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PubMed1986

Thermal inactivation of human immunodeficiency virus in lyophilised blood products evaluated by ID50 titrations.

Inactivation of human immunodeficiency virus (HIV) in lyophilised small pool cryoprecipitate, factor VIII concentrate, prothrombin complex and C1-esterase inhibitor concentrate by prolonged heat treatment (72 h, 60 degrees C) was studied. Plasma products, inoculated prior to lyophilisation, had infectious titres ranging from 10(7) to 10(10.5). Residual infectivity (TCID50) was assessed by multiple titrations on H9 cells in a macro system and subsequent detection of virus replication by determining reverse transcriptase activity. Kinetics of inactivation showed a biphasic pattern: during the first 8 h a variable TCID50 reduction up to 10(4.3) was observed, followed by an additional loss of 10(1)-10(2.7) during the next 64 h. Heat treatment for 72 h resulted in a mean TCID50 reduction of 10(5). It is concluded that prolonged heat treatment may lead to the adequate prevention of HIV transmission by lyophilised plasma products.

Acquired Immunodeficiency Syndrome↗
PubMed1977

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. 3. 1H and 31P nuclear-magnetic-resonance studies on the phosphocarrier protein HPr; tyrosine titration and denaturation studies.

The phosphocarrier protein HPr has been investigated by proton nuclear magnetic resonance (NMR) at 270 MHz in order to evaluate structural properties of the whole molecule and its active site. The titration behaviour of the three tyrosines of the HPr protein was analysed by monitoring the chemical shifts of the aromatic proton resonances of these residues as a function of pH. It was found that the HPr protein contains a lot of slowly exchanging NH backbone protons which suggested a relatively rigid secondary structure of the protein molecule itself although it contains no disulfide bridges. The HPr protein shows a sharp reversible denaturation behaviour at alkaline pH values. Between pH 10.8 and 11.1 two C-2 proton resonance peaks for the single histidine residue could be observed together with abrupt changes in the aromatic and aliphatic absorption region of the HPr protein which are due to chemical exchange processes. The NMR spectrum of the HPr protein is only changed a little upon raising the temperature from 14 degrees C to 70 degrees C. At 76 degrees C all resonances in the spectrum broaden and almost disappear. This process is irreversible.

Bacterial Proteins↗
PubMed1977

Small-angle X-ray titration study on the complex formation between 5-S RNA and the L18 protein of the Escherichia coli 50-S ribosome particle.

The 5-S RNA (A) and the L18 protein (B) from Escherichia coli ribosomes form one single AB complex in the concentration ranges supposed to prevail in vivo; at concentrations of L18 higher than 40 mM there is some indication for a minor species, most probably an AB2 species. This is indicated from the X-ray scattering titration data of the 5-S RNA/L18 system recorded at 21 degrees C in ribosomal reconstitution buffer. As a result of the 1:1 complex formation, there is a relatively small but defined increase in the radius of gyration from 3.61 to 3.85 nm. This result as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L18 model is quite far from the electron density centre and where protein L18 interacts with one or both of the minor arms of the supposed Y-shaped 5-S RNA molecule.

Escherichia coli↗
PubMed1979

The ternary 5-S RNA complex of proteins L 18 and L 25. A small-angle X-ray scattering titration study.

The 5-S RNA (A) and the proteins L 18 (B) and L25 (C) from Escherichia coli ribosomes form a ternary complex of the type ABC with a stepwise stability constant, log K111 approximately equal to 6.5. This is indicated from X-ray scattering titrations recorded at 21 degrees C in ribosomal reconstitutional buffer. When the ternary ABC complex forms there is only a limited change in the scattering curve compared to that of 5-S RNA, indicating that 5-S RNA does not undergo a major conformational change during the complex formation. The increase in the radius of gyration from 3.61 nm (5-S RNA) to 3.95 nm (ABC complex) as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L 18 and L25 models are quite far from the electron density centre and where the protein molecules interact mainly with the minor arms of the supposed Y-shaped 5-S RNA molecule.

Escherichia coli↗
PubMed1980

Binding of manno-oligosaccharides to concanavalin A. Substitution titration with a fluorescent-indicator ligand.

