Isolation of peroxisomes from frozen human liver samples.

This paper shows the successful isolation of peroxisomes from human liver samples that were kept frozen at -70 degrees C. Purification of these peroxisomes was obtained by a combination of two subcellular fractionation techniques: differential centrifugation and isopycnic fractionation in Nycodenz density gradients. Peroxisome integrity was evaluated by latency measurements and by ultrastructural observation. The procedure described here may be useful for the isolation of other subcellular organelles from frozen human samples.     

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungusPleurotus eryngii

Summary Production of extracellular hydrogen peroxide by fungal oxidases is been investigated as a requirement for lignin degradation. Aryl-alcohol oxidase activity is described in extracellular liquid and mycelium ofPleurotus eryngii and studied under non-limiting nitrogen conditions. This aryl-alcohol oxidase catalyses conversion of primary aromatic alcohols to the corresponding aldehydes and H₂O₂, showing no activity with aliphatic and secondary aromatic alcohols. The enzyme is stable at pH 4.0–9.0, has maximal activity at 45°–50°C and pH 6.0–6.5, is inhibited by Ag⁺, Pb²⁺ and NaN₃, and has aK _m of 1.2 mM using veratryl alcohol as substrate. A single protein band with aryl-alcohol oxidase activity was found in zymograms of extracellular and intracellular crude enzyme preparations fromP. eryngii.     

Effect of Glutathione and N-Acetylcysteine on in Vitro and in VIVO Cardiac Toxicity of Doxorubicin

The effects of two sulfhydryl compounds, glutathione (GSH) and N-acetylcysteine (NAC), on the cardiotoxicity of doxorubicin (DXR) were tested on in vitro and in vivo models. DXR was administered to rats as 4 weekly i.v. doses of 3mg/kg. GSH (1.5 mmoles/kg), given i.v. 10 min before and 1 hr after DXR, was found to prevent the development of the delayed cardiotoxic effects of DXR, as assessed by electrocardiographic and mechanical parameters, as well as by histological examination of left ventricular preparations. In contrast, equimolar oral doses of NAC (1 hr before and 2hrs after DXR) were found to be ineffective. Both GSH and NAC prevented the negative inotropic effect produced by DXR on isolated rat atria. A good correlation exists between the cardioprotective effects of the two agents and their ability to enhance the non-protein sulfhydryl group content of the myocardium. Differences observed in vivo between GSH and NAC might be accounted for by pharmacokinetic factors.     

The acidic polysaccharide from Palmaria decipiens (Palmariales,Rhodophyta)

Palmaria decipiens, one of the most abundant red seaweeds of the chilean Antarctic, was collected in King George Island. The hot water extract (26% yield) showed by acid hydrolysis to contain xylose, galactose and traces of glucose. Fractionation with cetrimide gave a soluble neutral xylan and an insoluble fraction. The insoluble fraction afforded an acidic polysaccharide that contained 4.8% of uronic acids, 2.8% of sulfate and 18.9% of protein. Polyacrylamide gel electrophoresis showed that it was homogeneous. The GLC and HPLC analysis of the total acidic hydrolysis products showed that the acidic polysaccharide was composed of the neutral sugars galactose and xylose in the molar ratio 8.2:1.0 and of galacturonic and glucuronic acid in the ratio 1.5:1.0. The second-derivative FT-IR spectrum showed the characteristic amide I, II and III bands of proteins. Alkaline cleavage with 0.1 M NaOH indicated the presence of a glycoprotein with O-glycosidic linkage.Results found in this work suggest that the acidic polysaccharide extracted from Palmaria decipiens is an acidic xylogalactan-protein complex.     

Repeated transfer of small RNA virus populations leading to balanced fitness with infrequent stochastic drift

The population dynamics of RNA viruses have an important influence on fitness variation and, in consequence, on the adaptative potential and virulence of this ubiquitous group of pathogens. Earlier work with vesicular stomatitis virus showed that large population transfers were reproducibly associated with fitness increases, whereas repeated transfers from plaque to plaque (genetic bottlenecks) lead to losses in fitness. We demonstrate here that repeated five-plaque to five-plaque passage series yield long-term fitness stability, except for occasional stochastic fitness jumps. Repeated five-plaque passages regularly alternating with two consecutive large population transmissions did not cause fitness losses, but did limit the size of fitness gains that would otherwise have occurred. These results underscore the profound effects of bottleneck transmissions in virus evolution.     

Changes in the lower premolar-size sequence during hominid evolution. Physlogenetic implications

The size relationship between the crown area of the lower canines (Cs), third (P3s) and fourth premolars (P4s), and first molars (M1s) in hominids is examined by means of the regression analysis. The lower P3 seem to be under the influence of those factors that control both the size of the anterior and posterior teeth, and the P4:P3 size ratio is related to the relative size of the anterior and posterior dentitions. So, the P4>P3 sequence is associated with the megadontia and hipermegadontia of the posterior teeth, whereas the expansion of the anterior teeth produces the P3>P4 sequence. We consider the P4:P3 size ratio as an excellent indicator of the taxonomic and philogenetic status of fossil hominids.     

Establishment of transformed root cultures of Perezia cuernavacana producing the sesquiterpene quinone perezone

Summary The sesquiterpene quinone currently known as perezone is abundantly produced by the roots of Perezia cuernavacana. This compound is of biotechnological interest since it may be used as a pigment and has several pharmacological properties. In this work we demonstrate that perezone is also produced in transformed root cultures of P. cuernavacana. Hairy roots were induced by inoculation of internodal segments of sterile plants of P. cuernavacana with Agrobacterium rhizogenes AR12 strain. The axenic liquid MS medium cultures of the hairy roots isolated from the internodes showed active growth in the absence of growth regulators. The transformed nature of the tissue was confirmed by genomic integration (PCR and slot blot hybridization) and expression (enzyme activity) of the marker gus-gene. The production of perezone by a transformed root culture was evidenced by IR spectroscopy. Our results offer an alternative for enhanced production of perezone and represent an advantage over its extraction from natural plant populations which present problems in their agronomic culture.