Scyllatoxin-based peptide design for E. coli expression and HIV gp120 binding.

Targeting the hydrophobic Phe43 pocket of HIV's envelope glycoprotein gp120 is a critical strategy for antiviral interventions due to its role in interacting with the host cell's CD4. Previous inhibitors, including small molecules and CD4 mimetic peptides based on scyllatoxin, have demonstrated significant binding and neutralization capabilities but were often chemically synthesized or contained non-canonical amino acids. Microbial expression using natural amino acids offers advantages such as cost-effectiveness, scalability, and efficient production of fusion proteins. In this study, we enhanced the previous scyllatoxin-based synthetic peptide by substituting natural amino acids and successfully expressed it in E. coli. The peptide was optimized by mutating the C-terminal amidated valine to valine and glutamine, and by reducing the disulfide bonds from three to two. Circular dichroism confirmed proper secondary structure formation, and fluorescence polarization analysis revealed specific, concentration-dependent binding to HIV gp120, supported by molecular dynamics simulations. These findings indicate the potential for scalable microbial production of effective antiviral peptides, with significant applications in pharmaceutical development for HIV treatment.

Systemic C-peptide supplementation ameliorates retinal neurodegeneration by inhibiting VEGF-induced pathological events in diabetes.

Diabetic retinopathy (DR) is caused by retinal vascular dysfunction and neurodegeneration. Intraocular delivery of C-peptide has been shown to be beneficial against hyperglycemia-induced microvascular leakage in the retina of diabetes; however, the effect of C-peptide on diabetes-induced retinal neurodegeneration remains unknown. Moreover, extraocular C-peptide replacement therapy against DR to avoid various adverse effects caused by intravitreal injections has not been studied. Here, we demonstrate that systemic C-peptide supplementation using osmotic pumps or biopolymer-conjugated C-peptide hydrogels ameliorates neurodegeneration by inhibiting vascular endothelial growth factor-induced pathological events, but not hyperglycemia-induced vascular endothelial growth factor expression, in the retinas of diabetic mice. C-peptide inhibited hyperglycemia-induced activation of macroglial and microglial cells, downregulation of glutamate aspartate transporter 1 expression, neuronal apoptosis, and histopathological changes by a mechanism involving reactive oxygen species generation in the retinas of diabetic mice, but transglutaminase 2, which is involved in retinal vascular leakage, is not associated with these pathological events. Overall, our findings suggest that systemic C-peptide supplementation alleviates hyperglycemia-induced retinal neurodegeneration by inhibiting a pathological mechanism, involving reactive oxygen species, but not transglutaminase 2, in diabetes.

Simultaneous attenuation of hyperglycemic memory-induced retinal, pulmonary, and glomerular dysfunctions by proinsulin C-peptide in diabetes.

BACKGROUND: Hyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic complications. Although coincident diabetic complications are reported, research on their development and treatment is limited. Thus, we investigated whether C-peptide can simultaneously inhibit HGM-induced retinal, pulmonary, and glomerular dysfunctions in diabetic mice supplemented with insulin. METHODS: Insulin-treated diabetic mice were supplemented with human C-peptide by subcutaneous implantation of K9-C-peptide depots for 4 weeks, and reactive oxygen species (ROS) generation, transglutaminase (TGase) activity, and vascular leakage were examined in the retina, lung, and kidney. RESULTS: We found hyperglycemia-induced persistent ROS generation and TGase activation after blood glucose normalization in the retina, lung, and kidney of insulin-supplemented diabetic mice. These pathological events were inhibited by systemic supplementation of human C-peptide via subcutaneous implantation of a thermosensitive biopolymer-conjugated C-peptide depot. ROS generation and TGase activation were in a vicious cycle after glucose normalization, and C-peptide suppressed the vicious cycle and subsequent endothelial permeability in human retinal endothelial cells. Moreover, C-peptide supplementation ameliorated HGM-induced retinal vascular leakage and neurodegeneration, pulmonary vascular leakage and fibrosis, and glomerular adherens junction disruption and vascular leakage. CONCLUSIONS: Overall, our findings demonstrate that C-peptide supplementation simultaneously attenuates vascular and neuronal dysfunctions in the retina, lung, and glomerulus of insulin-supplemented diabetic mice.