The association constants for binding of methyl alpha-D-mannopyranoside (I), mannobiose (II) and mannotriose (III) to concanavalin A were determined in the temperature range 285-313 K by a substitution titration, using 4-methylumbelliferyl alpha-D-mannopyranoside as a carbohydrate-specific and fluorescent indicator. All binding equilibria are simple, but establish extremely slowly with II and III. At 298.3 K, K increases moderately from I to III: (6.4 +/- 0.5) x 10(3), (1.2 +/- 0.1) x 10(4) and (1.10 +/- 0.05) x 10(5) M-1. For binding of I, II and III, the - delta H degree values are constant (36 +/- 2 kJ mol-1) and equal to the average value (36.1 +/- 0.6 kJ mol-1) obtained for the three corresponding 4-methylumbelliferyl alpha-D-manno-oligosaccharides [Van Landschoot, A., Loontiens, F. G., and De Bruyne, C. K (1978) Eur. J. Biochem. 83, 277-285]. The data are interpreted as arising from specific binding to a single mannopyranosyl residue in (alpha 1 leads to 2)-linked manno-oligosaccharides.

Concanavalin A↗
PubMed1991

N-Butyl-N-methyl-4-nitrophenyl carbamate as a specific active site titrator of bile-salt-dependent lipases.

The effect of a series of synthetic carbamates on the human (milk or pancreatic) bile-salt-dependent lipase (cholesterol esterase) was examined. N-isopropyl-O-phenyl, N-methyl-O-phenyl, N-butyl-(4-nitrophenyl), N-phenyl-(4-nitrophenyl), N-butyl-N-methyl and N-pentyl-O-phenyl carbamates were inhibitors of the enzyme activity, while O-isopropyl-N-phenyl, O-methyl-N-phenyl, O-benzyl-N-isopropyl and O-cyclohexyl-N-phenyl carbamates were not even recognized by the enzyme. The N-alkyl chain length is essential for the enzyme inhibition and N-butyl-(4-nitrophenyl) or N-pentyl-O-phenyl carbamates are more potent inhibitors than N-methyl-O-phenyl or N-isopropyl carbamates. The inhibition by reactive carbamates fits the criteria for mechanism-based inhibition: the inhibition is first-order with time, shows saturation kinetics with increasing carbamate concentration and leads to an inactive stoichiometric enzyme-inhibitor complex; the enzyme activity can be protected by a competitive inhibitor. Evidence is shown that the enzymatic nucleophilic attack of carbamates is directed at the carbonyl carbon atom and not the nitrogen atom. The inhibition of bile-salt-dependent lipase does not occur consecutive to the formation of a reactive isocyanate derivative of carbamate but via a tetrahedral intermediate involving essential residues implicated in the enzyme catalytic site. This intermediate evolves by liberation of alcohol (or phenol) and formation of an inactive carbamyl enzyme. Among the carbamates tested, N-butyl-N-methyl-(4-nitrophenyl) carbamate specifically inhibits the bile-salt-dependent lipase; the release of 4-nitrophenol from this carbamate is directly proportional to the enzyme inhibition and it may be defined as a specific active-site titrator for bile-salt-dependent lipases.

Animals↗
PubMed1991

Sensitive titration microcalorimetric study of the binding of Salmonella O-antigenic oligosaccharides by a monoclonal antibody.

The binding of several oligosaccharide haptens by a monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-antigen was studied by titration microcalorimetry. In the software developed by Wiseman et al. [Wiseman, T., Williston, S. & Brandts, J.F. (1989) Anal. Biochem. 17, 131-137] the number of binding sites/macromolecule is one of the optional regression parameters in the non-linear least-squares analysis of the calorimetric data. Instead, an approach was adopted in which the concentration of binding sites was treated as a regression parameter, obviating the requirement for precise values of antibody absorption coefficients and minimizing effects due to partially inactive antibody preparations. Furthermore, performing the least-squares analysis in two steps, first using a differential heat mode and then an integral heat mode, was shown to yield the most accurate results. The technique gave accurate results using not more than 1-2 mumol ligand and less than 7 mg antibody. Haptens 2-5 were oligomers of the O-antigenic repeating unit varying in chain length by 2-5 repeating units and a trisaccharide glycoside 1, which filled the binding site. The latter hapten exhibited a favourable entropy contribution to binding (delta Go = -31 kJ.mol-1; delta Ho = -21 kJ.mol-1 and -T delta So -10 kJ.mol-1), while all four oligomers 2-5 showed a constant binding energy delta Go = -33 kJ.mol-1, composed of increasingly stronger enthalpy forces compensated by an increasingly unfavourable entropy contribution. These observations are compared with results from enzyme immunoassays and a high-resolution crystal structure for the dodecasaccharide 3 bound to the Fab derived from Se155-4.

