A new RNase-based immunoconjugate selectively cytotoxic for ErbB2-overexpressing cells

We report a new tumor-directed immunoRNase, a chimeric protein made up of an antibody fragment (single-chain Fv fragment) directed to ErbB2, a cell surface receptor, and a non-toxic, human ribonuclease, which upon cell internalization becomes cytotoxic. The immunoRNase is active as a ribonuclease, specifically binds and selectively kills ErbB2-positive cells. ErbB2 is one of the most specific tumor-associated antigens identified so far, overexpressed on tumor cells of different origin. Its choice as target antigen and that of a non-toxic, human RNase as the killer moiety makes this immunoRNase a new, potentially attractive anticancer agent.     

Determination of the triterpenoid,betulinic acid,in Doliocarpus schottianus by HPLC

The isolation, detection and quantification of betulinic acid in Doliocarpus schottianus are described. The isolation from the plant extract was made by column chromatography and centrifugal TLC, and betulinic acid was characterized by spectrometric methods. The detection and quantification were made by HPLC using a C18 column eluted with acetonitrile: water and detected at 210 nm. The results showed that the metabolite accumulates in the bark of the plant, but very small concentrations were also found in the leaves and wood.     

Influence of forward leaning and incentive spirometry on inspired volumes and inspiratory electromyographic activity during breathing exercises in healthy subjects

Breathing exercises (BE), incentive spirometry and positioning are considered treatment modalities to achieve lung re-expansion. This study evaluated the influence of incentive spirometry and forward leaning on inspired tidal volumes (V_T) and electromyographic activity of inspiratory muscles during BE. Four modalities of exercises were investigated: deep breathing, spirometry using both flow and volume-oriented devices, and volume-oriented spirometry after modified verbal instruction. Twelve healthy subjects aged 22.7 ± 2.1 years were studied. Surface electromyography activity of diaphragm, external intercostals, sternocleidomastoid and scalenes was recorded. Comparisons among the three types of exercises, without considering spirometry after modified instruction, showed that electromyographic activity and V_T were lower during volume-oriented spirometry (p = 0.000, p = 0.054, respectively). Forward leaning resulted in a lower V_T when compared to upright sitting (p = 0.000), but electromyographic activity was not different (p = 0.606). Inspired V_T and electromyographic activity were higher during volume-oriented spirometry performed after modified instruction when compared with the flow-oriented device (p = 0.027, p = 0.052, respectively). In conclusion BE using volume-oriented spirometry before modified instruction resulted in a lower work of breathing as a result of a lower V_T and was not a consequence of the device type used. Forward leaning might not be assumed by healthy subjects during situations of augmented respiratory demand.     

Structure of CcmG/DsbE at 1.14 A resolution: high-fidelity reducing activity in an indiscriminately oxidizing environment

CcmG is unlike other periplasmic thioredoxin (TRX)-like proteins in that it has a specific reducing activity in an oxidizing environment and a high fidelity of interaction. These two unusual properties are required for its role in c-type cytochrome maturation. The crystal structure of CcmG reveals a modified TRX fold with an unusually acidic active site and a groove formed from two inserts in the fold. Deletion of one of the groove-forming inserts disrupts c-type cytochrome formation. Two unique structural features of CcmG-an acidic active site and an adjacent groove-appear to be necessary to convert an indiscriminately binding scaffold, the TRX fold, into a highly specific redox protein.     

Characterization of the hypotensive effect of S-nitroso-N-acetylcysteine in normotensive and hypertensive conscious rats.

S-Nitrosothiols (RSNOs) are potent vasodilators found naturally in vivo. A variety of synthetic RSNOs have been considered as potential nitric oxide (NO) donors for biomedical applications. We have characterized the hypotensive effect of the RSNO S-nitroso-N-acetylcysteine (SNAC) in normotensive and hypertensive conscious rats. SNAC reduced the medium arterial pressure in a dose-response manner in both normotensive and hypertensive animals. At the same doses (EC(50) of SNAC), SNAC showed a vasodilator effect in normotensive rats more potent and more prolonged than that of sodium nitroprusside (SNP). The hypotensive effect of SNAC was also more potent in methylene blue-treated rats, where the cGMP-dependent pathway had been blockaded. These data indicate that SNAC acts by both cGMP-dependent and cGMP-independent pathways. It was also shown that the thiol N-acetylcysteine (NAC) potentiates the action of SNP in hypertensive rats, pointing to the mediation of thiols in the vasodilator action of SNP in this condition. Such mediation may involve the formation of a more potent thiol complex with the nitroprusside anion or the transfer of NO to NAC, generating SNAC as a primary vasoactive species. The kinetic monitoring of the decomposition reactions of SNAC and SNP showed that both compounds are quite stable under the infusion conditions used. Therefore, their vasodilator action cannot be assigned to their breakdown with release of free NO in solution. As the two compounds are unlikely to cross the plasmalemma of smooth muscle cells, their actions are probably associated with the mediation of endogenous thiols in transnitrosation reactions.     

