PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)(6)-Hb, that carries approximately six PEG5K chains/Hb - HexaPEGylated Hb. PEGylation increased the O(2) affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)(6)-Hb, its molecular volume, O(2) affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)(2)-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)(6)-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)(2)-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.
Intracorporal injection of plasmids encoding opiorphins into retired breeder rats can result in animals developing a priapic-like condition. Microarray analysis demonstrated that following intracorporal gene transfer of plasmids expressing opiorphins the most significantly upregulated gene in corporal tissue was the ornithine decarboxylase gene (ODC). Quantitative RT-PCR confirmed the upregulation of ODC, as well as other genes involved in polyamine synthesis, such as arginase-I and -II, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT), and S-adenosylmethionine decarboxylase. Western blot analysis demonstrated upregulation of arginase-I and -II, ODC, and SAT at the protein level. Levels of the polyamine putrescine were upregulated in animals treated with opiorphin-expressing plasmids compared with controls. A direct role for the upregulation of polyamine synthesis in the development of the priapic-like condition was supported by the observation that the ODC inhibitor 1,3-diaminopropane, when added to the drinking water of animals treated with plasmids expressing opiorphins, prevented experimental priapism. We also demonstrate that in sickle cell mice, another model of priapism, there is increased expression of the mouse opiorphin homologue in corporal tissue compared with the background strain at a life stage prior to evidence of priapism. At a life stage when there is onset of priapism, there is increased expression of the enzymes involved in polyamine synthesis (ODC and arginase-I and -II). Our results suggest that the upregulation of enzymes involved in the polyamine synthetic pathway may play a role in the development of experimental priapism and represent a target for the prevention of priapism.
Intaglietta. Microvascular PO 2 during extreme hemodilution with hemoglobin site specifically PEGylated at Cys-93() in hamster window chamber. Am J Physiol Heart Circ Physiol 287: H1609 -H1617, 2004. First published June 10, 2004 10.1152/ajpheart.00146.2004.-The oxygen transport capacity of nonhypertensive polyethylene glycol (PEG)-conjugated hemoglobin solutions were investigated in the hamster chamber window model. Microvascular measurements were made to determine oxygen delivery in conditions of extreme hemodilution [hematocrit (Hct) 11%]. Two isovolemic hemodilution steps were performed with a 6% Dextran 70 (70-kDa molecular mass) plasma expander until Hct was 35% of control. Isovolemic blood volume exchange was continued using two surface-modified PEGylated hemoglobins (P5K2, P 50 ϭ 8.6, and P10K2, P50 ϭ 8.3; P50 is the hemoglobin PO2 corresponding to its 50% oxygen saturation) until Hct was 11%. P5K2 and P10K2 are PEG-conjugated hemoglobins that maintain most of the hemoglobin allosteric properties and have a cooperativity index of n ϭ 2.2. The effects of these molecular solutions were compared with those obtained in a previous study using MP4, a PEG-modified hemoglobin whose P 50 was 5.4 and cooperativity was 1.2 (Tsai et al., Am J Physiol Heart Circ Physiol 285: H1411-H1419, 2003). Tissue oxygen levels were higher after P5K2 (7.0 Ϯ 2.5 mmHg) and P10K2 (6.3 Ϯ 2.3 mmHg) versus MP4 (1.7 Ϯ 0.5 mmHg) or the nonoxygen carrier Dextran 70 (1.3 Ϯ 1.2 mmHg). Microvascular oxygen delivery was higher after P5K2 and P10K2 (2.22 and 2.34 ml O 2/dl blood) compared with MP4 (1.41 ml O2/dl blood) or Dextran 70 (0.90 ml O2/dl blood); however, all these values were lower than control (7.42 ml O2/dl blood). The total hemoglobin in blood was similar in all cases; therefore, the improvement in tissue PO 2 and oxygen delivery appears to be due to the increased cooperativity of the new molecules.surface-modified hemoglobin; functional capillary density; oxygencarrying capacity; blood substitutes; tissue oxygen delivery; hemoglobin cooperativity THE DEVELOPMENT of blood substitutes [oxygen-carrying plasma expanders (OCPEs)] has traditionally focused on devising fluids whose transport properties are equivalent to those of blood. This requires using an oxygen carrier that provides an oxygen transport capacity and release properties similar to those of erythrocytes, a task for which hemoglobin (Hb) placed inside the red blood cell (RBC) is ideally suited.
What’s known on the subject? and What does the study add? Diabetes is a common precursor for ladder pathology, including detrusor overactivity and cystopathy. There is preliminary, but increasing evidence, suggesting that oxidative stress plays a significant role in the development of diabetic complications including its affect on the bladder. In the present study we investigated the effect of streptozotocin induced‐diabetes in rats on the global expression of genes in the rat bladder using microarray analysis, and combined this data with our previously reported study looking at changes in protein levels using proteomics. This analysis demonstrated that markers of oxidative stress were significantly increased in the diabetic bladder. Overall, our work adds to the growing body of evidence that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways which may contribute to a deterioration in bladder function. OBJECTIVE To investigate the role that oxidative stress plays in the development of diabetic cystopathy. MATERIALS AND METHODS Comparative gene expression in the bladder of non‐diabetic and streptozotocin (STZ)‐induced 2‐month‐ old diabetic rats was carried out using microarray analysis. Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S‐transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. The activity of protein degradation pathways was assessed using Western blot analysis. RESULTS Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ‐induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P= 1.27 × 10−10). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up‐regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0‐fold, respectively, and the apoptosis inducing protein, BAX, was up‐regulated by 6.76 ± 0.76‐fold. CONCLUSION Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function.
Priapism is a debilitating disease for which there is at present no clinically accepted pharmacologic intervention. It has been estimated that priapism lasting more than 24 hours in patients is associated with a 44–90% rate of erectile dysfunction (ED). In this investigation we determined in two animal models of priapism (opiorpin-induced priapism in the rat and priapism in a mouse model of sickle cell disease) if there is evidence for an increase in markers of oxidative stress in corporal tissue. In both animal models we demonstrate that priapism results in increased levels of lipid peroxidation, glutathione S-transferase activity, and oxidatively damaged proteins in corporal tissue. Using Western blot analysis we demonstrated there is up regulation of the ubiquitination ligase proteins, Nedd-4 and Mdm-2, and the lysososomal autophage protein, LC3. The anti-apoptotic protein, Bcl-2, was also up regulated. Overall, we demonstrate that priapism is associated with increased oxidative stress in corporal tissue and the activation of protein degradation pathways. Since oxidative stress is known to mediate the development of ED resulting from several etiologies (for example ED resulting from diabetes and aging) we suggest that damage to erectile tissue resulting from priapism might be prevented by treatments targeting oxidative stress.
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