Strain-insensitive and multiplexed potentiometric ion sensors via printed PMMA molecular layer.

Wearable biomimetic electronics have aroused tremendous attention due to their capability to continuously detect and deliver real-time dynamic physiological signals pertaining to the wearer's environment. However, upon close contact with the human skins, a wearable sensor undergoes mechanical strain which inevitably degrades the electrical performance. To address this issue, we demonstrate a universal design approach for stretchable and multiplexed biosensors that can yield unaltered ion sensing performance under variable mechanical tensile strains, which is achieved by introducing a PMMA molecular layer between stretchable substrate and ion sensors. Such design demonstrates reliable multiplexed ion sensing capability and provides high sensitivity (>50 mV/decade), reliable selectivity, as well as wide working range (0.1-100 mM) for sodium, ammonium, potassium and calcium ions in complex sweat biomarkers. Via this introduced PMMA molecular layer, our sensor even exhibits 95 % electrical performance maintained up to 30 % tensile strain, whereas the mechanical tensile property is far superior to original sensor performance. Besides, the sensors were also utilized for real-time monitoring of ions in sweat to validate its biomedical electronics applications. This sensing platform can be easily extended to other biomimetic sensors to enable stable signal acquisition for biomedical electronics.

Application Effect of Transparent Supervision Based on Informatization in Prevention and Control of Carbapenem-Resistant Klebsiella pneumoniae Nosocomial Infection.

Objective: To explore the effect of transparent supervision model on the prevention and control of carbapenem-resistant Klebsiella pneumoniae (CRKP) nosocomial infection and the value of the autoregressive integrated moving average (ARIMA) model in predicting the incidence of CRKP infection. Methods: A total of 46,873 inpatients from Jiawang District People's Hospital of Xuzhou between January 2019 and December 2019 (prior to COVID-19 prevention and control) were selected as the preintervention group and 45,217 inpatients from January 2020 to December 2020 (after the COVID-19 prevention and control) as the postintervention group. We performed transparent supervision on CRKP patients detected by the real-time monitoring system for nosocomial infection. Incidence and detection rate of CRKP, utilization rate of special grade hydrocarbon enzyme alkene antibiotics, hand hygiene compliance rate, qualified rate of ATP tests on surface of environmental objects, and execution rate of CRKP core prevention and control were compared between the two groups. Results: Transparent supervision of CRKP-infected patients was conducted daily from January to December 2020, which resulted in the following: (a) the infection rate of CRKP decreased in a fluctuating manner, and the actual value of hydrocarbon alkene use rate was basically the same as the predicted value with an overall decreasing trend; (b) after the intervention, hand hygiene compliance rate increased from 53.30% to 70.24% (P < 0.001) and the ATP qualified rate increased from 53.77% to 92.24% (P < 0.001); (c) the fitted value of the ARIMA model was in good agreement with the actual value. The incidence of CRKP infection and the utilization rate of carbene antibiotics were also in good agreement with the predicted value. The average relative errors were 11% and 10.78%. Conclusions: During the COVID-19 outbreak in 2020, the ARIMA model effectively fit and predicted the CRKP infection rate, thereby providing scientific guidance for the prevention and control of CRKP infection. In addition, the transparent supervision intervention model improved the hand hygiene compliance and environmental hygiene qualification rates of medical staff, effectively reducing CRKP cross-infection in the hospital.

Current understanding on the role of CCT3 in cancer research.

Chaperonin containing TCP1 Subunit 3 (CCT3) is an important member of the chaperone protein family, providing a favorable environment for the correct folding of proteins in cell division, proliferation, and apoptosis pathways, which is involved in a variety of biological processes as well as the development and invasion of many malignant tumors. Many malignancies have been extensively examined with CCT3. It is presently used as a possible target for the treatment of many malignancies since it is not only a novel biomarker for the screening and diagnosis of different tumors, but it is also closely associated with tumor progression, prognosis, and survival. Recent studies have shown that the expression of CCT3 is up-regulated in some tumors, such as liver cancer, breast cancer, colon cancer, acute myeloid leukemia, etc. In this paper, we review the role of CCT3 in various tumors.

