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.     

Screening for mutations in the neurofibromatosis type 2 (NF2) gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. They are usually sporadic but can also occur associated with the neurofibromatosis type 2 (NF2) syndrome. The gene responsible for NF2, recently isolated from chromosome 22, encodes a membrane-organizing protein that shows high sequence homology to a protein family thought to link the cytoskeleton with membrane proteins. Mutations of the NF2 gene have been described in sporadic meningiomas, exclusively in tumors that show loss of heterozygosity (LOH) of 22q. These preliminary results indicate that the NF2 gene is involved in the pathogenesis of at least a subset of meningiomas, where it does indeed behave as a tumor suppressor gene. In order to characterize better the role of the NF2 gene in the genesis of meningiomas we have examined the entire coding sequence of the gene in 125 meningiomas by single-strand conformational polymorphism analysis; furthermore, LOH analysis for markers of 22q has been carried out. Inactivating mutations were identified in 30% of our samples, all of which also showed LOH of 22q. The majority of mutations identified were frameshifts and nonsense mutations, which are predicted to produce a truncated or non-functional protein. We also found two missense and three in-frame deletions that may pinpoint specific regions of the protein critical to its function. Furthermore, the distribution of mutations throughout the gene, suggested that exons 2, 3, 5, 11 and 13 are more frequently involved. Our results reconfirm the importance of the NF2 gene in the pathogenesis of meningiomas and also suggest that there may be a nonrandom clustering of mutations throughout the gene.     

Minor pollinosis fromCryptomeria japonica in Varese,northern Italy. Aerobiological monitoring and main clinical features

In the last 2 years we have conducted an aerobiological monitoring program ofCryptomeria japonica, a plant belonging to the family of Taxodiaceae that sometimes causes pollinosis in the period from February to April. Throughout 1994, we checked the incidence of its sensitization and the clinical effects in 85 subjects with correlated seasonal symptoms, who gave a positive skin prick test (SPT) for Betulaceae and/or Corylaceae. Twenty-five patients (29.4%; 19 M; 6 F; mean age, 38.8 years) all with oculorhinitis, were SPT positive to an allergenic extract ofCryptomeria. RAST confirmed this positivity in 44% of the cases. No patients showed monosensitization forCryptomeria to either SPT or RAST. Two subjects gave a positive result on specific nasal provocation. RAST inhibition showed no cross-reaction betweenCryptomeria and birch pollen. During the pollen season each patient made a list, scoring symptoms and specifying any drugs used, so we could correlate these elements with aerobiological observations. The pollen concentration probably exceeded the allergizing threshold forCryptomeria on 8 days during 3 months of recording. The intensity and duration of symptoms seemed to be mainly influenced by sensitization to Betulaceae and Corylaceae. It is thus possible that a combination of minor pollinosis may produce seasonal symptoms in allergic patients.     

Use of the 0.22 {micro}m Millipore membrane for light-transmission measurements of aquatic particles

The use of the 0.22 µm Millipore cellulose membrane forthe measurement of the in vivo absorption of aquatic particlesby the light-transmission method has been explored relativeto the performance of the currently used 0.7 µm WhatmanGF/F glass-fiber filter. The standard solvent extraction processfor pigment removal has been replaced by a newly developed processbased on NaClO bleaching. The tests carried out indicate thatthe two filters exhibit comparable performances. It has beenshown that a double measurement, performed first in the particlesretained in the GF/F filter and then on the sample obtainedpassing the GF/F filtrate through a Millipore membrane, allowsfor the selective determination of the absorption of the particlefraction with diameter in the 0.22–0.7 µm range.     

Biotreatment of hydrocarbons from petroleum tank bottom sludges in soil slurries

Summary Biotreatment of oil wastes in aqueous slurries prepared with sandy loam soil and inoculated with selected soil cultures was evaluated. After 90 days, oil removal was 47%. Removal of each hydrocarbon class was 84% for saturates, 20% for aromatics, and 44% for asphaltenes. Resins increased by 68%. The use of a soil with a lower level of fine particles or minor organic matter content, or reinoculation with fresh culture did not improve oil elimination. Residual oil recovered from slurries was biotreated. Oil removal was 22%. Slurry-phase biotreatment showed less variability and faster oil removal than solid-phase biotreatment.     

Biochemical and immunocytochemical identification of conglutin γ, a lupin seed lectin,in the roots of young germinating seedlings

Summary Polyclonal antibodies directed against polypeptide epitopes of conglutin , a glycosylated lectin in lupin seed, have been used to identify and quantify this protein in root extracts of germinating lupins. The highest conglutin content was found in protein extracts of root elongation zones after 5 to 7 days germination. Root conglutin showed the same subunit composition, glycosylation pattern, isoelectric point, and lectin activity as the cotyledonary one. Immunolocalization experiments on root thin sections demonstrated that conglutin is chiefly present in the intercellular spaces of the cortical parenchyma, where it forms large aggregates. Labelling of the Golgi complexes and the area between the plasmalemma and cell wall revealed the conglutin pathway from post-synthetic processing to excretion via the secretory system.Abbreviations EDTA ethylene diamino tetraacetic acid - IEF isoelectric focusing - LRW London resin white - NC nitrocellulose - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - P_i inorganic phosphate - SDS sodium dodecylsulphate - TEM transmission electron microscopy     

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment #1), we analyzed two antigen lots of two human isolates (Bt1 & Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh & Morton medium) in SDS-PAGE and in two immunological tests (imunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment #2), we compared the antigenic profile of three antigen batches from three human isolates (Bt1, Bt2 & Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment #1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment #2, the reference system for P. brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.     