Dopamine ameliorates hyperglycemic memory-induced microvascular dysfunction in diabetic retinopathy.

Dopamine is a neurotransmitter that mediates visual function in the retina and diabetic retinopathy (DR) is the most common microvascular complication of diabetes and the leading cause of blindness; however, the role of dopamine in retinal vascular dysfunction in DR remains unclear. Here, we report a mechanism of hyperglycemic memory (HGM)-induced retinal microvascular dysfunction and the protective effect of dopamine against the HGM-induced retinal microvascular leakage and abnormalities. We found that HGM induced persistent oxidative stress, mitochondrial membrane potential collapse and fission, and adherens junction disassembly and subsequent vascular leakage after blood glucose normalization in the mouse retinas. These persistent hyperglycemic stresses were inhibited by dopamine treatment in human retinal endothelial cells and by intravitreal injection of levodopa in the retinas of HGM mice. Moreover, levodopa supplementation ameliorated HGM-induced pericyte degeneration, acellular capillary and pericyte ghost generation, and endothelial apoptosis in the mouse retinas. Our findings suggest that dopamine alleviates HGM-induced retinal microvascular leakage and abnormalities by inhibiting persistent oxidative stress and mitochondrial dysfunction.

Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes.

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2-/- mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.

Inhibitory effects of the atypical antipsychotic, clozapine, on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

To investigate the adverse effects of clozapine on cardiovascular ion channels, we examined the inhibitory effect of clozapine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Clozapine-induced inhibition of Kv channels occurred in a concentration-dependent manner with an half-inhibitory concentration value of 7.84 ± 4.86 µM and a Hill coefficient of 0.47 ± 0.06. Clozapine did not shift the steady-state activation or inactivation curves, suggesting that it inhibited Kv channels regardless of gating properties. Application of train pulses (1 and 2 Hz) progressively augmented the clozapine-induced inhibition of Kv channels in the presence of the drug. Furthermore, the recovery time constant from inactivation was increased in the presence of clozapine, suggesting that clozapine-induced inhibition of Kv channels is use (state)-dependent. Pretreatment of a Kv1.5 subtype inhibitor decreased the Kv current amplitudes, but additional application of clozapine did not further inhibit the Kv current. Pretreatment with Kv2.1 or Kv7 subtype inhibitors partially blocked the inhibitory effect of clozapine. Based on these results, we conclude that clozapine inhibits arterial Kv channels in a concentrationand use (state)-dependent manner. Kv1.5 is the major subtype involved in clozapine-induced inhibition of Kv channels, and Kv2.1 and Kv7 subtypes are partially involved.

The inhibitory effect of ziprasidone on voltage-dependent K+ channels in coronary arterial smooth muscle cells.

We investigated the effect of ziprasidone, a widely used treatment for schizophrenia, on voltage-dependent K+ (Kv) channels of coronary arterial smooth muscle cells using the patch-clamp technique. Ziprasidone dose-dependently inhibited Kv channels with an IC50 value of 0.39 ± 0.06 µM and a Hill coefficient of 0.62 ± 0.03. Although ziprasidone had no effect on the steady-state inactivation kinetics of the Kv channels, the steady-state activation curve shifted towards a more positive potential. These results suggest that ziprasidone inhibits Kv channels by targeting their voltage sensors. The recovery time constant of Kv channel inactivation was increased in the presence of ziprasidone. Furthermore, application of train steps (of 1 and 2 Hz) in the presence of ziprasidone led to a progressive increase in the blockade of Kv currents, suggesting that ziprasidone-induced inhibition of Kv channels is use (state)-dependent. Pretreatment with Kv1.5, Kv2.1, and Kv7 subtype inhibitors partially suppressed the ziprasidone-induced inhibition of Kv currents. These results suggest that ziprasidone inhibits vascular Kv channels through its effect on gating properties. The Kv channel-inhibiting action of ziprasidone is concentration- and use (state)-depedent.

