficacy of cytotoxic chemotherapy (P < 0.0001). A significant relationship between clinical regression and pretreatment proliferative activity was also observed, with 31/35 (89%) responders in the high proliferation group (S-phase fraction > 5% or BrdU labelling index > 3.3%) compared to 20/36 (56%) in the low proliferation group (P < 0.002). For patients undergoing chemotherapy including doxorubicin, a high incidence of G2M accumulation was observed (33%), a modification which was rare (4.5%) for a regimen with no anthracycline, for which S-phase was the most frequently modified cell cycle compartment (64%). The measurement of the pretreatment tumour proliferative activity as well as the early kinetic modifications, as indicators of response, may prove interesting parameters for the future management of neoadjuvant chemotherapy.

Antineoplastic Combined Chemotherapy Protocols↗

Exploration of the effects of linker chain modifications on anti-HIV activities in a series of cosalane analogues.

The effects of linker chain modifications were investigated in a series of cosalane analogues. The modifications investigated included: (1) shortening the three-carbon linker chain between the dichlorodisalicylmethane and the cholestane moiety by one carbon atom; (2) lengthening the linker chain by one carbon; (3) hydrogenation of the double bond in the linker chain; (4) changing the point of attachment of the linker chain from C-3 to C-6; (5) insertion of a phosphate between the steroid and the linker chain. With the exception of the phosphate modification, which abolished anti-HIV activity and increased cytotoxicity, the linker chain modifications produced relatively minor changes in anti-HIV activity and increased cytotoxicity, the linker chain modifications produced relatively minor changes in anti-HIV potency. The steroid and attached linker chain of cosalane therefore appear only to provide a general lipophilic appendage for the dichlorodisalicylmethane pharmacophore.

Anti-HIV Agents↗

Modulation of collagen proteolysis by chemical modification of amino acid side-chains in acellularized arteries.

In this study, we have examined the effects of specific chemical modifications of amino acid side-chains on the in vitro degradation of "native" collagen obtained from acellular matrix (ACM)-processed arteries. Two monofunctional epoxides of different size and chemistry were used to modify lysine, with or without methylglyoxal modification of arginine. Biochemical, thermomechanical, tensile mechanical, and multi-enzymatic (collagenase, cathepsin B, acetyltrypsin, and trypsin) degradation analyses were used to determine the effects of modifications.Collagen solubilization by enzymes was found to depend upon the size and chemistry of epoxides used to modify lysine residues. In general, the solubilization of native ACM collagen by collagenase, cathepsin B, trypsin, and acetyltrypsin was either unaltered or decreased after modification with glycidol. In contrast, n-butylglycidylether (n-B) treatment increased solubilization by all enzymes. Subsequent arginine modification significantly reduced collagen solubilization by acetyltrypsin for glycidol-treated ACM arteries, whereas increased collagen solubilization was observed for n-B-treated ACM arteries with all enzymes. Gel chromatographic analyses of collagen fragments solubilized by trypsin revealed that both the amount and sites of cleavage were altered after lysine and arginine modification. The ability to modulate the enzymatic degradation of tissue-derived materials as demonstrated in this study may facilitate the design of novel engineering scaffolds for tissue regeneration or collagen-based drug delivery systems.

Amino Acid Sequence↗

A novel dry method for surface modification of SU-8 for immobilization of biomolecules in Bio-MEMS.

SU-8 has been primarily used for structural elements and microfludics components in MEMS. Microsystems for biological applications require immobilization of biomolecules on the MEMS structures. In order to functionalize SU-8 for such purposes, the surface needs to be modified. In this paper, we report a novel dry method of surface modification of SU-8 which is compatible with standard microfabrication techniques. The surface obtained by spin coating SU-8 (2002) on silicon wafer was modified by grafting amine groups using pyrolytic dissociation of ammonia in a hotwire CVD setup. To demonstrate the presence of amine groups on modified SU-8 surface, the surface characteristic after modification was assessed using Fourier transform infrared spectroscopy. The change in SU-8 surface morphology before and after surface modification was investigated using atomic force microscopy. To show the utility of this process for application in Bio-MEMS, SU-8 microcantilevers were fabricated and subjected to the same surface modification protocol. Following this, the cantilevers were incubated first in a suspension of human immunoglobulin (HIgG) and then in FITC tagged goat anti-human IgG in order to demonstrate the utility of the surface modification performed. The efficacy of the process was assessed by observing the cantilevers under a fluorescence microscope.

Acrylic Resins↗

Retinoic acid receptor beta2 is epigenetically silenced either by DNA methylation or repressive histone modifications at the promoter in cervical cancer cells.

To elucidate the silencing mechanism of retinoic acid receptor beta2 (RAR beta2) in cervical carcinogenesis, we investigated RAR beta2 expression and the status of both DNA methylation and histone modifications at the promoter in cervical cancer cell lines. RAR beta2 was frequently repressed in cancer cell lines and in primary cancers of the cervix. Although the majority of RAR beta2-negative cancers had methylated promoter, RAR beta2 was repressed with hypomethylated promoter in a substantial fraction of the cancers. The RAR beta2-negative cells with hypomethylated promoters showed a repressive histone modification pattern at the promoter. RAR beta2 was reactivated by a histone deacetylase inhibitor, accompanied by formation of active histone modifications. The repressive modification was also observed in cells repressed with hypermethylated promoter, but RAR beta2 was reactivated only by DNA demethylating agent and not by histone deacetylase inhibitor. Our results suggest that RAR beta2 is silenced by either of the two key epigenetic pathways, DNA methylation or repressive histone modifications, depending on the individual cancer cells.

Base Sequence↗