Antibodies, Monoclonal↗
PubMed1995

NMR studies and redox titration of the tetraheme cytochrome c3 from Desulfomicrobium baculatum. Identification of the low-potential heme.

The tetraheme cytochromes c3 isolated from two strains of Desulfomicrobium baculatum were studied by monitoring the spectral changes undergone during redox titrations followed by 1H NMR. The evolution of the three-protein intensity signals at low field allowed the partial identification of the heme methyl resonances in the spectrum of the fully oxidized state. The chemical shift variation shown by the protons of the aromatic sidechains as well as of the substituents of the higher-potential heme HIII [Coutinho, I. B., Turner, D. L., LeGall, J. & Xavier, A. V. (1993) Biochem. J. 294, 899-908] yielded the assignment of the lower midpoint redox potential to heme HII in the three-dimensional structure. This cross-assignment is achieved by comparing the chemical shifts of the resonances in the spectra obtained at intermediate oxidation levels with the pseudocontact shifts predicted to arise from the three lower-potential hemes. The cross-assignment for the cytochromes from these two strains is different from that of the cytochromes from Desulfovibrio vulgaris and Desulfovibrio gigas.

Cytochrome c Group↗
PubMed1997

Redox titration of two [4Fe-4S] clusters in the photosynthetic reaction center from the anaerobic green sulfur bacterium Chlorobium vibrioforme.

Anaerobic green sulfur bacteria contain photosynthetic reaction centers analogous to photosystem I (PS I) of plants and cyanobacteria. These reaction centers, termed type I, are characterized by the presence of bound iron-sulfur clusters as the terminal electron acceptors. In this work, the iron-sulfur clusters in Chlorobium vibrioforme were studied using selective light-induced reduction protocols, spin quantifications, and chemical redox titrations coupled with EPR detection. Illumination of a dark-frozen sample at 12 K results in the appearance of a spectrum termed signal I. Chemical reduction in darkness at solution potentials between -414 mV and -492 mV results in the appearance of a different spectrum termed signal II. Illumination of these chemically poised samples at 12 K results in the appearance of signal I such that the sum of the intensity of signal I + signal II is nearly constant for every ratio of signal I/signal II. As the solution potential is lowered to -545 mV, the spectrum shifts to yet a third set of resonances, termed signal III. Concomitant with this shift is a loss of low temperature light-induced reduction of signal I. Photoaccumulation of a sample poised at a solution potential of -50 mV results also in the appearance of signal III at nearly the same spin concentration as the chemically reduced sample. Spin quantifications imply that signals I and II are both derived from the reduction of one iron-sulfur cluster, termed center I; signal III is derived from simultaneous reduction of two iron-sulfur clusters, centers I and II. By measuring the EPR signal intensities over a range of solution potentials, centers I and II were shown to have Em (pH 10.0) values of -446 mV and -501 mV, respectively. The observations are consistent with a structural and functional analogy of centers I and II with F(A) and F(B) of PS I.

Anaerobiosis↗
PubMed1998

A dose titration study on the effect of virginiamycin on gilt/sow and piglet performance.

Virginiamycin (VM), a compound with proven performance-promoting properties in pigs, can also be considered as sow performance enhancer. The present dose titration study investigates the long-term effects of VM on gilt/sow and litter performance. A total of 140 healthy gilts, in 35 sets of 2 + 2 siblings each, were used in this trial; gilts in each set were randomly allocated to the four treatments (35 gilts per each treatment group): VM0 = negative control, virginiamycin 0 mg/kg, VM20 = virginiamycin 20 mg/kg, VM40 = virginiamycin 40 mg/kg and VM60 = virginiamycin 60 mg/kg of feed. VM was added to the gilt/sow feed for a period starting from 6 months of age up to conception of the fourth parity, covering three complete breeding cycles (pregnancy, lactation and weaning-to-conception interval). The feed given did not contain any other antibacterial agent or performance enhancer. Piglets were given exactly the same quantity of creep feed from the fifth day onwards; it did not contain any antibacterial agent or performance enhancer. The lactation period was the same in all gilts/sows (21 days). Results indicate that VM supplementation of the feed improved gilt/sow performance by: (i) increasing (P < 0.05) the mean body weight at each farrowing, (ii) decreasing (P < 0.05) the mean weight loss from farrowing to weaning, and (iii) decreasing (P < 0.05) the mean weaning-to-conception intervals. VM supplementation of gilt/sow feed also resulted in improved litter size and litter performance in terms of: (i) a higher (P < 0.05) mean number of piglets born alive at each birth, (ii) a higher (P < 0.05) mean number of piglets weaned, (iii) a higher (P < 0.05) mean piglet body weight at each birth, and (iv) a higher (P < 0.05) mean piglet body weight at each weaning. These beneficial effects of VM were more pronounced in the VM40 group.