Activity-expression profiling of glucose-6-phosphate dehydrogenase in tissues of normal and diabetic mice.

Glucose-6-phosphate dehydrogenase (G6PD) plays a principal role in the regulation of oxidative stress by modulating the nicotinamide adenine dinucleotide phosphate pool and is expected to be associated with metabolic diseases such as diabetes mellitus (DM). However, it is unclear whether hyperglycemia increases G6PD activity levels in DM because suitable assays for quantifying the activity in a high-throughput manner are lacking. Using liquid droplet arrays tailored to analyze tissue lysates, we performed G6PD activity profiling in eight tissues of normal and diabetic mice: brain, heart, kidney, liver, lung, muscle, spleen, and thyroid. Diabetic mice exhibited significantly higher G6PD activities in the kidney, liver, spleen, and thyroid than normal mice; no significant difference was found in the brain, heart, lung, or muscle. We also performed G6PD expression profiling in the eight tissues using Western blot analysis. Diabetic mice showed significantly elevated G6PD expression levels in the kidney, lung, spleen, and thyroid compared with normal mice; no significant difference was found in the brain, heart, liver, or muscle. An analysis of G6PD activity-expression profiles demonstrated tissue-specific changes in response to hyperglycemia. Thus, our approach would be helpful for understanding the role of G6PD in tissue-based pathogenesis of diabetic complications.

The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory-induced endothelial dysfunction.

Clinical trials suggested that the vascular system can remember episodes of poor glycemic control through a phenomenon known as hyperglycemic memory (HGM). HGM is associated with long-term diabetic vascular complications in type 1 and type 2 diabetes, although the molecular mechanism of that association is not clearly understood. We hypothesized that transglutaminase 2 (TGase2) and intracellular reactive oxygen species (ROS) play a key role in HGM-induced vascular dysfunction. We found that hyperglycemia induced persistent oxidative stress, expression of inflammatory adhesion molecules, and apoptosis in the aortic endothelium of HGM mice whose blood glucose levels had been normalized by insulin supplementation. TGase2 activation and ROS generation were in a vicious cycle in the aortic endothelium of HGM mice and also in human aortic endothelial cells after glucose normalization, which played a key role in the sustained expression of inflammatory adhesion molecules and apoptosis. Our findings suggest that the TGase2-ROS vicious cycle plays an important role in HGM-induced endothelial dysfunction.-Lee, J.-Y., Lee, Y.-J., Jeon, H.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory-induced endothelial dysfunction.

Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

C-peptide has a beneficial effect against diabetic complications, but its role in hyperglycemia-induced metastasis is unknown. We investigated hyperglycemia-mediated pulmonary vascular leakage and metastasis and C-peptide inhibition of these molecular events using human pulmonary microvascular endothelial cells (HPMVECs) and streptozotocin-induced diabetic mice. VEGF, which is elevated in the lungs of diabetic mice, activated transglutaminase 2 (TGase2) in HPMVECs by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels. VEGF also induced vascular endothelial (VE)-cadherin disruption and increased the permeability of endothelial cells, both of which were prevented by the TGase inhibitors monodansylcadaverine and cystamine or TGM2-specific small interfering RNA. C-peptide prevented VEGF-induced VE-cadherin disruption and endothelial cell permeability through inhibiting ROS-mediated activation of TGase2. C-peptide supplementation inhibited hyperglycemia-induced ROS generation and TGase2 activation and prevented vascular leakage and metastasis in the lungs of diabetic mice. The role of TGase2 in hyperglycemia-induced pulmonary vascular leakage and metastasis was further demonstrated in diabetic Tgm2-/- mice. These findings demonstrate that hyperglycemia induces metastasis, and C-peptide prevents the hyperglycemia-induced metastasis in the lungs of diabetic mice by inhibiting VEGF-induced TGase2 activation and subsequent vascular leakage.-Jeon, H.-Y., Lee, Y.-J., Kim, Y.-S., Kim, S.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

N-Terminal Modification of the Tetrapeptide Arg-Leu-Tyr-Glu, a Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) Antagonist, Improves Antitumor Activity by Increasing its Stability against Serum Peptidases.

The tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a vascular endothelial growth factor (VEGF) receptor-2 antagonist, has been used previously either alone or in combination with chemotherapeutic drugs for treating colorectal cancer in a mouse model. We analyzed the half-life of the peptide and found that because of degradation by aminopeptidases B and N, it had a short half-life of 1.2 hours in the serum. Therefore, to increase the stability and potency of the peptide, we designed the modified peptide, N-terminally acetylated RLYE (Ac-RLYE), which had a strongly stabilized half-life of 8.8 hours in serum compared with the original parent peptide. The IC50 value of Ac-RLYE for VEGF-A-induced endothelial cell migration decreased to approximately 37.1 pM from 89.1 pM for the parent peptide. Using a mouse xenograft tumor model, we demonstrated that Ac-RLYE was more potent than RLYE in inhibiting tumor angiogenesis and growth, improving vascular integrity and normalization through enhanced endothelial cell junctions and pericyte coverage of the tumor vasculature, and impeding the infiltration of macrophages into tumor and their polarization to the M2 phenotype. Furthermore, combined treatment of Ac-RLYE and irinotecan exhibited synergistic effects on M1-like macrophage activation and apoptosis and growth inhibition of tumor cells. These findings provide evidence that the N-terminal acetylation augments the therapeutic effect of RLYE in solid tumors via inhibition of tumor angiogenesis, improvement of tumor vessel integrity and normalization, and enhancement of the livery and efficacy of the coadministered chemotherapeutic drugs. SIGNIFICANCE STATEMENT: The results of this study demonstrate that the N-terminal acetylation of the tetrapeptide RLYE (Ac-RLYE), a novel vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor, significantly improves its serum stability, antiangiogenic activity, and vascular normalizing potency, resulting in enhanced therapeutic effect on solid tumors. Furthermore, the combined treatment of Ac-RLYE with the chemotherapeutic drug, irinotecan, synergistically enhanced its antitumor efficacy by improving the perfusion and delivery of the drug into the tumors and stimulating the conversion of the tumor-associated macrophages to an immunostimulatory M1-like antitumor phenotype.

NF-κB-responsive miRNA-31-5p elicits endothelial dysfunction associated with preeclampsia via down-regulation of endothelial nitric-oxide synthase.

Inflammatory cytokines, including tumor necrosis factor-α (TNFα), were elevated in patients with cardiovascular diseases and are also considered as crucial factors in the pathogenesis of preeclampsia; however, the underlying pathogenic mechanism has not been clearly elucidated. This study provides novel evidence that TNFα leads to endothelial dysfunction associated with hypertension and vascular remodeling in preeclampsia through down-regulation of endothelial nitric-oxide synthase (eNOS) by NF-κB-dependent biogenesis of microRNA (miR)-31-5p, which targets eNOS mRNA. In this study, we found that miR-31-5p was up-regulated in sera from patients with preeclampsia and in human endothelial cells treated with TNFα. TNFα-mediated induction of miR-31-5p was blocked by an NF-κB inhibitor and NF-κB p65 knockdown but not by mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase inhibitors, indicating that NF-κB is essential for biogenesis of miR-31-5p. The treatment of human endothelial cells with TNFα or miR-31-5p mimics decreased endothelial nitric-oxide synthase (eNOS) mRNA stability without affecting eNOS promoter activity, resulting in inhibition of eNOS expression and NO/cGMP production through blocking of the functional activity of the eNOS mRNA 3'-UTR. Moreover, TNFα and miR-31-5p mimic evoked endothelial dysfunction associated with defects in angiogenesis, trophoblastic invasion, and vasorelaxation in an ex vivo cultured model of human placental arterial vessels, which are typical features of preeclampsia. These results suggest that NF-κB-responsive miR-31-5p elicits endothelial dysfunction, hypertension, and vascular remodeling via post-transcriptional down-regulation of eNOS and is a molecular risk factor in the pathogenesis and development of preeclampsia.