Skin Barrier Damage due to Prolonged Mask Use among Healthcare Workers and the General Population during the COVID-19 Pandemic: A Prospective Cross-Sectional Survey in China.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has resurged in localized areas in China. Individuals wear masks to prevent the spread of droplets. However, skin barrier damage occurs because of the prolonged use of masks. OBJECTIVE: To investigate the prevalence and associated risk factors of skin injuries among healthcare workers (HCWs) and the general population during the COVID-19 outbreak. METHODS: A multicenter cross-sectional study of skin barrier damage caused by wearing masks was conducted using an online questionnaire between December 10 and December 31, 2020. Data regarding demographics, characteristics of facial skin damage, and information on masks were registered. Multivariate logistic regression was used to analyze factors associated with skin barrier damage, and odds ratios (OR) with 95% confidence intervals (CI) were used to establish correlation strength. RESULTS: A total of 1,538 responses were retrieved from 1,700 questionnaires (response rate, 90.47%), and 1,409 questionnaires were valid (effective response rate, 91.61%). The respondents comprised 567 HCWs (40.24%) and 842 individuals from the general population (59.76%). The prevalence of skin injuries was 46.03% among HCWs and 46.20% among the general population. History of chronic skin disease (OR, 6.01; 95% CI, 4.75-7.75), type of mask used (OR, 2.77; 95% CI, 1.95-3.93), daily wearing time (OR, 1.57; 95% CI, 1.36-1.82), and mask replacement cycle (OR, 0.76; 95% CI, 0.68-0.86) were associated with skin barrier damage. CONCLUSION: There was a high incidence of skin barrier damage due to prolonged mask use among HCWs and the general population, and treatment and prevention were inadequate. Attention needs to be given to strengthening comprehensive health education and popularization of science.

Progress in the application of reflectance confocal microscopy in dermatology.

Reflectance confocal microscopy (RCM) is abbreviated as skin three-dimensional computed tomography, which can help clearly observe the structure of the epidermis and superficial dermis. It is a non-invasive skin disease examination method and provides fast access to real-time, dynamic skin micro-anatomical images. Therefore, RCM is widely used in the clinical diagnosis of skin diseases. For example, the RCM features of vitiligo are as follows: pigment loss or partial pigment loss in the lesion area, loss of the basal layer pigment ring. The RCM findings of Riehl melanosis are as follows: basal cell liquefaction and degeneration. The RCM results for verruca plana show: the Rose-like structure. The characteristics of psoriasis under RCM include: hyperkeratosis, parakeratosis, thickening of the spinous layer, capillary dilatation and hyperaemia, peripheral inflammatory cell infiltration. Epidermal brain-like structure was observed under RCM of seborrheic keratosis. With RCM, image acquisition and preservation of the skin is convenient, and the technique is convenient for comparing the development of lesions during long-term follow-up observation. Therefore, it helps to understand disease development in real time and dynamically and can be used to evaluate the curative effect. In this article, we briefly review the technical principles, diagnostic criteria for RCM application and RCM-related research progress in the diagnosis of pigmentary diseases, inflammatory diseases, skin tumours, and other common skin diseases.

Dermatoscopy and Reflective Confocal Microscopy for Facial Seborrheic Keratosis, Verruca Plana, and Nevus Pigmentosus.

