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Structure and assembly of calf hoof keratin filaments.

Keratin filament polypeptides were purified from calf hoof stratum corneum with the aim of studying the in vitro assembly process and determining structural parameters of reconstituted filaments. Anion exchange chromatography was used to obtain the most complete fractionation and identification of the acidic and basic components in the purified polypeptide mixture to date. The reassembly products of the fractionated components were investigated by electron microscopy. Fully reconstituted filaments yield homogeneous solutions, and values of 9.8 nm for the filament diameter and 25 kDa/nm for the mass per unit length (M/L) were obtained by X-ray solution scattering. The structures formed in solution at various stages of filament assembly were not sufficiently homogeneous to be studied by this technique. X-ray diffraction patterns from native stratum corneum display strong maxima at 3.6 and 5.4 nm. Contrary to previous reports, these maxima do not appear to be due to lipids since they are also observed with delipidated rehydrated specimens. A series of weak maxima is also detected in the patterns of dry tissue. The absence of these features in the patterns of reconstituted filaments suggests that, in contrast to some electron microscopic observations, there are no prominent regularities in the structure of calf hoof keratin filaments.

Animals↗

Control of the pathogenic conformational change of the prion protein by an attractor of low order.

The molecular vibrations of the infectious part of the prion protein has been studied by the methods of nonlinear dynamics using NMR-data of the protein in solution to test a new hypothesis for the nature of the pathogenic isomerization. The result was that the conformational change accompanying the conversion of the physiological form (PrP(c)) to the pathological (PrP(Sc)) one displays the characteristic properties of an attractor of the dimension 2.7+/-0.2, whereas the dimensions of the C-terminal, potentially infectious half of the physiological and the pathological forms are 5.3+/-0.3 and 3.9+/-0.3, resp. A plot of the average RMS of the vibrations per atom of the amino acids along the peptide chain suggests a pivotal role of E(167) and D(196). The vibrations of these residues are strongly dampened by the pathogenic conformational change suggesting that the neutralization to the hydrophobic form facilitates the isomerisation. This observation lends credence to the hypothesis that the pathological conformational change is released by the charge neutralization of the abundant, basic side chains lining the cleft in PrP(c) by a pK-shift caused by a transient ion flux from an action potential or by an RNA ligand. The attractor ensures that only the two conformers PrP(c) and PrP(Sc)-monomer, which are switching the polyadenylation of mRNA for synaptic proteins on and off, prevail. Some xenogenic PrP(Sc) seem to have a tendency to polymerize irreversibly, which is likely to inhibit the translation, thus killing neurons. The autocorrelation function is dampened (long correlation length for the 3 cases), the Poincaré plot seems to show the cross-section of a dense attractor for PrP(Sc) and a loose one for PrP(c), the Lyapunov exponent is positive and the power spectrum is broad. The Hurst plots of PrP(Sc) and PcP(c) show monofractality. The attractor hypothesis offers a supplement, or an alternative, to the current, inconclusive ideas of the nature of the PrP isomerization. The identification of the control factors may permit the reversal of the fatal conformational change. The dynamic parameters were checked by the interpoint distance method of Judd and by an analysis of the Fourier-transformed data.

Animals↗

Kinetic study of photocatalytic degradation of carbamazepine, clofibric acid, iomeprol and iopromide assisted by different TiO2 materials--determination of intermediates and reaction pathways.

The light-induced degradation of clofibric acid, carbamazepine, iomeprol and iopromide under simulated solar irradiation has been investigated in aqueous solutions suspended with different TiO2 materials (P25 and Hombikat UV100). Kinetic studies showed that P25 had a better photocatalytic activity for clofibric acid and carbamazepine than Hombikat UV100. For photocatalytic degradation of iomeprol Hombikat UV100 was more suitable than P25. The results can be explained by the higher adsorption capacity of Hombikat UV100 for iomeprol. The study also focuses on the identification and quantification of possible degradation products. The degradation process was monitored by determination of sum parameters and inorganic ions. In case of clofibric acid various aromatic and aliphatic degradation products have been identified and quantified. A possible multi-step degradation scheme for clofibric acid is proposed. This study proves the high potential of the photocatalytic oxidation process to transform and mineralize environmentally relevant pharmaceuticals and contrast media in water.