TNF-α elicits phenotypic and functional alterations of vascular smooth muscle cells by miR-155-5p-dependent down-regulation of cGMP-dependent kinase 1.

cGMP-dependent protein kinase 1 (PKG1) plays an important role in nitric oxide (NO)/cGMP-mediated maintenance of vascular smooth muscle cell (VSMC) phenotype and vasorelaxation. Inflammatory cytokines, including tumor necrosis factor-α (TNFα), have long been understood to mediate several inflammatory vascular diseases. However, the underlying mechanism of TNFα-dependent inflammatory vascular disease is unclear. Here, we found that TNFα treatment decreased PKG1 expression in cultured VSMCs, which correlated with NF-κB-dependent biogenesis of miR-155-5p that targeted the 3'-UTR of PKG1 mRNA. TNFα induced VSMC phenotypic switching from a contractile to a synthetic state through the down-regulation of VSMC marker genes, suppression of actin polymerization, alteration of cell morphology, and elevation of cell proliferation and migration. All of these events were blocked by treatment with an inhibitor of miR-155-5p or PKG1, whereas transfection with miR-155-5p mimic or PKG1 siRNA promoted phenotypic modulation, similar to the response to TNFα. In addition, TNFα-induced miR-155-5p inhibited the vasorelaxant response of de-endothelialized mouse aortic vessels to 8-Br-cGMP by suppressing phosphorylation of myosin phosphatase and myosin light chain, both of which are downstream signal modulators of PKG1. Moreover, TNFα-induced VSMC phenotypic alteration and vasodilatory dysfunction were blocked by NF-κB inhibition. These results suggest that TNFα impairs NO/cGMP-mediated maintenance of the VSMC contractile phenotype and vascular relaxation by down-regulating PKG1 through NF-κB-dependent biogenesis of miR-155-5p. Thus, the NF-κB/miR-155-5p/PKG1 axis may be crucial in the pathogenesis of inflammatory vascular diseases, such as atherosclerotic intimal hyperplasia and preeclamptic hypertension.

Dipeptidyl peptidase-4 inhibitor sitagliptin induces vasorelaxation via the activation of Kv channels and PKA.

The present study investigated the vasorelaxant effects of sitagliptin, which is a dipeptidyl peptidase-4 (DPP-4) inhibitor in aortic rings pre-contracted with phenylephrine (Phe). Sitagliptin induced vasorelaxation in a concentration-dependent manner but the inhibition of voltage-dependent K+ (Kv) channels by pretreatment with 4-aminopyridine (4-AP) effectively reduced this effect. By contrast, the inhibition of inward rectifier K+ (Kir) channels by pretreatment with barium (Ba2+), large-conductance calcium (Ca2+)-activated K+ (BKCa) channels with paxilline, and adenosine triphosphate (ATP)-sensitive K+ (KATP) channels with glibenclamide did not change this effect. Although the application of SQ 22536, which is an adenylyl cyclase inhibitor, also did not change this effect, treatment with KT 5720, a protein kinase A (PKA) inhibitor, effectively reduced the vasorelaxant effects of sitagliptin. ODQ, which is a guanylyl cyclase inhibitor, and KT 5823, a protein kinase G (PKG) inhibitor, did not impact the effect. Furthermore, neither the inhibition of Ca2+ channels by pretreatment with nifedipine nor the inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps by pretreatment with thapsigargin changed the effect. Similarly, the effects of sitagliptin were not altered by eliminating the endothelium, by pretreatment with a nitric oxide (NO) synthase inhibitor (L-NAME), or by inhibition of small- and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa) using apamin and TRAM-34. Taken together, these results suggest that sitagliptin induces vasorelaxation by inhibiting both membrane potential (Em)-dependent and -independent vasoconstriction and activating PKA and Kv channels independently of PKG signaling pathways, other K+ channels, SERCA pumps, and the endothelium.

REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation.

