Regulation of Cardiac Inward Rectifier Potassium Current (IK1) by Synapse-associated Protein-97

Synapse-associated protein-97 (SAP97) is a membrane-associated guanylate kinase scaffolding protein expressed in cardiomyocytes. SAP97 has been shown to associate and modulate voltage-gated potassium (Kv) channel function. In contrast to Kv channels, little information is available on interactions involving SAP97 and inward rectifier potassium (Kir2.x) channels that underlie the classical inward rectifier current, I_K1. To investigate the functional effects of silencing SAP97 on I_K1 in adult rat ventricular myocytes, SAP97 was silenced using an adenoviral short hairpin RNA vector. Western blot analysis showed that SAP97 was silenced by ∼85% on day 3 post-infection. Immunostaining showed that Kir2.1 and Kir2.2 co-localize with SAP97. Co-immunoprecipitation (co-IP) results demonstrated that Kir2.x channels associate with SAP97. Voltage clamp experiments showed that silencing SAP97 reduced I_K1 whole cell density by ∼55%. I_K1 density at −100 mV was −1.45 ± 0.15 pA/picofarads (n = 6) in SAP97-silenced cells as compared with −3.03 ± 0.37 pA/picofarads (n = 5) in control cells. Unitary conductance properties of I_K1 were unaffected by SAP97 silencing. The major mechanism for the reduction of I_K1 density appears to be a decrease in Kir2.x channel abundance. Furthermore, SAP97 silencing impaired I_K1 regulation by β₁-adrenergic receptor (β1-AR) stimulation. In control, isoproterenol reduced I_K1 amplitude by ∼75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that β1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and β1-AR. SAP97 immunolocalizes with protein kinase A and β1-AR in the cardiac myocytes. Our results suggest that in cardiac myocytes SAP97 regulates surface expression of channels underlying I_K1, as well as assembles a signaling complex involved in β1-AR regulation of I_K1.     

Apoptosis in mosquito midgut epithelia associated with West Nile virus infection

The mosquito Culex pipiens pipiens is a documented vector of West Nile virus (WNV, Flaviviridae, Flavivirus). Our laboratory colony of C. p. pipiens, however, was repeatedly refractory to experimental transmission of WNV. Our goal was to identify if a cellular process was inhibiting virus infection of the midgut. We examined midguts of mosquitoes fed control and WNV-infected blood meals. Three days after feeding, epithelial cells from abdominal midguts of mosquitoes fed on WNV fluoresced under an FITC filter following Acridine Orange staining, indicating apoptosis in this region. Epithelial cells from experimental samples examined by TEM exhibited ultrastructural changes consistent with apoptosis, including shrinkage and detachment from neighbors, heterochromatin condensation, nuclear degranulation, and engulfment of apoptotic bodies by adjacent cells. Virions were present in cytoplasm and within cytoplasmic vacuoles of apoptotic cells. No apoptosis was detected by TEM in control samples. In parallel, we used Vero cell plaque assays to quantify infection after 7 and 10 day extrinsic incubation periods and found that none of the mosquitoes (0/55 and 0/10) which imbibed infective blood were infected. We propose that programmed cell death limits the number of WNV-infected epithelial cells and inhibits disseminated viral infections from the mosquito midgut.     

N-succinimidyl 3-[(131)I]iodo-4-phosphonomethylbenzoate ([(131)I]SIPMB), a negatively charged substituent-bearing acylation agent for the radioiodination of peptides and mAbs