Synaptosomes isolated from Goto-Kakizaki diabetic rat brain exhibit increased resistance to oxidative stress: role of vitamin E

Increased oxidative stress is believed to be an important factor in the development of diabetic complications. In this study, the effect of diabetes on the susceptibility of synaptosomes to oxidative stress, induced by the oxidizing system ascorbate/Fe2+, on the activity of antioxidant enzymes and on the levels of glutathione and vitamin E was investigated. Synaptosomes were isolated from brain of 29-weeks-old Goto-Kakizaki (GK) rats, a model of non-insulin dependent diabetes mellitus and from normal Wistar rats. Synaptosomes isolated from GK rats displayed a lower susceptibility to lipid peroxidation, as assessed by quantifying thiobarbituric acid reactive substances (TBARS), than normal rats (5.33 +/- 0.79 and 7.58 +/- 0.7 nmol TBARS/mg protein, respectively). In the absence of oxidants, no significant differences were found between the levels of peroxidation in synaptosomes of diabetic or control rats. Superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase activities were unaltered in the brain of diabetic rats. There were no statistically significant differences in fatty acid composition of total lipids and reduced glutathione levels in synaptosomes of diabetic and control rats. The decreased susceptibility to membrane lipid peroxidation of diabetic rats synaptosomes correlated with a 1.3-fold increase in synaptosomal vitamin E levels. Vitamin E levels in plasma were also higher in diabetic rats (21.32 micromol/l) as compared to normal rats (15.13 micromol/l). We conclude that the increased resistance to lipid peroxidation in GK rat brain synaptosomes may be due to the increased vitamin E content, suggesting that diabetic animals might develop enhanced defense systems against brain oxidative stress.     

Evaluation of novel beta-lactam antibiotics in comparison to cefotaxime on plant regeneration of <Emphasis Type="Italic">Citrus sinensis</Emphasis> L. Osb.

Identification of beta-lactam antibiotics that have negligible effects on plant regeneration is a critical step towards the establishment of a reliable Agrobacterium-mediated transformation protocol for perennial trees. In the present report, we have evaluated the effects of the novel beta-lactam antibiotics meropenem and timentin on plant regeneration of a perennial woody fruit plant, Citrus sinensis, in comparison with the commonly used beta-lactam cefotaxime. It was observed that, in contrast to cefotaxime, meropenem and timentin had a positive or no detrimental effect on the shoot regeneration from epicotyl explants. Residual effects of the beta-lactams from shoot regeneration medium also affected the subsequent ability of the roots to elongate. The addition of meropenem and/or timentin in the rooting medium mostly improved or did not affect the rooting ability of the adventitious shoots. These data indicated that meropenem and timentin can positively replace cefotaxime in Agrobacterium-mediated transformation of C. sinensis.     

Genetic composition,population structure and phylogeography of the loggerhead sea turtle: colonization hypothesis for the Brazilian rookeries

The loggerhead sea turtle, Caretta caretta, is the most common species of sea turtle nesting in Brazil and is listed as endangered by the IUCN. Our study characterizes the genetic structure of loggerheads in Brazil based on mitochondrial DNA control region variability and presents a hypothesis for the colonization of Brazilian rookeries. We analyzed 329 samples from Brazilian rookeries and an oceanic foraging ground, and we compared our results with previously published data for other loggerhead populations. Brazilian rookeries had four haplotypes, none of which have been reported for rookeries outside Brazil. Six haplotypes were found in the foraging aggregation. The presence of the CC-A4 haplotype at all sampled sites and the low nucleotide diversity suggest a common origin for all rookeries, with CC-A4 being the ancestral haplotype of the Brazilian populations. The occurrence of three haplotypes in the foraging aggregation that are known only from rookeries outside of Brazil is consistent with the transoceanic migratory behavior of loggerheads. Our results indicated that the colonization of Brazilian rookeries probably occurred from the southern USA stock. This recent colonization most likely followed a north to south route along the Brazilian coastline, influenced by the Brazilian warm current. Our results further suggest the existence of two genetic population units of loggerheads in Brazil and corroborate natal homing behavior in loggerheads.     

Efflux pumps expression and its association with porin down-regulation and β-lactamase production among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> causing bloodstream infections in Brazil

Background  

Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.