Regulated in development and DNA damage responses 1 (REDD-1), an inhibitor of mammalian target of rapamycin (mTOR), is induced by various cell stressors, including LPS, a major player in the pathogenesis of endotoxemic shock. However, the pathologic role of REDD-1 in endotoxemia is largely unknown. We found that LPS increased REDD-1 expression, nuclear transcription factor-κB (NF-κB) activation, and inflammation and that these responses were suppressed by REDD-1 knockdown and in REDD-1+/- macrophages. REDD-1 overexpression stimulated NF-κB-dependent inflammation without additional LPS stimulation. REDD-1-induced NF-κB activation was independent of 2 classic IKK-dependent NF-κB pathways and the mTOR signaling pathway; however, REDD-1, particularly its C-terminal region (178-229), interacted with and sequestered IκBα, to elicit atypical NF-κB activation during the delayed and persistent phases of inflammation after stimulation. Moreover, REDD-1 knockdown mitigated vascular inflammation and permeability in endotoxemic mice, resulting in decreases in immune cell infiltration, systemic inflammation, caspase-3 activation, apoptosis, and consequent mortality. We further confirmed the inflammatory and cytotoxic effects of REDD-1 in endotoxemic REDD-1+/- mice. Our data support the likelihood that REDD-1 exacerbates endotoxemic inflammation via atypical NF-κB activation by sequestering IκBα.-Lee, D.-K., Kim, J.-H., Kim, J., Choi, S., Park, M., Park, W., Kim, S., Lee, K.-S., Kim, T., Jung, J., Choi, Y. K., Ha, K.-S., Won, M.-H., Billiar, T. R., Kwon, Y.-G., Kim, Y.-M. REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation.

The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

We investigated the beneficial effects of midazolam against vascular endothelial growth factor (VEGF)-induced vascular leakage and its molecular mechanism of action in human retinal endothelial cells (HRECs) and the retinas of diabetic mice. Midazolam inhibited VEGF-induced elevation of intracellular Ca2+, generation of reactive oxygen species (ROS), and transglutaminase activation in HRECs; these effects were reversed by the GABA, type A (GABAA) receptor antagonist flumazenil but not by the translocator protein antagonist PK11195. Midazolam also prevented VEGF-induced disassembly of adherens junctions and in vitro permeability. Intravitreal injection of midazolam prevented hyperglycemia-induced ROS generation, transglutaminase activation, and subsequent vascular leakage in the retinas of diabetic mice, and those effects were reversed by flumazenil. The roles of flumazenil were further supported by identifying GABAA receptors in mouse retinas. Thus, midazolam prevents hyperglycemia-induced vascular leakage by inhibiting VEGF-induced intracellular events in the retinas of diabetic mice.-Lee, Y.-J., Kim, M., Lee, J.-Y., Jung, S.-H., Jeon, H.-Y., Lee, S.-A., Kang, S., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

The anticholinergic drug oxybutynin inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells.

This study demonstrates the inhibitory effect of anticholinergic drug oxybutynin on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Oxybutynin inhibited vascular Kv channels in a concentration-dependent manner, with an IC50 value of 11.51 ± 0.38 µmol/L and a Hill coefficient (n) of 2.25 ± 0.12. Application of oxybutynin shifted the activation curve to the right and the inactivation curve to the left. Pretreatment with the Kv1.5 subtype inhibitor DPO-1 and the Kv2.1 subtype inhibitor guangxitoxin suppressed the oxybutynin-induced inhibition of the Kv current. However, application of the Kv7 subtype inhibitor linopirdine did not affect the inhibition by oxybutynin of the Kv current. The anticholinergic drug atropine did not inhibit the Kv current nor influence oxybutynin-induced inhibition of the Kv current. From these results, we concluded that oxybutynin inhibited the vascular Kv current in a concentration-dependent manner by influencing the steady-state activation and inactivation curves independent of its anticholinergic effect.

Plasmodium vivax Merozoite Surface Protein 1 Paralog as a Mediator of Parasite Adherence to Reticulocytes.

