Selective monitoring of peptidase activities with synthetic polypeptide substrates and polyion-sensitive membrane electrode detection

A novel method to monitor specific peptidase activities in biological samples as complex as undiluted plasma/blood is described. The approach is based on the design of synthetic polypeptide substrates in which di- or triarginine sequences are linked to each other via one or more other amino acids recognized specifically by the peptidase to be determined. Detection of chymotrypsin and renin activities using synthetic substrates P4 (F-R-R-R-F-V-R-R-F-NH2) and P5 (R-R-R-L-L-R-R-L-L-R-R-R), respectively, serves to demonstrate the principles of this new assay system. A polyion-sensitive membrane electrode, prepared by doping polymer films with dinonylnaphthalene-sulfonate (DNNS), is shown to exhibit significant nonequilibrium electromotive force (EMF) responses toward these and other polycationic substrates at microgram/milliliter levels under physiological conditions. The same electrode, however, exhibits much smaller total EMF response toward the shorter fragments of the synthetic peptides generated by peptidase activity; hence, the addition of peptidase to a solution containing the synthetic substrate yields a change in electrode EMF response, the rate of which is proportional to the activity of peptidase present. Other synthetic polycationic peptides as well as natural polycationic peptides (e.g., protamine) that lack specific cleavage sites for chymotrypsin and renin, yet are detected by the DNNS-based membrane electrode, do not elicit any significant change in EMF response in the presence of the peptidases, confirming the feasibility and utility of the proposed bioanalytical method.     

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.     

Adolescent sexuality: Part 4. The practitioner''s role

As no systematic study has been done to get an accurate estimate of the incidence of return to oestrus after first insemination in sows in the Netherlands, the objectives of this investigation were: 1) to obtain an estimate of the incidence of return to oestrus after insemination at the herd level; 2) to investigate the association between incidence of return to oestrus after first insemination and reproduction characteristics in order to get an impression of the economic importance of reproductive failure. These objectives were investigated by using the reproduction results of 240 swine breeding herds in the Southern Netherlands in 1987. This information was obtained from CBK plus computerized herd management records. The average incidence rate of return to oestrus after first insemination at a herd level was 16.9 per 100 first inseminations. The occurrence of return to oestrus after first insemination was distinctly higher in the insemination months July and August compared to the rest of the year. An increased incidence, with 10 returns per 100 first inseminations corrected for confounders in a multiple linear regression model, was associated with a decrease of approximately 0.3 live born piglets/sow/year. A prospective longitudinal study was started in 1988 and 1989 in 37 sow herds. Individual sows were monitored from weaning to first insemination, to the occurrence of return to oestrus, or not, after first insemination, and to farrowing. The investigation focused in particular on the relationship between return to oestrus after first insemination and seroconversion against porcine parvovirus (PPV) and Leptospira interrogans serovar bratislava (L. bratislava).(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma osmolality and brain water content in a rat glioma model

Little is known about how intravenous fluids influence peritumoral edema formation. This experiment was designed to determine, in a rat glioma model, whether changes in plasma osmolality alter water content, as assessed by specific gravity (SpGr), in normal and neoplastic cerebral tissue. Cells cultured from an ethylnitrosourea-induced rat glioma were stereotactically implanted into the right striatum of Fischer 344 rats. A tumor growth interval of 21 days was allowed. In a second experiment, rats underwent a 60-second cortical freeze injury followed by 24 hours' recovery. In both experiments, rats were assigned to one of three groups: hypotonic (100 ml/kg of 0.2 mol/L NaCl in H2O, intraperitoneally; resultant plasma osmolality approximately 268 mOsm/kg); isotonic (no treatment; plasma osmolality approximately 298 mOsm/kg); or hypertonic (10 ml/kg of 1.0 mol/L NaCl in H2O, intraperitoneally; plasma osmolality approximately 342 mOsm/kg). Thirty minutes after fluid injection, regional SpGr was determined using a kerosene-bromobenzene gradient. In subsets of rats, the tissue morphology and blood-brain barrier permeability of Evans blue dye were assessed. Tissue within the freeze lesion was stained by Evans blue dye with sharp demarcation. Evans blue dye did not stain gliomatous tissue, and central necrosis was not histologically evident. In isotonic rats, glioma SpGr was reduced (1.0411 +/- 0.0012 g/ml) relative to the contralateral striatum (1.0437 +/- 0.0008 g/ml; P < 0.001). Despite this, a strong linear relation was observed for SpGr and plasma osmolality in both neoplastic and normal tissue. Within the freeze lesion in isotonic rats, SpGr was severely reduced (1.0335 +/- 0.0008 g/ml; P < 0.0001) compared with contralateral frontal cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some Properties of Electroless Ni-P,Co-P,and Ni-Co-P Deposits

The electroless deposition of Ni-P, Ni-Co-P and Co-P alloys has been investigated. The effects of pH on the chemical composition of the deposits and the induced stresses have been explored. The magnetic and electrochemical properties and the mechanical hardness of the deposits have been studied after annealing at various temperatures and are related to the crystal structures.     