Adsorption↗

Immunocytochemical localization of epidermal growth factor receptors in human testis from infertile subjects.

OBJECTIVE: To investigate the immunolocalization of the epidermal growth factor receptor (EGFR) in normal and pathological human testis by immunocytochemical technique. DESIGN: Cytologic specimens were obtained by bilateral fine needle aspiration (FNA) of the testis and stained in May Grünwald-Giemsa for the cytologic analysis; immunolocalization of EGFR was analyzed on duplicate slides from each testis using two anti-EGFR monoclonal antibodies and peroxidase-antiperoxidase technique. SETTING: Infertility center of an academic unit. PATIENTS: A total of 42 infertile patients, affected by various testicular diseases. The control group was made up of 10 normal sperm patients with autoimmune infertility and cytologic picture of normal spermatogenesis. INTERVENTIONS: Exogenous FSH was administered 75 IU IM on alternate days for 3 months on 16 of the infertile patients who showed oligospermia and normal FSH plasma levels. Semen analysis and testicular FNA (and after cytologic and immunocytochemical studies) were repeated at 3 months of treatment. MAIN OUTCOME MEASURES: Luteinizing hormone and FSH plasma levels were determined by RIA methods; qualitative and quantitative parameters for the cytologic evaluation are reported in our previous works. RESULTS: The cytologic analysis permitted identification of seven classes of infertile subjects, characterized by different cytologic pictures. Epidermal growth factor receptor immunostaining evidenced weak positivity on Sertoli and germ cells (with the exception of spermatozoa) in the presence of normal germ line and normal FSH plasma levels and strongly intense positivity in the presence of serious hypospermatogenesis, spermatogonial or spermatocytic arrest, and Sertoli cell-only syndrome. These conditions were characterized by higher FSH plasma levels than normal controls. All of the subjects who received exogenous FSH, with moderate hypospermatogenesis or spermatidic arrest, showed on Sertoli and germ cells a weak EGFR immunostaining before the treatment and intense immunostaining after the treatment. CONCLUSIONS: These results confirm recent demonstrations of EGFRs in human testis and evidence different EGFR immunostaining in the presence of various degrees of testicular damage, suggesting a role of this growth factor in growth and differentiation of the germ cells throughout spermatogenesis. The observation that intense EGFR immunostaining was found in subjects showing high FSH plasma levels and in all of the patients who received exogenous FSH, supports a possible role of this gonadotropin in the modulation of the EGFR expression.

Adult↗

Determination of quaternary ammonium pesticides by liquid chromatography-electrospray tandem mass spectrometry.

A method for the direct determination of paraquat, diquat, chlormequat and difenzoquat in water samples, using an on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry system was developed. No sample preparation was required and the detection limits were below the European Union maximum residue levels. The chromatographic separation was performed using an XTera MS C8 column. The concentration of the ion pair reagent, the pH and the gradient elution were optimized to give high recoveries and good chromatographic resolution between quats. The detection was carried out using an ion trap as mass analyzer. Parameters such as the magnitude and duration of the resonant excitation voltage and the magnitude of the trapping RF voltage for full scan tandem mass spectrometry (MS-MS) experiments were studied to establish the optimal experimental conditions. Moreover, the accurate optimization of these parameters allowed MS-MS experiments of low mass ions, below m/z 200, providing unambiguous peak identification. Finally, the reproducibility of the proposed method was shown by good run-to-run and day-to-day precision values and its applicability to the determination of quats in drinking water was evaluated using spiked samples.