Effects of plant size on photosynthesis and water relations in the desert shrub Prosopis glandulosa (Fabaceae)

The Jornada del Muerto basin of the Chihuahuan Desert of southern New Mexico, USA, has undergone a marked transition of plant communities. Shrubs such as mesquite (Prosopis glandulosa) have greatly increased or now dominate in areas that were previously dominated by perennial grasses. The replacement of grasses by shrubs requires an establishment phase where small shrubs must compete directly with similar-sized grass plants. This is followed by a phase in which large, established shrubs sequester nutrients and water within their biomass and alter soil resources directly under their canopy, creating “islands” of fertility. We hypothesized that these two phases were associated with shrubs having different physiological response capacities related to their age or size and the resource structure of the environment. As a corollary, we hypothesized that responses of small shrubs would be more tightly coupled to variation in soil moisture availability compared to large shrubs. To test these hypotheses, we studied gas exchange and water relations of small (establishing) and large (established) shrubs growing in the Jornada del Muerto as a function of varying soil moisture during the season. The small shrubs had greater net assimilation, stomatal conductance, transpiration, and xylem water potential than large shrubs following high summer rainfall in July, and highest seasonal soil moisture at 0.3 m. High rates of carbon assimilation and water use would be an advantage for small shrubs competing with grasses when shallow soil moisture was plentiful. Large shrubs had greater net assimilation and water-use efficiency, and lower xylem water potential than small shrubs following a dry period in September, when soil moisture at 0.3 m was lowest. Low xylem water potentials and high water-use efficiency would allow large shrubs to continue acquiring and conserving water as soil moisture is depleted. Although the study provides evidence of differences in physiological responses of different-sized shrubs, there was not support for the hypothesis that small shrubs are more closely coupled to variation in soil moisture availability than large shrubs. Small shrubs may actually be less coupled to soil moisture than large shrubs, and thus avoid conditions when continued transpiration could not be matched by equivalent water uptake.     

Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: Effect of surface charge

A series of charge-modified thaumatins with different values of surface charge were partitioned in aqueous two-phase systems (ATPS) to study the effect of surface charge as a single property on partitioning. Electrophoretic mobility of the proteins in titration curves was used as a measure of surface charge. Four modified proteins derived from thaumatin with the following values of isoelectric point: 8.70, 8.15, 5.60, and 4.50 were used for partitioning. The resolution of the systems in terms of protein surface charge was calculated. Partitioning of modified thaumatins in PEG 4000/dextran systems with phosphate buffer, Tris buffer, NaCl, KCl, and sulfate salts was carried out. Among the sulfate salts tested, the addition of 50 mM Li(2)SO(4) to the system buffered with phosphate gave the highest value of resolution for differences in surface protein charge (RSPC). It shows a decrease in the value of K (partition coefficient) with an increase in the protein's charge. The addition of 100 mM KCl to the system promoted the opposite effect on the RSPC value. Charge-modified proteins were partitioned in PEG/salt systems to investigate the ability of these systems for resolving differences in surface charge. The PEG/citrate system seemed to have almost no ability for resolving proteins on the basis of surface charge differences; PEG/phosphate systems had some capability for resolving differently charged proteins. The more negative proteins tended to have higher values of K than the more positively charged fractions. The use of charge-modified proteins allowed the investigation of the effect of protein surface charge on partitioning in aqueous two-phase systems independently from other protein parameters as they were prepared from a common parent protein thaumatin. This technique provides an interesting novel tool to investigate the effect of protein surface charge on partitioning in ATPS taking protein charge as an independent parameter. (c) 1996 John Wiley & Sons, Inc.     

Immunomicroscopical localization of human preformed natural antibodies against pig tissues in xenogeneic transplantation

Summary The targets of preformed natural antibodies need to be identified whenever the use of pig organs is considered for human transplantation. In this study we used extracorporeal perfusion of pig organs with human blood, immunocytological techniques and immuno-electron microscopy to identify the targets and the nature of human preformed natural antibodies against pig antigens. The antibodies were found to be mainly of the IgG and IgM type and directed not only against endothelial cells, but also against mesenchymal and epithelial structures. To reproduce an in vivo situation, a Bio-pump was used to xenoperfuse pig kidneys and livers with human fresh oxygenated blood at 37°C, drawn from polycythaemic patients. Biopsies showed a deposition of human IgG and IgM on tubuli and glomeruli of pig kidneys and on endothelial cells of pig livers. Preperfusion of pig liver with human blood for 45 minutes before perfusion of kidneys significantly reduced the deposition of the natural antibodies.