Renal responses to hemorrhage are age dependent in conscious sheep.

Experiments were carried out in conscious, chronically instrumented lambs (n = 8) and young adult sheep (n = 11) to investigate age-dependent renal responses to hemorrhage. Various parameters of renal function were measured for 1 h before and 1 h after either 10% hemorrhage (experiment 1) or 20% hemorrhage (experiment 2). The two experiments were carried out in random order at intervals of 2-5 days. There were no effects of 10-20% hemorrhage on renal plasma flow in either age group. Blood pressure decreased after 20% but not 10% hemorrhage in both age groups. Glomerular filtration rate and filtration fraction decreased after 20% hemorrhage in both age groups, the decrease being greater in lambs than young adult sheep. In response to 20% hemorrhage, urinary flow rate and urinary Na+ excretion rate decreased by 40 min after hemorrhage in young adult sheep but not lambs and remained decreased for 60 min; urinary chloride excretion rate showed a similar response. In lambs but not young adult sheep, free water clearance increased by 20 min after 20% hemorrhage and remained above control at 60 min. Urinary osmolality decreased at 20 min after 20% hemorrhage in young adult sheep but not lambs, returning to control levels by 40 min. These data provide new information that renal responses to hypotensive hemorrhage appear to be developmentally regulated.     

Combined Genetic and Genealogic Studies Uncover a Large BAP1 Cancer Syndrome Kindred Tracing Back Nine Generations to a Common Ancestor from the 1700s

We recently discovered an inherited cancer syndrome caused by BRCA1-Associated Protein 1 (BAP1) germline mutations, with high incidence of mesothelioma, uveal melanoma and other cancers and very high penetrance by age 55. To identify families with the BAP1 cancer syndrome, we screened patients with family histories of multiple mesotheliomas and melanomas and/or multiple cancers. We identified four families that shared an identical BAP1 mutation: they lived across the US and did not appear to be related. By combining family histories, molecular genetics, and genealogical approaches, we uncovered a BAP1 cancer syndrome kindred of ~80,000 descendants with a core of 106 individuals, whose members descend from a couple born in Germany in the early 1700s who immigrated to North America. Their descendants spread throughout the country with mutation carriers affected by multiple malignancies. Our data show that, once a proband is identified, extended analyses of these kindreds, using genomic and genealogical studies to identify the most recent common ancestor, allow investigators to uncover additional branches of the family that may carry BAP1 mutations. Using this knowledge, we have identified new branches of this family carrying BAP1 mutations. We have also implemented early-detection strategies that help identify cancers at early-stage, when they can be cured (melanomas) or are more susceptible to therapy (MM and other malignancies).     

Functional comparison of hemoglobin purified by different methods and their biophysical implications

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS–PAGE, SEC, and ESI‐MS), susceptibility to oxidation (k_ox), responses to physiological conditions (pH, [Cl^?], [IHP], and T), and ligand binding kinetics (O₂, NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF‐purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb‐based O₂ carrier synthesis. Biotechnol. Bioeng. 2010; 106: 76–85. © 2010 Wiley Periodicals, Inc.     

Iron mobilization from ferritin by chelating agents

The release of iron from horse spleen ferritin by the chelating agents desferrioxamine B, rhodotorulic acid, 2,3-dihydroxybenzoate, 2,2′-bipyridyl and pyridine-2-aldehyde-2-pyridyl hydrazone (Paphy) has been studied in vitro. Ferritin prepared by classical procedures involving thermal denaturation releases its iron less effectively than ferritin isolated by a modified procedure that avoids this step. Desferrioxamine B and rhodotorulic acid are the most effective in releasing iron from both preparations of ferritin. When FMN is added, iron release by desferrioxamine B, rhodotorulic acid, and 2,3-dihydroxybenzoate was effectively blocked, whereas both bipyridyl and Paphy showed a marked simulation. A substantial increase in iron release was also observed for bipyridyl and Paphy with ascorbate; a less important increase was noted for rhodotorulic acid. EDTA exerted a marked inhibition of iron release from ferritin with rhodotorulic acid, 2,3-dihydroxybenzoate, bipyridyl, and Paphy. The effects of citrate and oxalate on iron release by the chelators was small. The effect of the concentration of flavin on iron release from ferritin by bipyridyl and desferrioxamine B have been studied. Desferrioxamine is unable to mobilize FeII from ferritin following reduction by reduced FMN, whereas bipyridyl can rapidly complex the ferrous iron. The results are discussed in the context of our current concepts of storage iron mobilization in the treatment of iron overload.     