Reductive electrochemical remediation of emerging and regulated disinfection byproducts

Long-term exposure to low concentrations of disinfection byproducts (DBPs) in drinking water has been associated with increased human-health risks of bladder cancer and adverse reproductive outcomes. In this study, we investigated electrochemical reduction utilizing a resin-impregnated graphite cathode for the degradation of 17 DBPs (i.e. halomethanes, haloacetonitriles, halopropanones, chloral hydrate and trichloronitromethane) at low μg L⁻¹ concentration levels. The reduction experiments were potentiostatically controlled at cathode potentials −700, −800 and −900 mV vs Standard Hydrogen Electrode (SHE) during 24 h. At the lowest potential applied (i.e. −900 mV vs SHE), the disappearance of DBPs from the solution after 24 h of reduction was >70%, except for chloroform (32%), 1,1-dichloropropanone (48%), and chloral hydrate (31%). Due to the participation of several removal mechanisms (e.g. electrochemical reduction, adsorption, volatilization and/or hydrolysis) it was not possible to distinguish the removal efficiencies of electrochemical reduction of individual compounds. Adsorption of the more hydrophilic DBPs (i.e. haloacetonitriles, chloral hydrate, and 1,1-dichloropropanone) onto the electrode seems to be affected by the cathode polarization, as the removals observed in the open circuit experiments were significantly higher than the ones obtained in electrochemical reduction under the same conditions. The overall efficiency of reduction was estimated based on the analyses of the released Cl⁻, Br⁻ and I⁻ ions. Nearly complete C-I bond cleavage was achieved at all three potentials applied, and from the theoretically predicted release of I⁻ ions, calculated based on the removed DBPs, 86 ± 9 to 92 ± 1% was measured in the catholyte solution at −700 to −900 mV vs SHE. Debromination efficiencies obtained were 74 ± 3, 79 ± 6 and 68 ± 4% at −700, −800 and −900 mV vs SHE, while for C-Cl bond cleavage the obtained values were 69 ± 1, 72 ± 1 and 76 ± 4%, respectively. Nevertheless, dechlorination efficiencies are to be considered as approximate, since an increase in Cl⁻ concentration was observed in the open circuit experiments due to the hydrolysis of some of the chlorine-containing DBPs. Although the Coulombic efficiencies for DPBs dehalogenation were only 1.9 ± 0.3 (−900 mV vs SHE) -4.1 ± 0.2% (−700 mV vs SHE), relatively low energy consumption of the process was observed, estimated at 72 ± 2 Wh m⁻³ at −900 mV vs SHE for the concentration range of DBPs in this study (i.e. 65.3-129.7 μg L⁻¹). The study demonstrated that reductive electrochemical treatment has the potential to be a modern remediation technology for the removal of low concentrations of halogenated DBPs in water.     

Lens design for a white-light cosine-transform achromat

We describe the lens design for a twin-imaging white-light interferometer in which the interference pattern at the exit-pupil plane is the cosine transform of the spatial-intensity distribution of the object. The achromatic condition in terms of optical power is derived. The analysis of the transform aberration shows that the even aberrations, e.g., spherical aberration and field curvature, do not degrade the cosine transform and need not be corrected. This significant simplification permits us to design systems with good performance and uncomplicated lens structures. We present a lens design with three elements and a length of 320 mm. The system is capable of resolving more than 10(6) pixels with an operating spectral bandwidth of 100 nm. The results of an experiment with an early four-element design are also presented.     

Confirmation of fenthion metabolites in oranges by IT-MS and QqTOF-MS

Identification of degradation products of the organophophorous pesticide fenthion formed in two orange varieties, Valencia Navel and Navel Late, under field conditions has been assessed using liquid chromatography quadrupole time-of-flight mass spectrometry and ion trap mass spectrometry. The structural elucidation of the metabolites was accomplished by the accurate mass measurements provided by the quadrupole time-of-flight mass spectrometer in MS and MS/MS modes. This instrument achieved elemental composition diagnosis for the precursor and product ions with absolute mass error of <5 ppm, which unambiguously establishes the identity of the metabolites even at low concentration. The presence of these compounds was also confirmed by electrospray ionization-ion trap mass spectrometry, performing successive fragmentation steps (MS(n)). Once identified, each molecule was confirmed by comparison with its analytical standard, also used to explore the quantitative capabilities of both mass analyzers. The extraction method was evaluated because it predetermines the metabolites that can be found (e.g., according to their polarity). Recoveries ranged from 70% for fenoxon sulfoxide (the most polar) to 101% for fenthion (the most apolar), which also indicates the method's facility to extract other more polar metabolites if present. Satisfactory linear range (r > 0.99) of more than 2 orders of magnitude was obtained with both analyzers for standards prepared in methanol and in untreated orange extracts. However, the matrix-matched standards showed suppression of the mass signal due to the matrix effect, especially for fenoxon sulfoxide and sulfone. The limits of quantification ranged from 0.005 to 0.015 mg/kg. The QqTOF-MS provided better quantification limits for fenthion and its sulfoxide and sulfone than the IT-MS. The resulting fenthion degration curves in oranges indicated that it was mainly degraded by sunlight photolysis to its sulfoxide and sulfone. However, hydrolysis was also observed by the appearance of fenoxon, fenoxon sulfoxide, and fenoxon sulfone, but always in low concentrations, which can be related to the rain events.     

The mechanical properties of model-compacted tablets

In this study, the compressive strength of tablets made with salt, starch and fat was investigated. The strength was found to increase with compaction pressure, up to a maximum value where further increase in the compaction pressure led to no increase in the strength. The maximum strength corresponded to the point where zero porosity was obtained during the compaction process. However, because of the elastic rebound of the tablets after ejection, the maximum strength corresponded to non-zero final tablet porosities which varied between the materials. For this reason, the use of the density occurring during the compaction process appeared to provide a more reliable comparison between the materials. A simple linear mixing rule did not hold in characterising the strength in the salt:starch:fat systems. However, two regimes were observed depending on the salt volume fraction. At low salt volume fractions, the effect of the salt was negligible. After a certain critical salt volume fraction, increasing the salt led to an increase in the strength. Finite element simulations based on X-ray microtomography images of the tablets suggested that in the first regime, the stresses due to the salt particles were localised but in the second regime, stress-bearing networks were formed between the salt particles.

---