Chlormequat↗

Phytochemical and pharmacological studies on Orthosiphon stamineus Benth. (Lamiaceae) hydroalcoholic extracts.

The main components of Orthosiphon stamineus Benth. leaves and extracts are the pharmacologically active polyphenols: the polymethoxylated flavonoids and the caffeic acid derivatives. Two tinctures, having different alcohol concentration, were studied from phytochemical and pharmacological point of view. The main polyphenols were identified and quantitatively determined by HPLC. Comparison of the retention parameters and UV-Vis spectra of standards and those of the separated compounds performed the identification of caffeic-, cichoric- and rosmarinic acids, respectively, of sinesetine and eupatorine. The quantitative determination was performed by external standard method. The diuretic, saluretic and uricosuric actions of the studied tinctures were compared by experiments on rats.

Animals↗

Echo correlation analysis and the acoustic evidence in the Kennedy assassination revisited.

A Dallas Police Department recording contemporaneous with the Kennedy assassination contains five impulsive sounds that have the acoustic waveform of Dealey Plaza gunfire. One of the sounds matches the echo pattern of a test shot fired from the Grassy Knoll. The shock wave precedence associated with this pattern is consistent with the muzzle velocity of a .30 calibre rifle. Criticism of the acoustic identifications on statistical grounds is based on erroneous assumptions concerning the assignment of values to the parameters that determine the probability that random noises could resemble gunshot patterns. A conservative estimate of the true value of the probability that the putative Grassy Knoll shot is attributable to random radio noise is no greater than 0.037. Alleged asynchroneity of the sounds with the time of the assassination stemmed from several incorrect assumptions. Whatever their origin, the gunshot-like sounds occur exactly synchronous with the time of the shooting.

Journal Article↗

Historical variation of elements with respect to different geochemical fractions in recent sediments from Pigeon Lake, Alberta, Canada.

Geochemical analysis of elements and organic matter were conducted on vertical profiles of the recent sediments from Pigeon Lake, Alberta, Canada, to determine historical variations in elemental content of the sediments as related to their geochemical fractions. The elements are grouped according to their affinity with different geochemical fractions, by using cluster analysis and sequential extraction experiments. As a result, four elemental fractions were identified: clastic mineral detritus; carbonate; organic; and elements that show less similarity to the previous groups perhaps due to anthropogenic input or the influence of other fractions, such as oxyhydroxides. Following the identification of geochemical fractions in the sediments, a three-step normalizing method was applied using parameters that represent each geochemical fraction. These normalizing techniques appear to be important in verifying whether the variation of elements is indeed the result of anthropogenic and/or natural activities. The normalized data are correlated with the recent history of human activity and natural events near Pigeon Lake. Given the age of the lake sediments, this correlation indicates that the depth profiles of elements after being normalized to the organic and carbonate fractions reflect the variation of detrital input into the lake. However, the former mainly corresponds to the coarse-grained clastic minerals originating from high-energy erosion as the result of natural events (e.g., flooding), whereas the latter corresponds to the low-energy erosion of the fine particles (enriched in lithophile elements) due to deforestation in the drainage basin. Normalizing to the clastic mineral detritus fraction results in the increase of heavy metals in the uppermost part of the sediment profiles, which coincides with industrial activities during the past two decades in central Alberta. However, the concentration of these elements is negligible, as compared to the quantities released by geogenic processes (erosion).

Alberta↗

Hydrogen-exchange stability analysis of Bergerac-Src homology 3 variants allows the characterization of a folding intermediate in equilibrium.

