Aspects of neural plasticity in the central nervous system—V. Studies on a model of transient forebrain ischemia in male Sprague-Dawley rats

A morphological and functional characterization of the four-vessel occlusion model of transient (30 min) forebrain ischemia has been carried out. The rats were classified as fully ischemic when an isoelectric pattern of electroencephalographic activity was present within 5 min of the occlusion of carotid arteries. Otherwise they were considered as partially ischemic rats. The modifications of cerebral blood content during and after the ischemic insult were assessed by a histochemical method which visualizes red blood cells in cerebral vessels. The periods of increase and decrease of red blood cell content were found to correspond to previous reports of post-ischemic hyper- and hypoperfusion. Neuronal damage was assessed by a quantitative analysis of Nissl stained preparations of cingulate cortex, dorsal hippocampus and striatum. The signs of morphological damage were quantified by means of computer-assisted image analysis of Nissl preparations. The highest vulnerability to the ischemic insult was demonstrated in the pyramidal layer of the hippocampal CA1 field and in the lateral striatum. Arterial blood pressure measurements were performed during the ischemic and post-ischemic periods, demonstrating a peak increase of arterial blood pressure within 2 min after carotid artery occlusion, followed by a slow decrease towards basal levels during the ischemic period and a full recovery within 15 min of reperfusion. Ischemic rats were tested in a neurological test battery and in a passive avoidance task. While a full recovery of the relatively simple tasks of the neurological test battery was attained within 14 days of reperfusion, a highly significant impairment of passive avoidance behavior was still present 15 days after the ischemic insult. Finally, a discriminant analysis was applied to separate, on the basis of non-invasive techniques (neurological tests and hot plate), the group of completely ischemic rats from that of partially ischemic rats.     

Enzymatic preparation of 20 beta-hydroxysteroids in a two-phase system.

The behavior of 20 beta-hydroxysteroid dehydrogenase in a two-phase system consisting of an emulsion of water with an immiscible organic solvent was investigated. The effect of several organic solvents on the stability, activity, and kinetic profile of the enzyme was considered. The most suitable system for carrying out the enzymatic reaction proved to be water-butyl acetate. The production of high quantitied of 20 beta-hydroxysteroids in 100% yield using catalytic amount of cofactor was achieved by coupling the 20 beta-hydroxysteroid dehydrogenase-and the alcohol dehydrogenase-catalyzed reactions.     

Molecular organization in bacterial cell membranes II. Reevaluation and identification of some chemical components of Micrococcus lysodeikticus membranes

Standard membranes of Micrococcus lysodeikticus were prepared by protoplastlysis in the presence of 50 mM Mg²⁺ and by repeated washings in the absence of this cation. Different washing procedures with EDTA-30 mM Tris (pH 7.5) and low-ionic-strength Tris buffers (pH 7.5) yielded three distinct depleted membranes. RNA and carbohydrates were reevaluated in all these membranes after extraction and/or partial fractionation of the membrane complexes. Values higher than 10% of membrane dry weight have been found for both components in the standard membranes. Figures between 6–12% for carbohydrates and 0.8–16% for RNA were found in the three depleted membranes. The protein: lipid ratio of depleted membranes is lower than that of the standard membrane.The existence of RNA has been confirmed by polyacrylamide gel electrophoresis and by sensitivity to ribonuclease (EC 2.7.7.16). Different patterns of proteins, RNAs and carbohydrate-containing material were revealed by gel electrophoresis in sodium dodecyl sulphate of each type of M. lysodeikticus membrane. These results confirm and extend previous findings obtained with a depleted membrane of M. lysodeikticus (Estrugo, S. F., Larraga, V., Corrales, M. A.; Duch, C. and Munõz, E. (1972) Biochim. Biophys. Acta 255, 960–973). They suggest the existence of specific interactions between membrane components which are broken down and/or altered by different membrane treatments. They also suggest the likely significance in bacterial membranes of components other than lipids and proteins.     

Membrane ATPase of Escherichia coli K 12 Selective solubilization of the enzyme and its stimulation by trypsin in the soluble and membrane-bound states

ATPase (EC 3.6.1.3) of Escherichia coli has been solubilized from two morphologically distinct membranes (vesicles and “ghosts”). Maximum ATPase release is attained with 3 mM EDTA in NH₄HCO₃, pH 9.0, and depends on protein concentration. After solubilization, the total enzyme activity is increased by 300% with respect to the membrane-bound enzyme. The released soluble ATPase accounts for more than 90% of this activity. Its specific activity is at least 10 times higher than the original value. Membrane treatment with buffers of various ionic strengths without EDTA and detergents is less selective. The molecular sieving properties (gel electrophoresis and Sephadex G-200 filtration) confirm the soluble nature of the preparation. A molecular weight close to 300 000 has been estimated for it.The membrane-bound ATPase is stimulated by trypsin by 70–100%. Most of the soluble ATPase maintains a trypsin activation of the same order. Exceptions are the preparations obtained at high protein dilution and extracted with sodium dodecyl sulphate and deoxycholate. The soluble ATPase is more labile than the membrane-bound enzyme. Its sensitivity to different temperatures depends upon protein concentration and pH during storage. Inactivation seems to result from dissociation and/or proteolysis.We suggest an ATPase link to the membrane through ionic divalent cation bridges. We also suggest that the enzyme possesses self-regulatory properties which would account for trypsin stimulation.     

Spectroscopic properties and related functions of the stigma measured in living cells of Euglena gracilis

The spectroscopic properties of stigma inside green and dark-grown cells and of isolated stigma globules have been studied by means of a microspectrophotometer built in the Laboratory. On the base of these results and of the analysis of the absorption spectra of a stigma suspension, cell suspension and cell methanolic extracts, it can be inferred that pigments localized in the stigma are free carotenoids which are not closely packed and do not show an ordered arrangement. Furthermore, the efficiency of the stigma as shading device is duscussed.     

Two new fluorescent derivatives of progesterone to use in fluoroimmunoassay

Synthesis, fluorometric and immunological properties of two new fluorescent derivatives of progesterone are reported. Both compounds were obtained from 11 alpha-hydroxyprogesterone 11-hemisuccinate; the fluorescent molecules were joined to the steroid by bifunctional arms. The first of these is cysteamine whose thiol group was reacted with N-(3-fluoranthenyl) maleimide, and the second is tyramine whose phenolic group was reacted with 1-nitroso-2 naphthol.     

Localization and quantitation of anionic charge sites in fetal and neonatal alveolar basement membranes

A method utilizing biopsy sized samples of lung for anionic charge site localization in alveolar and capillary basement membranes in human tissue is discussed. Tissue fixed in either paraformaldehyde-lysine-periodate or 1% paraformaldehyde with 0.05% glutaraldehyde, cut into 30 mu sections, and incubated with the cationic probe, polyethyleneimine, was processed for electron microscopic analysis using standard techniques. Anionic charge sites were identified and regularly distributed in increments of approximately 40-50 nm in the lamina rara externa of the alveolar basement membrane, with lesser amounts found in the lamina rara interna and lamina densa. Anionic charge sites were also demonstrated in the interstitial portion of the capillary basement membrane and on cell surfaces. These methods can be used to more broadly define the localization of anionic charge sites in human lung tissue in both normal and pathologic states.     

An improved HPLC assay of the substrate and products of the isopenicillin N synthase

Summary An improved HPLC method is described to separate isopenicillin N from its penicilloic acid and from the reduced and oxidized forms of -(L--amino-adipyl)-L-cysteinyl-D-valine and of DTT. When applied to assay the activity of isopenicillin N synthase only isopenicillin N and its penicilloic acid could be detected.