Animals↗
PubMedDate not supplied

Clinical evaluation of titration of hepatitis C virus core antibody and its subclasses.

Titrations of anti-hepatitis C core (anti-HCc) immunoglobulin G (IgG) antibodies and its subclasses were studied in 90 patients with acute and chronic hepatitis C virus (HCV) infection, including 27 patients who underwent interferon (IFN) therapy. The positivity rates for each anti-HCc subclass were as follows: 95.2% for IgG1, 12.0% for IgG2, 69.9% for IgG3 and 19.3% for IgG4. The total anti-HCc IgG titre correlated well with the IgG1, titre, indicating that IgG1 was the main virus-specific IgG. Changes of IgG1 production mainly contributed to fluctuations of the anti-HCc IgG titre and corresponded well to positivity for HCV-RNA during and after IFN therapy. IgG3 was detected prior to IgG1 during the early phase of acute hepatitis in some cases and also appeared with relapse after IFN therapy. The serial assay of anti-HCc subclasses showed the patients' humoral immune response to HCV infection, and might be useful for evaluation of anti-viral immunity influenced by IFN therapy.

Acute Disease↗
PubMed2006

Up-titration of vardena fi l dose from 10 mg to 20 mg improved erectile function in men with spinal cord injury.

AIM: Vardenafil is a highly selective phosphodiesterase type-5 inhibitor for the treatment of erectile dysfunction (ED). Efficacy of vardenafil has been demonstrated in various ED populations, but that in Japanese patients with spinal cord injury (SCI) has not been assessed. METHODS: This was an open-label, multicenter, flexible dose, 12-week study in patients with ED due to SCI. Following a 4-week observation period, patients received vardenafil 10 mg for 4 weeks, and based on efficacy, tolerability and patient preference, doses for the remaining 8 weeks were decided by investigators. The primary efficacy parameter was erectile function domain score of the International Index of Erectile Function. RESULTS: Ten patients took 10 mg all through the study, while 22 patients took 20 mg after completing 4 weeks' treatment with 10 mg. The erectile function domain score increased from 12.2 at baseline to 25.0 at Last Observation Carried Forward (LOCF) in the former group and from 10.3 to 22.5 in the latter group, respectively. Importantly, there was a 5.0 point increase in erectile function domain score after up-titration in the latter group. Drug-related adverse events were observed in 22% of patients including hot flushes (9%) and headache (6%), but these were transient and mild in intensity. Serious adverse events and adverse events leading to discontinuation of the study drug were not reported. CONCLUSIONS: Vardenafil 10 and 20 mg was well tolerated and improved erectile function in patients with SCI. Of interest, erectile function was further improved by 20 mg in patients who were not sufficiently treated with 10 mg.

Adult↗
PubMed1986

Dose titration in clinical trials. An example using emepronium carrageenate in detrusor instability.

Seventy-two women with proven detrusor instability completed a novel clinical trial of the new anticholinergic emepronium carrageenate (Cetiprin novum), which allowed for dose titration. The tolerated dose has been shown to correlate with the severity of symptoms. Statistically significant improvement was achieved in symptomatic and urodynamic parameters without serious side effects.

Adult↗
PubMed2006

Attempted forced titration of blood pressure to <130/85 mm Hg in type 2 diabetic hypertensive patients in clinical practice: the diastolic cost.

The authors assessed the practicality and results of forced titrating of blood pressure to <130/85 mm Hg based on guidelines of the sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure in the setting of a clinical practice in 257 diabetic, hypertensive patients. Goal diastolic pressure was achieved in 90% of the patients, but goal systolic pressure was achieved in only 33%. In 57% of the patients, the attained diastolic pressure was < or =70 mm Hg, and in 20% of the cohort diastolic pressure was reduced to <70 mm Hg (mean, 60+/-1 mm Hg). Patients with final diastolic pressure <70 mm Hg were older, had a higher prevalence of coronary artery disease, and higher initial systolic and pulse pressures compared with patients with final diastolic pressure of 71-85 mm Hg. Thus, attempted lowering of blood pressure to <130/85 mm Hg is associated with excessive lowering of diastolic pressure in a significant number of patients. Whether the benefits of tight systolic control outweigh the risks of excessive diastolic reduction requires further prospective assessment.