Amide hydrogendeuterium exchange rates have been determined for two mutants of alpha-spectrin Src homology 3 domain (WT), containing an elongated stable (SHH) and unstable (SHA) distal loop. SHA, similarly to WT, follows a two-state transition, whereas SHH apparently folds via a three-state mechanism. Native-state amide hydrogen exchange is effective in ascribing energetic readjustments observed in kinetic experiments to species stabilized within the denatured base and distinguishing those from high-energy barrier crossings. Comparison of DeltaG(ex) and m(ex) parameters for amide protons of these mutants demonstrates the existence of an intermediate and allows the identification of protons protected in this state. The consolidation of a form containing a prefolded long beta-hairpin induces the switch to a three-state mechanism in an otherwise two-state folder. It can be inferred that the unbalanced high stability of individual elements of secondary structure in a polypeptide could ultimately complicate its folding mechanism.

Amino Acid Sequence↗

Long-term prognosis of acute otitis media in infancy: determinants of recurrent acute otitis media and persistent middle ear effusion.

OBJECTIVES: Children under 2 years of age with acute otitis media are known to have a relatively poor prognosis. The objective of this study was to assess whether known determinants for recurrent acute otitis media and persistent middle ear effusion after an episode of acute otitis media during childhood also apply to children under 2 years. METHODS STUDY DESIGN: prospective study of 210 children under 2, with identification of potential prognostic determinants. Univariate and multivariate logistic regression analyses were applied to evaluate which parameters independently contributed to the prediction of both outcome measures (recurrent acute otitis media and persistent middle ear effusion). A prognostic function was developed, and the area under the receiving operating characteristic (ROC) was used to estimate the predictive ability of the prognostic models. POPULATION: children under 2 years of age with an episode of acute otitis media in family practice. OUTCOMES MEASURED: recurrent acute otitis media and persistent middle ear effusion. RESULTS: For the outcome recurrent acute otitis media data from 210 children were used and winter season, male sex, passive smoking and persistent symptoms for more than 10 days at presentation were independent prognostic determinants. For the outcome persistent middle ear effusion data from 190 children were used and winter season, bilateral disease at entry, a sibling history of recurrent acute otitis media, and a previous episode of acute otitis media independently predicted the outcome. No sufficiently discriminatory prognostic model could be constructed for either outcome measure. CONCLUSION: Prediction of recurrent acute otitis media or persistent middle ear effusion in individual young children remains poor.

Acute Disease↗

History and evolution of methods for solving the inverse problem.

This article serves as an introduction to the other articles in this issue devoted to the problem of the localization of neural generators. Elements of the theory of electric volume conduction are briefly introduced, as far as these apply to the interpretation of observed scalp potentials. First, some basic methods for display of the different aspects of the spatiotemporal information are described. Next, the most prominent source and volume conductor models that have been postulated for the involved forward problem are summarized. The problems of source identification and source localization, known as the inverse problem, are then formulated in terms of a parameter estimation procedure. The importance of introducing a priori information in the inverse problem, aimed at stabilizing (regularizing) the obtained solution, is emphasized. Methods for imposing such constraints are briefly outlined.

Biophysics↗

P300 component identification using source analysis techniques: reduced latency variability.

P300 latency variability in normal subjects is a complicating factor in clinical event-related potential studies because it limits diagnostic applicability. The current study was conducted to determine whether identification of P300 (P3A and P3B) components using source analysis techniques can reduce variability in P300 parameters. Data were recorded with a 128-channel EEG system in 18 healthy subjects. The authors used a standard, auditory two-tone oddball paradigm with targets of 2000 Hz and standards of 1000 Hz. Two simple source analysis models with one or two rotating dipoles were applied to grand average data and individual data. Dipole time courses were combined with mapping results to extract P3A and P3B component latencies. Latencies obtained with conventional P300 analysis were compared with source analysis results. The source analysis method identified both P3A and P3B components in a substantially larger percentage of subjects (88% vs. 33%) than the conventional method. The source analysis method yielded a later mean P3B latency (357 msec vs. 323 msec, P < 0,001) with a smaller standard deviation (9 msec vs. 23 msec, P = 0,003) than the conventional P300 method. The relative contribution of the temporally separate P3A and P3B components to the P300 complex amplitude is highly variable. This explains the larger latency standard deviation in conventional P300 analysis. The source analysis method was able to identify P300 components in a large percentage of the cases. The result is a considerable reduction of P300 latency variability in normal subjects. This could have important consequences for clinical event-related potential research, because diagnostic sensitivity and specificity of P300 latency may improve with this method.