Plasmodium vivax parasites preferentially invade reticulocytes in human beings. P. vivax merozoite surface protein 1 (PvMSP1) and PvMSP1 paralog (PvMSP1P) may have important functions in reticulocyte adherence during invasion. These proteins share similar structures, including the presence of two epidermal growth factor (EGF)-like and glycosylphosphatidylinositol (GPI)-anchored domains at the C terminus. However, there have been no reports concerning the functional activity of PvMSP1P in reticulocyte adherence during P. vivax invasion. In this study, the ability of PvMSP1P-19 to bind to reticulocytes and normocytes was analyzed. The reticulocyte binding activity of PvMSP1P-19 was 4.0-fold higher than its normocyte binding activity. The binding of PvMSP1P-19 to reticulocytes and normocytes was inhibited in a dose-dependent manner by antibodies from immunized rabbits and by antibodies from vivax parasite-infected patients. Consistently, antibodies against PvMSP1P inhibited parasite invasion during short-term in vitro cultivation. Similar to the case for PvDBPII binding activity, PvMSP1P-19 binding activity was reduced in chymotrypsin-treated reticulocytes. However, no significant difference between the binding of PvMSP1P-19 to Duffy-positive and Duffy-negative erythrocytes was found. The minimal binding motif of PvMSP1P-19 was characterized using synthetic peptides. The results showed that the residues at amino acid positions 1791 to 1808 may have an important function in mediating merozoite adherence to reticulocytes. The positively charged residues within the EGF-like domain were shown to constitute a key binding motif. This work presents strong evidence supporting the role of PvMSP1P in host target cell selection and invasion of Duffy-independent pathway in P. vivax Moreover, PvMSP1P-19-specific antibodies may confer protection against P. vivax reinvasion.

Alterations of ATP-sensitive K+ channels in human umbilical arterial smooth muscle during gestational diabetes mellitus.

We investigated the alterations of ATP-sensitive K+ (KATP) channels in human umbilical arterial smooth muscle cells during gestational diabetes mellitus (GDM). The amplitude of the KATP current induced by application of the KATP channel opener pinacidil (10 µM) was reduced in the GDM group than in the control group. Pinacidil-induced vasorelaxation was also predominant in the normal group compared with the GDM group. Reverse transcription polymerase chain reaction and Western blot analysis suggested that the expression of KATP channel subunits such as Kir6.1, Kir6.2, and SUR2B were decreased in the GDM group relative to the normal group. The application of forskolin and adenosine, which activates protein kinase A (PKA) and thereby KATP channels, elicited KATP current in both the normal and GDM groups. However, the current amplitudes were not different between the normal and GDM groups. In addition, the expression levels of PKA subunits were not altered between the two groups. These results suggest that the reduction of KATP current and KATP channel-induced vasorelaxation are due to the decreased expression of KATP channels, not to the impairment of KATP-related signaling pathways.

High-throughput investigation of transglutaminase 2 kinase regulation using a novel cysteine-modified peptide array.

Transglutaminase 2 (TGase2) kinase has emerged as an important regulator of apoptosis as well as chromatin structure and function; however, details about the pathophysiological functions of TGase2 kinase have been limited because of the lack of a suitable activity assay for systematic investigation of TGase2 kinase regulation in a high-throughput manner. Thus, we developed a novel on-chip TGase2 kinase activity assay using a cysteine-modified insulin-like growth factor-binding protein-3-derived peptide (CMI peptide) on an array platform. This peptide array-based activity assay was reproducible, with a detection limit of 2.127 µg/ml. We successfully applied this assay to investigate the effects of thiol-reactive compounds and divalent cations on TGase2 kinase by determining the half maximal inhibitory concentrations (IC50). Thiol-reactive compounds inhibited TGase2 kinase activity in a concentration-dependent manner, with IC50 values ranging from 0.125 to 5.550 mM. Divalent metal cations also showed a concentration-dependent inhibition, with IC50 values ranging from 0.005 to 1.937 mM; however, Ca2+ had no effect on TGase2 kinase activity. Thus, this novel kinase activity assay using the CMI peptide array described here is suitable for systematic investigation of TGase2 kinase regulation and may be useful for investigating the roles of TGase2 kinase in pathogenesis of kinase-mediated diseases.