OBJECTIVE: To describe the features of facial seborrheic keratosis, verruca plana, and nevus pigmentosus by dermatoscopy and reflective confocal microscope (RCM). STUDY DESIGN: Cross-sectional observational study. PLACE AND DURATION OF STUDY: Dermatology Department of The Affiliated Hospital of Xuzhou Medical University from January 2017 to January 2019. METHODOLOGY: Patients of either gender and age, clinically diagnosed as seborrheic keratosis, verruca plana, and nevus pigmentosus without any prior treatment, were enrolled. Patients with extremities and trunk involvement were excluded. One typical skin lesion was chosen from each patient and subjected to dermatoscopy and RCM separately; imaging features were recorded and analysed. RESULTS: A total of 402 patients (183 men and 219 women) between 6 and 88 years of age (mean age 41.8 years) were inducted. The duration of disease was between one week and 10 years. Seborrheic keratosis on dermatoscopy presented as acne-like openings (122/172; 70.93%, milia-like cysts (113/172; 65.70%), hairpin-like vessels (108/172; 62.79%, brain-like structures (103/172; 59.88%, worm-like pharyngeal margins (17/172; 9.88%), and fingerprint-like structures (8/172; 4.65%). On RCM, it showed epidermal cerebral gyrus structure (165/172; 95.93%), superficial vasodilatation and hyperemia (81/172; 47.09%), and keratinous cysts (73/172; 42.44%). Verruca plana on dermatoscopy showed a number of punctate hemorrhages scattered against a light-red background (108/114; 94.74%); on RCM it showed rose-like concentric structures (89/114; 78.07%). The features of nevus pigmentosus observed by dermatoscopy were homogeneous mode (23/52; 44.23%), light brown color (30/52; 57.69%) for intradermal nevus; mesh mode (18/41; 43.90%, dark brown color, little black dots and spherical structure (both 18/41; 43.90%) were visible for junctional nevus; globular mode, reticular and cobblestone structures (both 11/23; 47.83%) for compound nevus. On RCM, there were a few nevus cells visible within the dermal papilla (52/52; 100%) for intradermal nevus; bright pebble-like structures accumulated in the basal layer, nested round and elliptical nevus cells (40/41; 97.56%) for junctional nevus; nevus cells in the epidermis and dermis (21/23; 91.30%) for compound nevus. CONCLUSION: Used in combination with clinical manifestations, the application of dermatoscopy and RCM may help distinguish seborrheic keratosis, verruca plana, and nevus pigmentosus on the basis of their respective dermatoscopic and RCM features. Key Words: Dermatoscopy, Reflective confocal microscope, Seborrheic keratosis, Verruca plana, Nevus pigmentosus, Brain-like structures, Rose-like structure.

Recent developments in dermoscopy for dermatology.

BACKGROUND: Dermoscopy is considered to be a bridge between clinical observation and histopathological examination, allowing the in vivo examination of skin microstructures that are not visible to the naked eye, from the epidermis to the superficial dermis. Dermoscopy has undergone rapid development, witnessing the history from natural light to polarized light, from handheld dermoscopy to videodermoscopy, and from classic dermoscopy to digital dermoscopy. Its application extends from the initial differential diagnosis of pigmented skin diseases (melanocytic and nonmelanocytic) to general dermatology, including appendage (nail and hair) abnormalities and diseases related to infection and inflammation. AIMS: We aimed to provide the latest developments in dermoscopy from the perspective of handheld dermoscopy, videodermoscopy, fluorescence-advanced videodermatoscopy, polarized transilluminating dermoscopy, and digital dermoscopy. METHODS: In this review, we searched the PubMed, Embase, Web of Science, and Cochrane Library databases for reviews, case reports, and observational studies on dermoscopy. RESULTS: We provided an updated review of dermoscopy based on published literature. CONCLUSION: Dermoscopy is an indispensable diagnostic tool in dermatology, and it is expected to be further developed in the future.

Potential New Cancer Immunotherapy: Anti-CD47-SIRPα Antibodies.

CD47 belongs to immunoglobulin superfamily and is widely expressed on the surface of cell membrane, while another transmembrane protein SIRPα is restricted to the surface of macrophages, dendritic cells, and nerve cells. As a cell surface receptor and ligand, respectively, CD47 and SIRPα interact to regulate cell migration and phagocytic activity, and maintain immune homeostasis. In recent years, studies have found that immunoglobulin superfamily CD47 is overexpressed widely across tumor types, and CD47 plays an important role in suppressing phagocytes activity through binding to the transmembrane protein SIRPα in phagocytic cells. Therefore, targeting CD47 may be a novel strategy for cancer immunotherapy, and a variety of anti-CD47 antibodies have appeared, such as humanized 5F9 antibody, B6H12 antibody, ZF1 antibody, and so on. This review mainly describes the research history of CD47-SIRPα and focuses on macrophage-mediated CD47-SIRPα immunotherapy of tumors.