Adult↗

Population pharmacokinetics of APOMINE: a meta-analysis in cancer patients and healthy males.

AIMS: 1) To characterize the population pharmacokinetics of apomine in healthy males and in male and female patients with solid tumours and 2) to understand more fully the influence of induction and between- and within-subject variability on exposure to drug using Monte Carlo simulation. METHODS: Apomine was administered once- or twice-daily with or without food in single and multiple oral doses of 30-2100 mg to healthy males (n = 19) and patients with solid tumours (n = 19). The data were divided into model development and validation sets. Models were developed using standard population methods. These were the identification of an appropriate base model, calculation of the empirical Bayes estimates of the primary pharmacokinetic parameters, covariate screening, forward stepwise addition of covariates using the likelihood ratio test as a model selection criteria, and backwards elimination to obtain the final model. To study the influence of data from individual subjects, the model development dataset was subjected to the delete-1 jack-knife and the final model was fitted to each jack-knifed dataset. Principal components analysis of the jack-knifed matrix of model parameters identified two influential subjects who were removed from the dataset, and the final model contained data from the remaining subjects. Model validation was examined using goodness of fit statistics and relative error measures using independent datasets from cancer patients. The model provided a reasonable approximation to the pharmacokinetic measurements in the validation datasets. Computer simulations were undertaken to understand further the pharmacokinetics of apomine in otherwise healthy females, a population not yet studied. RESULTS: Apomine pharmacokinetics were complex and consistent with a two-compartment model with a lag-time. Apparent oral clearance at baseline and apparent volume of distribution at steady-state were larger in healthy males than in cancer patients (41 ml h(-1) and 14.1 l vs 10 ml h(-1) and 8.9 l, respectively, for a 75 kg person). Clearance was time-variant showing a maximal increase with full induction of 320 ml h(-1), independent of patient type. The time to reach 50% maximal induction was about 2 days. The fraction of drug absorbed was relatively constant at doses less than 100-200 mg once daily but decreased at higher doses. Food also decreased relative bioavailability by 36%. Patient characteristics had no effect on apomine pharmacokinetics except for weight, which was proportional to the volume of the central compartment. Between-subject variability (68% for clearance, 30% for central volume, and 141% for peripheral volume) was moderate to large and independent of patient type. Inter-occasion variability was small (18% for both clearance and central volume). Residual variability was modelled with an additive and proportional error model. Cancer patients had slightly higher plasma concentrations than healthy males but this difference was probably not clinically significant. Steady-state was reached in about 3-4 days after once-daily drug administration. The half-life of apomine after three weeks of once-daily dosing was 41 h in cancer patients and 32 h in healthy males. CONCLUSIONS: A population model for apomine has been developed has been developed that characterizes its pharmacokinetics in cancer patients and healthy subjects under a variety of conditions.

Adolescent↗

Acute phase reactant proteins in prostatic cancer.

A profile of acute phase reactant proteins has been studied in patients with cancer of the prostate as an aid to diagnostic staging and therapy control. A linear discriminant function analysis incorporating serum acid phosphatase, prealbumin, alpha1 antitrypsin, alpha1 acid glycoprotein and haptoglobin allows the correct identification of metastatic disease in 88.6% of patients. The profile may also serve to augment other parameters in the assessment of the physiological effect of oestrogen treatment and show whether the prescribed medication is being taken.

Acid Phosphatase↗

Hemodialysis grafts: color Doppler flow imaging correlated with digital subtraction angiography and functional status.

