Phase Identification in Multi-Phase Cu-Zn/Cu-Al Alloys with Macroscopic Concentration Gradients

In order to study concentration invariant phase transitions in Cu-Zn-Al alloys which occur in a very narrow concentration range samples with concentration gradients were produced. Cylinders made of brass and Cu-Al bronze were joined by diffusion bonding and subsequently annealed. The samples exhibit multi-phase regions (α, β, γ1, martensite) with changing compositions and very different chemical properties. Standard techniques for the metallographic preparation of Cu alloys usually target specific phases within limited concentration ranges. To evaluate the microstructure, a two-stage chemical etching method using sodium hydroxide solution and subsequent selective colour etching was successfully used to gain an overview of the phases present in different areas along the concentration gradient. For the characterization of martensite, its birefringent optical properties were utilized. In polarized light, details of the martensitic structure are revealed which would otherwise only be accessible by scanning electron methods.

2-Hydroxy-N-phenylbenzamides and Their Esters Inhibit Acetylcholinesterase and Butyrylcholinesterase

The development of novel inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) represents a viable approach to alleviate Alzheimer’s disease. Thirty-six halogenated 2-hydroxy-N-phenylbenzamides (salicylanilides) with various substitution patterns and their esters with phosphorus-based acids were synthesized in yields of 72% to 92% and characterized. They were evaluated for in vitro inhibition of AChE from electric eel and BuChE from equine serum using modified Ellman’s spectrophotometric method. The benzamides exhibited a moderate inhibition of AChE with IC50 values in a narrow concentration range from 33.1 to 85.8 µM. IC50 values for BuChE were higher (53.5–228.4 µM). The majority of derivatives inhibit AChE more efficiently than BuChE and are comparable or superior to rivastigmine—an established cholinesterases inhibitor used in the treatment of Alzheimer’s disease. Phosphorus-based esters especially improved the activity against BuChE with 5-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl diethyl phosphite 5c superiority (IC50 = 2.4 µM). This derivative was also the most selective inhibitor of BuChE. It caused a mixed inhibition of both cholinesterases and acted as a pseudo-irreversible inhibitor. Several structure-activity relationships were identified, e.g., favouring esters and benzamides obtained from 5-halogenosalicylic acids and polyhalogenated anilines. Both 2-hydroxy-N-phenylbenzamides and esters share convenient physicochemical properties for blood-brain-barrier penetration and thus central nervous system delivery.

TLC Determination of Mebendazol and Pentoxifylline as Residues on Pharmaceutical Equipment Surfaces

This paper presents the applicability of thin-layer chromatographic methods with a subsequent densitometric or video densitometric quantitation for determination of residues in controlling pharmaceutical equipment cleanliness. Analytical methods were developed for monitoring residues of pentoxifylline at 10 mg/m2 and mebendazol at 1 mg/m2 on stainless steel surfaces. Simulated samples were prepared by addition of a calculated amount of pharmaceutical (as a solution) on a 35 × 35 cm stainless steel surface. After evaporation of solvent, the residues were wiped with wetted cotton. The cotton was extracted with dichloromethan–methanol (1 + 1). Filtered extract was concentrated by vacuum evaporation and an aliquot applied to the plate, where standards were also applied. In the narrow concentration range near the acceptable residue limits, linear calibration curve could be obtained for both substances. The mean recovery (n = 4) obtained by densitometric quantitation was 93.4% for pentoxifylline and 85.6% for mebendazol, with coefficients of variation of 3.5 and 8.3%, respectively. Results of video densitometric quantitation did not differ significantly. However, data acquisition and evaluation is faster compared with densitometry and allows better archiving possibilities as required by the regulatory authorities. Both quantitation modes can be applied to routine control of pharmaceutical equipment cleanliness.

Threonine synthesis from aspartate in Escherichia coli cell-free extracts: pathway dynamics

We have developed an experimental model of the whole threonine pathway that allows us to study the production of threonine from aspartate under different conditions. The model consisted of a desalted crude extract of Escherichia coli to which we added the substrates and necessary cofactors of the pathway: aspartate, ATP and NADPH. In this experimental model we measured not only the production of threonine, but also the time dependence of all the intermediate metabolites and of the initial substrates, aspartate, ATP and NADPH. A stoichiometric conversion of precursors into threonine was observed. We have derived conditions in which a quasi steady state can be transiently observed and used to simulate physiological conditions of functioning of the pathway in the cell. The dependence of threonine synthesis and of the aspartate and NADPH consumption on the initial aspartate and threonine concentrations exhibits greater sensitivity to the aspartate concentration than to the threonine concentration in these non-steady-state conditions. A response to threonine is only observed in a narrow concentration range from 0.23 to 2mM.