Confocal laser scanning microscopy, a diagnostic alternative for five pigmented lesions on the face: An observational study.

OBJECTIVE: To describe the confocal laser scanning microscopy (CLSM) features of five different facial hyperpigmentation diseases and to highlight the features that can be used to differentiate between the diseases. MATERIALS AND METHODS: Confocal laser scanning microscopy features of skin lesions of 406 patients with different facial hyperpigmentation diseases (chloasma, naevus fusco-caeruleus zygomaticus, naevus of Ota, freckles and Riehl melanosis) were retrospectively analysed. All patients were diagnosed clinically. The features of each layer of the skin in the involved regions and at the junction of the lesion with normal skin were studied, and the characteristic features of each disease were identified. The CLSM probe was applied perpendicular to the skin surface. Scanning was performed with medical ultrasonic coupling agent applied between the adhesive window and skin and between the lens and adhesive window. The skin was scanned layer by layer, and the best cross-sectional images were stored in the computer for analysis. RESULTS: Chloasma lesions showed significantly increased pigment content in the epidermal basal layer and, in some cases, varying degrees of pigment particle deposition in the upper dermis. Naevus fusco-caeruleus zygomaticus and naevus of Ota lesions showed normal epidermal pigment content, with cord-like high-refractive pigment masses scattered in the dermis; cord-like or dendritic melanocytes were seen between collagen fibre bundles in the upper and middle dermis, but no inflammatory cell infiltration was seen. Freckles lesions showed increased numbers of pigment particles in the basal layer, but no abnormal changes in the dermis. Riehl melanosis was characterised by liquefaction degeneration of the basal cells, accompanied by considerable monocyte and melanophage infiltration in the dermal papilla. CONCLUSIONS: Used in combination with clinical manifestations, CLSM can be a useful auxiliary method for diagnosis of naevus fusco-caeruleus zygomaticus, naevus of Ota, chloasma, freckles and Riehl melanosis.

Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells.

NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5) gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs) are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT) and hypertensive subjects (HT). We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and 2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold greater in HT than NT (P=<0.001, n=6-14/group). Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA), effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (-45.1±3.2% vs. mock-siRNA, n=6-8) in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (-32.5±1.8%) than HT (-14.8±1.8). In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.

Alterations of reproductive hormones and receptors of male rats at the winter and summer solstices and the effects of pinealectomy.

BACKGROUND: Photoperiodic changes mediate certain physiological or pathological alterations in organisms. Solstices represent either the longest or shortest photoperiod of a year. OBJECTIVES: Intact and pinealectomized rats were used to investigate the potential changes of reproductive hormones in the hypothalamus-pituitary-testis (HPT) axis including GnRH, FSH, LH, testosterone and melatonin, and their receptors at summer solstice (SS) and winter solstices (WS). METHODS: The levels of reproductive hormones in HPT axis and the binding characteristics of their receptors were examined using radioimmunoassay and radioreceptor assay techniques, respectively. RESULTS: The results indicate that in the intact male rat, GnRH, LH and testosterone are higher at the SS than at the WS. However, FSH exhibited no significant seasonal changes. In the testis, Bmax and Kd of LH receptors are higher at the WS than at the SS while those of FSH receptors are higher at the SS than at the WS. In addition, the melatonin in HPT axis appeared significant differences between WS and SS. Bmax and Kd of melatonin receptors in the hypothalamus and pituitary also showed higher at the WS than at the SS. Moreover, reproductive hormone production lost their seasonal rhythms after pinealectomy. CONCLUSION: The most important discovery in this study is that we first reported that pinealectomy had profound effects on the binding characteristics of melatonin with its subtype receptors. Especially at the hypothalamus, the dominated melatonin receptors shifted from MT1 to MT2 after pinealectomy at the two solstices.

D5 dopamine receptor decreases NADPH oxidase, reactive oxygen species and blood pressure via heme oxygenase-1.