An important criterion in design of acylation agents for the radioiodination of internalizing monoclonal antibodies (mAbs) is to maximize the retention of radioiodine in the tumor following mAb intracellular processing. We have previously shown that labeling methods that generate positively charged catabolites have enhanced tumor retention. Herein we have extended this strategy to investigate the potential utility of labeling internalizing mAbs with an acylation agent that yielded labeled catabolites that would be negatively charged at lysosomal pH. The negatively charged acylation agent, N-succinimidyl 3-[(131)I]iodo-4-phosphonomethylbenzoate ([(131)I]SIPMB), was prepared from its tin precursor, N-succinimidyl 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoate (tBu-SPMTB), in 40% radiochemical yield. The free acid, 3-[(131)I]iodo-4-phosphonomethylbenzoic acid ([(131)I]IPMBA), was also prepared from the corresponding precursor, 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoic acid (tBu-PMTBA), in 80% radiochemical yield. The rapidly internalizing mAb L8A4 was conjugated to [(131)I]SIPMB in 25-40% yield with preservation of its immunoreactivity. Internalization and processing in the U87DeltaEGFR glioma cell line was studied in a paired label format with L8A4 labeled with (125)I using the Iodogen method. Retention of initially bound radioactivity in these cells at 24 h from [(131)I]SIPMB-labeled mAb was approximately 6-fold higher than that for directly labeled mAb. Catabolite analysis demonstrated that this difference reflected an order of magnitude higher retention of low molecular weight species in these cells. The [(131)I]SIPMB-L8A4 conjugate was intact over the first 2 h; thereafter, lysine-[(131)I]SIPMB was the predominant catabolite. In contrast, L8A4 labeled using Iodogen rapidly gave rise to mono-[(125)I]iodotyrosine within 2 h, which then cleared rapidly from the cells. These results suggest that SIPMB could be a potent candidate for labeling internalizing mAbs and warrant further study.     

Synthesis,characterization and DNA binding studies of a ruthenium(II) complex

[Ru(bzimpy)(2)]Cl(2), where bzimpy is 2,6-bis(benzimidazol-2-yl) pyridine was synthesized and characterized by ESI-MS, UV-Visible, (1)H NMR and fluorescence spectra. Absorption titration and thermal denaturation experiments indicate that the complex binds to DNA with moderate strength. Viscosity measurement shows that the mode of binding could be surface binding. Fluorescence study shows that the fluorescence intensity of the complex decreases with increasing concentrations of DNA, which is due to the photoelectron transfer from guanine base to (3)MLCT of the complex. Photoexcitation of the complex in the MLCT region in the presence of plasmid DNA has been found to give rise to nicking of DNA.     

Studies onplant aspartate transcarbamylase: Regulatory properties of the enzyme from mung bean (Phaseolus aureus) seedlings

Aspartate transcarbamylase (EC 2·1·3·2) purified from mung bean seedlings was used as a model to understand the mechanism of allosteric regulation. The enzyme exhibited homotropic interactions with carbamyl phosphate. Preincubation of the enzyme with aspartate abolished the sigmoidicity of the carbamyl phosphate saturation curve. UMP was the most potent inhibitor of the reaction and was noncompetitive with respect to aspartate. The sigmoidicity of carbamyl phosphate saturation curves increased with increase in UMP concentration. These results were analysed by an iterative least squares procedure. There was no change inV _max values with increase in the UMP concentration, although theK _0·5 values (concentration of carbamyl phosphate required to reach half maximal velocity) increased. This implied that the effect of UMP was on the binding of carbamyl phosphate only and not on the catalytic function of the enzyme. The allosteric properties of the enzyme could be explained in terms ofK system of the symmetry model. The values of the allosteric constantsn, L andc calculated for mung bean enzyme, making use of the Monod equation accounted for all the observed properties. The enzyme appeared to be a tetramer (n=4) and in the absence of ligands was predominantly in theT form (L _o= 2·25). Carbamyl phosphate bound preferentially to theR form (c= 10_?3), while UMP bound preferentially to theT form and hence these two ligands exhibited the typical heterotropic interactions as expected of antagonistic ligands.     

A new colorimetric method for the estimation of resorcinol

A new, rapid, simple, and sensitive colorimetric method for the estimation of resorcinol in microgram amounts is described.     

Alterations in the activities of the enzymes of proline metabolism in Ragi (Eleusine coracana) leaves during water stress

Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase. These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved and also highlight the role of these enzymes in proline accumulation during water stress.