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy

Objective

There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1).

Materials and Methods

pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis.

Results

After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation.

Conclusion

This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.     

Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.     

Genetic variability within isolates of Colletotrichum lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil

Colletotrichum lindemuthianum, the causal agent of anthracnose in the common bean (Phaseolus vulgaris), presents a wide genetic and pathogenic variability that gives rise to complications in the development of resistant bean cultivars. The aim of this study was to identify the variability within race 65 of C. lindemuthianum, the race most commonly encountered in Brazil, through randomly amplified polymorphic DNA (RAPD) and anastomosis analyses. Thirteen isolates of race 65, collected in different years and from various host cultivars located in diverse areas of the state of Minas Gerais, Brazil, were investigated. Twenty-four RAPD primers were employed and 83 polymorphic bands amplified. Genetic similarities were estimated from the Sorensen-Dice coefficient and ranged from 0.54 to 0.82. The dendrogram obtained by cluster analysis classified the isolates into 11 separate groups. For the purposes of the analysis of anastomosis, isolates were considered to be compatible when the fusion of hyphae from different isolates could be observed. The proportion of compatible reactions for each isolate was estimated and similarity estimates, based on the Russel & Rao coefficient, ranged from 0.28 to 0.85. Isolates were classified into 11 anastomosis groups, 10 of which were formed by only one isolate. Although isolates LV61, LV73 and LV58 were classified in the same anastomosis group, they were genetically distinct according to RAPD analysis. Results from both RAPD and anastomosis analyses revealed great variability within C. lindemuthianum race 65.     

Coactivation during gait as an adaptive behavior after stroke

The aims of the present study were to quantify the impairment in ankle coactivation on the paretic and non-paretic sides of subjects with hemiparesis and to examine the relationship of ankle coactivation with postural instability, motor deficit of the paretic lower extremity and locomotor performance. Electromyography of the medial gastrocnemius (MG) and tibialis anterior (TA) muscles were recorded bilaterally during gait in 30 subjects (62.1±9.9 years) who had suffered a recent stroke (<6 months) as well as on one side of 17 healthy controls (59.3±9.1 years) walking at very slow speed. Ankle muscle coactivation was calculated by dividing the time of overlap between MG and TA signals (threshold of 20 μV) by the duration of the gait phases of interest: stance, swing, first and second double support sub-phases and single support sub-phase. The time spent in single support and the peak plantarflexor moment of force on the paretic side were used to measure, respectively, postural stability and dynamic strength of the paretic plantarflexors. The subjects with hemiparesis demonstrated less coactivation on the paretic side during the single support sub-phase (p<0.01) and more coactivation during first and second double support sub-phases on the non-paretic side (p<0.001) compared to control values. The patients with coactivation patterns that differed the most from controls were the patients with the more severe impairments and disabilities. While the reduced coactivation on the paretic side may contribute to poor postural stability and poor locomotor performance, the presence of excessive coactivation on the non-paretic side when both limbs were in ground contact may be an adaptation to help maintain postural stability during gait.     

Xanthan gum production and rheological behavior using different strains of Xanthomonas sp.

The proposal of the present study was to select and carry out the molecular characterization of strains of Xanthomonas sp. in order to correlate with gum production and determine possible genetic alterations during the study. The gums produced were also evaluated rheologically. Ten strains of Xanthomonas were used in the screening and the best ones in terms of productivity were Xanthomonas campestris pv. mangiferaeindicae 1230 (8.93 g/L), X. campestris pv. campestris 254 (9.49 g/L) and X. campestris pv. campestris 1078 (9.67 g/L). The gum produced by X. campestris pv. mangiferaeindicae presented the best apparent viscosity. The results for the profiles of the bands produced by RAPD showed considerable genetic variability amongst the evaluated strains, making not possible to neither group the strains according to pathovar or species, nor correlate the band profile with the productivity obtained. According to the RAPD analysis, no detectable mutations occurred in these bacteria during the study.