Twenty-two patients with hemodialysis grafts were prospectively evaluated with color Doppler flow imaging and digital subtraction angiography (DSA). Eighteen patients had normal functional parameters during hemodialysis, and four had increased venous pressure during hemodialysis. Color Doppler flow imaging allowed identification of nine macroaneurysms related to wall degeneration, two cases of spontaneous fistula formation between the graft and peripheral veins, and 20 stenoses. Use of color Doppler flow imaging led to overestimation of the degree of stenosis at the venous anastomosis when compared with use of angiography. Three cases of subclavian venous stenosis were identified only at angiography. Color Doppler flow imaging appears accurate in the detection of stenoses and seems sufficient for follow-up of normally functioning grafts. However, because of its low sensitivity for identification of proximal stenoses and the necessity of obtaining an angiogram to plan surgical or percutaneous correction, DSA remains the technique of choice.

Angiography, Digital Subtraction↗

Identifying marker genes in transcription profiling data using a mixture of feature relevance experts.

Transcription profiling experiments permit the expression levels of many genes to be measured simultaneously. Given profiling data from two types of samples, genes that most distinguish the samples (marker genes) are good candidates for subsequent in-depth experimental studies and developing decision support systems for diagnosis, prognosis, and monitoring. This work proposes a mixture of feature relevance experts as a method for identifying marker genes and illustrates the idea using published data from samples labeled as acute lymphoblastic and myeloid leukemia (ALL, AML). A feature relevance expert implements an algorithm that calculates how well a gene distinguishes samples, reorders genes according to this relevance measure, and uses a supervised learning method [here, support vector machines (SVMs)] to determine the generalization performances of different nested gene subsets. The mixture of three feature relevance experts examined implement two existing and one novel feature relevance measures. For each expert, a gene subset consisting of the top 50 genes distinguished ALL from AML samples as completely as all 7,070 genes. The 125 genes at the union of the top 50s are plausible markers for a prototype decision support system. Chromosomal aberration and other data support the prediction that the three genes at the intersection of the top 50s, cystatin C, azurocidin, and adipsin, are good targets for investigating the basic biology of ALL/AML. The same data were employed to identify markers that distinguish samples based on their labels of T cell/B cell, peripheral blood/bone marrow, and male/female. Selenoprotein W may discriminate T cells from B cells. Results from analysis of transcription profiling data from tumor/nontumor colon adenocarcinoma samples support the general utility of the aforementioned approach. Theoretical issues such as choosing SVM kernels and their parameters, training and evaluating feature relevance experts, and the impact of potentially mislabeled samples on marker identification (feature selection) are discussed.

Acute Disease↗

Applications of biochemical epidemiology in the study of human carcinogenesis.

The measurement of biochemical parameters in epidemiologic studies can contribute to a better understanding of mechanisms of chemical carcinogenesis. Through identification and quantitation of chemicals in tissues or body fluids or through markers of "effective dose" such as DNA adducts, mechanisms of cancer etiology and pathogenesis can be elucidated. Different approaches and the associated methodologic problems are discussed along with examples. As a comprehensive case study, the relationship between aromatic amine exposure and bladder cancer is considered in light of different biochemical aspects which could be the target of epidemiologic studies.

Amines↗

Utilization of substrate-induced quenching for screening targets promoting NADH and NADPH consumption.

Oxidation of reduced nicotinamide adenine dinucleotides is a common event for many biochemical reactions. However, its exploitation for ultrahigh-throughput screening purposes is not an easy task and is affected by various drawbacks. It is known that such nucleotides induce quenching on the fluorescence of several dyes and that this quenching disappears with oxidation of the nucleotide. We have made use of this property to develop an assay for high-throughput screening with NADH and NADPH-dependent reductases. Full screening campaigns have been run with excellent assay quality parameters, and interesting hits have been identified. The method is amenable to miniaturization and allows easy identification of false positives without needing extra secondary assays. Although it is based on monitoring substrate consumption, it is demonstrated that the effect of fractional conversion on assay sensitivity is negligible.

Biological Assay↗