Aged↗
PubMed2007

Titration of HCTZ to 50 mg daily in individuals with stage 2 systolic hypertension pretreated with an angiotensin receptor blocker.

The authors studied the combination of hydrochlorothiazide (HCTZ) 50 mg/d plus olmesartan medoxomil (OM) 40 mg/d in stage 2 systolic hypertension during an extension phase of an open-label 12-week dose titration study. Subjects whose blood pressure remained above 120/80 mm Hg (n=105) on OM 40/HCTZ 25 mg/d subsequently received OM 40/HCTZ 50 mg/d for 4 weeks. Increasing HCTZ from 25 mg/d to 50 mg/d decreased systolic blood pressure by 3.6 mm Hg, increased BP control rates (<140/90 mm Hg) from 70.4% to 77.5%, and increased BP normalization rates (<120/80 mm Hg) from 15.4% to 27.8%. The combination was well tolerated. Compared with OM 40 mg/d monotherapy, neither dose of HCTZ affected serum potassium, but both increased serum glucose by about 5%. There was a dose-dependent increase in uric acid but no acute gout attacks. OM 40/HCTZ 50 mg/d is an effective strategy for managing stage 2 systolic hypertension.

Angiotensin II Type 1 Receptor Blockers↗
PubMed2000

Gabapentin versus vigabatrin as first add-on for patients with partial seizures that failed to respond to monotherapy: a randomized, double-blind, dose titration study. GREAT Study Investigators Group. Gabapentin in Refractory Epilepsy Add-on Treatment.

PURPOSE: Our objective was to compare the efficacy and safety of gabapentin and vigabatrin as first-line add-on treatment in patients with partial epilepsy. METHODS: This was a multicenter, double-blind, randomized dose titration study. After baseline assessment and randomization, the dose could be increased if seizures persisted and reduced if side effects occurred. Health-related quality of life was assessed at baseline and at the end of the study. By a protocol amendment post hoc, all randomized patients were offered a standardized perimetry examination at the end of the study. Improvement rate was the proportion of patients with a reduction of seizure frequency of at least 50% during an 8-week period without any adverse events causing withdrawal. RESULTS: One hundred two patients were randomized and analyzed on an intent-to-treat basis. The improvement rate was 48% in the gabapentin group and 56% in the vigabatrin group. The improvement rate, when per protocol criteria were fulfilled, was 57% in the gabapentin group and 59% in the vigabatrin group. The proportion of seizure-free patients was 31% in the gabapentin group and 39% in the vigabatrin group. There was no difference in quality-of-life scores between the groups. Perimetry after termination of the study on 64 patients showed abnormal results in 3 of 32 patients in the vigabatrin group. CONCLUSION: Approximately one third of the patients in both groups became seizure-free. Although no major differences were seen in terms of the improvement rate between the groups, equivalence between the two drugs was not found.

Acetates↗
PubMed2000

Identification of iron-regulated genes of Helicobacter pylori by a modified fur titration assay (FURTA-Hp).

The Escherichia coli-based Fur titration assay (FURTA), although a powerful tool for identification of genes regulated by the ferric uptake regulator (Fur), was unsuccessful for the gastric pathogen Helicobacter pylori. The FURTA was modified by construction of an E. coli indicator strain producing H. pylori Fur only. The promoter regions of the ferric citrate receptor homolog fecA2 and the riboflavin synthesis gene ribBA were both positive in the modified FURTA, but negative in the original FURTA. Transcription of fecA2 and ribBA was demonstrated to be iron-repressed in H. pylori. This type of modification should allow FURTA analysis for bacteria with Fur binding sequences poorly recognized by E. coli Fur.

Amino Acid Sequence↗
PubMed1982

An improved foot-shock titration procedure in rats for centrally acting analgesics.

A foot-shock titration method for the measurement of antinociceptive activity in the rat has been developed. A new grid design is described which makes the use of scrambled shocks unnecessary. Thresholds for responses elicited at different levels of integration within the central nervous system are measured: Detection threshold, flexor reflex, coordinated jumping and vocalization. These thresholds were found to be stable over time in saline-treated rats. The two opioid analgesics morphine and d-propoxyphene increased these thresholds, each in a characteristic way. Morphine was more selective in its action and significantly more effective in increasing the vocalization threshold than the threshold for jumping, whereas d-propoxyphene increased both these thresholds to the same extent. Neither drug affected the detection threshold in low to moderate doses. The difference in pharmacodynamic profile is discussed in terms of heterogeneity of opioid receptor-effector mechanisms at different levels of the neuroaxis.

Analgesics↗