D5 dopamine receptor (D5R) knock-out mice (D5(-/-)) have a higher blood pressure (BP) and higher reactive oxygen species (ROS) production than their D5R wild-type littermates (D5(+/+)). We tested the hypothesis that the high BP and increased ROS production in D5(-/-) mice may be caused by decreased heme oxygenase-1 (HO-1) expression and activity. We found that renal HO-1 protein expression and HO enzyme activity were decreased (65 and 50%, respectively) in D5(-/-) relative to D5(+/+) mice. A 24 h of administration of hemin, an HO-1 inducer, increased HO-1 expression and HO activity (6.8- and 1.9-fold, respectively) and normalized the increased ROS production and BP in D5(-/-) mice. Expression of HO-1 protein and HO activity were increased (2.3- and 1.5-fold, respectively) in HEK cells that heterologously expressed human wild-type D5R (HEK-hD5R), but not the empty vector-transfected HEK-293 cells. Fenoldopam (Fen), a D5R agonist, increased HO activity (3 h), HO-1 protein expression, HO-1 and D5R colocalization and co-immunoprecipitation in HEK-hD5R cells. Cellular NADPH oxidase activity was decreased by 35% in HEK-hD5R that was abrogated with silencing of the heme oxygenase 1 gene (HMOX1). HMOX1 siRNA also impaired the ability of Fen to decrease NADPH oxidase activity in HEK-hD5R cells. In summary, the D5R positively regulates HO-1 through direct protein/protein interaction in the short-term and by increasing HO-1 protein expression in the long-term. The impaired D5R regulation of HO-1 and ROS production contributes to the pathogenesis of hypertension in D5(-/-) mice.

Identification of RanBP 9/10 as interacting partners for protein kinase C (PKC) gamma/delta and the D1 dopamine receptor: regulation of PKC-mediated receptor phosphorylation.

We reported previously that ethanol treatment regulates D(1) receptor phosphorylation and signaling in a protein kinase C (PKC) delta- and PKCgamma-dependent fashion by a mechanism that may involve PKC isozyme-specific interacting proteins. Using a PKC isozyme-specific coimmunoprecipitation approach coupled to mass spectrometry, we report the identification of RanBP9 and RanBP10 as novel interacting proteins for both PKCgamma and PKCdelta. Both RanBP9 and RanBP10 were found to specifically coimmunoprecipitate with both PKCgamma and PKCdelta; however, this association did not seem to mediate the ethanol regulation of the PKCs. It is noteworthy that the D(1) receptor was also found to specifically coimmunoprecipitate with RanBP9/10 from human embryonic kidney (HEK) 293T cells and with endogenous RanBP9 from rat kidney. RanBP9 and RanBP10 were also found to colocalize at the cellular level with the D(1) receptor in both kidney and brain tissue. Although overexpression of RanBP9 or RanBP10 in HEK293T cells did not seem to alter the kinase activities of either PKCdelta or PKCgamma, both RanBP proteins regulated D(1) receptor phosphorylation, signaling, and, in the case of RanBP9, expression. Specifically, overexpression of either RanBP9 or RanBP10 enhanced basal D(1) receptor phosphorylation, which was associated with attenuation of D(1) receptor-stimulated cAMP accumulation. Moreover, treatment of cells with select PKC inhibitors blocked the RanBP9/10-dependent increase in basal receptor phosphorylation, suggesting that phosphorylation of the receptor by PKC is regulated by RanBP9/10. These data support the idea that RanBP9 and RanBP10 may function as signaling integrators and dictate the efficient regulation of D(1) receptor signaling by PKCdelta and PKCgamma.

Upregulation of renal sodium transporters in D5 dopamine receptor-deficient mice.