How external osmolarity affects the activity of the contractile vacuole complex, the cytosolic osmolarity and the water permeability of the plasma membrane in Paramecium multimicronucleatum

The rate of fluid expulsion, R(CVC), from the contractile vacuole complex (CVC) of Paramecium multimicronucleatum was estimated from the volume of the contractile vacuoles (CVs) immediately before the start of fluid discharge and from the time elapsing between discharges. The R(CVC) increased when the cell was exposed to a strongly hypotonic solution and decreased in a weakly hypotonic solution. When the cell was exposed to an isotonic or a hypertonic solution, R(CVC) fell to zero. The time constant, tau, used to describe the change in R(CVC) in response to a change in external osmolarity shortened after a short-term exposure to a strongly hypotonic solution and lengthened after a short-term exposure to a less hypotonic solution. A remarkable lengthening of tau occurred after a short-term exposure to isotonic or hypertonic solution. Under natural conditions, mechanisms for controlling R(CVC) are effective in maintaining the cytosolic osmolarity hypertonic within a narrow concentration range despite changes in the external osmolarity, which is normally hypotonic to the cytosol. Cells exposed to an isotonic or hypertonic solution resumed CV activity when left in the solution for 12 h. The cytosolic osmolarity was found to increase and to remain hypertonic to the external solution. This will permit cells to continue to acquire water. The increase in the cytosolic osmolarity occurred in a stepwise fashion, rather than linearly, as the external osmolarity increased. That is, the cytosolic osmolarity first remained more-or-less constant at an increased level until the external osmolarity exceeded this level. Thereupon, the cytosolic osmolarity increased to a new higher level in 12 h, so that the cytosol again became hypertonic to the external solution and the cells resumed CV activity. These results imply that the cell needs to maintain water segregation activity even after it has been exposed to an isotonic or hypertonic environment. This supports the idea that the CVC might be involved not only in the elimination of excess cytosolic water but also in the excretion of some metabolic waste substances.

Belousov-Zhabotinskii oscillation reaction with glyceraldehyde

An oscillation system of Belousov-Zhabotinskii type with aldehyde as substrate is described for the first time. Oscillations of the concentration of Mn(III) ions can be followed spectrophotometrically or potentiometrically with a Pt electrode in a relatively narrow concentration range of the substrate. In contrast to the analogous reaction with malonic acid, no oscillations of the concentration of Br- ions were detected by a bromide ion selective electrode. This together with the fact that the concentration of Mn(III) ions oscillates even at relatively high bromine concentrations suggests that oscillations of the concentration of the redox catalyst Mn(II)/Mn(III) are probably controlled by organic radicals rather than by Br- ions.

Effects of detergents and cytochrome c binding on scalar and vectorial proton ejection by proteoliposomes containing cytochrome oxidase

The detergent lauryl maltoside abolishes respiratory control and proton ejection by cytochrome c oxidase-containing proteoliposomes over a narrow concentration range. Expression of cryptic activity (inward-facing oxidase) is released over the same concentration range. Catalytic functions (Vmax. and Km) of the enzyme are not changed by the detergent. Lipid micelles containing detergent bind approximately the same amount of cytochrome c as do vesicles containing an equivalent amount of lipid. Uncoupler-insensitive proton release is seen when proteoliposomes are pulsed with ferrocytochrome c at low ionic strength. Such uncoupler-insensitive acidification is not seen at higher ionic strength, nor with oxygen pulses of anaerobic solutions previously incubated with cytochrome c. Vesicles at low ionic strength catalyse cytochrome c autoxidation; this process can mimic proton re-equilibration in systems that have pumped protons from inside to the bulk phase. Proton re-equilibration following a pulse of cytochrome c or oxygen is multiphasic. The slowest phases are attributed to vesicle heterogeneity, some internal alkali being retained within vesicles of low intrinsic proton permeability. This can be overcome by the addition of either very low levels of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone or high levels of valinomycin.

Properties of chitin synthase from mucoraceous hosts of a mycoparasite

Crude chitin synthase extracted from young (24 h) hyphae of Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts, respectively, to the mycoparasite, Piptocephalis virginiana, was identified and characterized by measuring the incorporation of the substrate [14C]UDP – N-acetylglucosamine into chitin. The enzyme activity was mainly associated with the mixed membrane fraction. Properties of the enzyme preparation such as activation with proteases, N-acetylglucosamine, magnesium, inhibition with polyoxin D, Vmax, apparent Km value for UDP – N-acetyl-D-glucosamine (UDP-GlcNAc), and response to pH were examined. Enzyme activity from both fungi displayed basically the same features as the corresponding enzymes reported from other mucoraceous fungi. However, the two enzyme preparations (from P. articulosus and C. cucurbitarum) differed from each other in their response to various proteases and storage at 4 °C. Enzyme preparation from P. articulosus was activated by all proteases, whereas the C. cucurbitarum preparation was activated by acid protease, slightly activated by trypsin over a narrow concentration range, and was inhibited by neutral protease. Enzyme preparation from C. cucurbitarum showed a rapid decrease in activity within the first 5 h of storage at 4 °C and also exhibited relatively higher activity of endogenous proteases than that from P. articulosus.