D(5) dopamine receptor (D(5)R)-deficient (D(5)(-/-)) mice have hypertension that is aggravated by an increase in sodium intake. The present experiments were designed to test the hypothesis that a dysregulation of renal sodium transporters is related to the salt sensitivity in D(5)(-/-) mice. D(5)R was expressed in the renal proximal tubule, thick ascending limb, distal convoluted tubule, and cortical and outer medullary collecting ducts in D(5)(+/+) mice. On a control Na(+) diet, renal protein expressions of NKCC2 (sodium-potassium-2 chloride cotransporter), sodium chloride cotransporter, and alpha and gamma subunits of the epithelial sodium channel were greater in D(5)(-/-) than in D(5)(+/+) mice. Renal renin abundance and urine aldosterone levels were similar but renal angiotensin II type 1 receptor (AT(1)R) protein expression was increased in D(5)(-/-) mice. An elevated Na(+) diet increased further the elevated blood pressure of D(5)(-/-) mice but did not affect the normal blood pressure of D(5)(+/+) mice. The increased levels of NKCC2, sodium chloride cotransporter, and alpha and gamma subunits of the epithelial sodium channel persisted with the elevated Na(+) diet and unaffected by chronic AT(1)R blockade (losartan) in D(5)(-/-) mice. The expressions of proximal sodium transporters NHE3 (sodium hydrogen exchanger type 3) and NaPi2 (sodium phosphate cotransporter type 2) were increased by the elevated Na(+) diet in D(5)(-/-) mice; the increased expression of NHE3 but not NaPi2 was abolished by AT(1)R blockade. Our findings suggest that the increased protein expression of sodium transporters/channels in distal nephron segments may be the direct consequence of the disruption of D(5)R, independent of the renin-angiotensin aldosterone system.

D3 dopamine receptor regulation of ETB receptors in renal proximal tubule cells from WKY and SHRs.

BACKGROUND: The dopaminergic and endothelin systems, by regulating sodium transport in the renal proximal tubule (RPT), participate in the control of blood pressure. The D(3) and ETB receptors are expressed in RPTs, and D(3) receptor function in RPTs is impaired in spontaneously hypertensive rats (SHRs). Therefore, we tested the hypothesis that D(3) receptors can regulate ETB receptors, and that D(3) receptor regulation of ETB receptors in RPTs is impaired in SHRs. METHODS: ETB receptor expression in RPT cells was measured by immunoblotting and reverse transcriptase-PCR and ETB receptor function by measuring Na(+)-K(+) ATPase activity. D(3)/ETB receptor interaction was studied by co-immunoprecipitation. RESULTS: In Wistar-Kyoto (WKY) RPT cells, the D(3) receptor agonist, PD128907, increased ETB receptor protein expression, effects that were blocked by removal of calcium in the culture medium. The stimulatory effect of D(3) on ETB receptor mRNA and protein expression was also blocked by nicardipine. In contrast, in SHR RPT cells, PD128907 decreased ETB receptor expression. Basal D(3)/ETB receptor co-immunoprecipitation was three times greater in WKY than in SHRs. The absolute amount of D(3)/ETB receptor co-immunoprecipitation induced by a D(3) receptor agonist was also greater in WKY than in SHRs. Stimulation of ETB receptors decreased Na(+)-K(+) ATPase activity in WKY but not in SHR cells. Pretreatment with PD128907 augmented the inhibitory effect of BQ3020 on Na(+)-K(+) ATPase activity in WKY but not in SHR cells. CONCLUSIONS: D(3) receptors regulate ETB receptors by physical receptor interaction and govern receptor expression and function. D(3) receptor regulation of ETB receptors is aberrant in RPT cells from SHRs.

D1-like receptors regulate NADPH oxidase activity and subunit expression in lipid raft microdomains of renal proximal tubule cells.

NADPH oxidase (Nox)-dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D(1)-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58+/-3.3%) than in hypertensive rats (31+/-5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67(phox) in cells from normotensive rats. D(1)-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl-beta-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats.

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells.

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

Response gene to complement 32, a novel regulator for transforming growth factor-beta-induced smooth muscle differentiation of neural crest cells.

We previously developed a robust in vitro model system for vascular smooth muscle cell (VSMC) differentiation from neural crest cell line Monc-1 upon transforming growth factor-beta (TGF-beta) induction. Further studies demonstrated that both Smad and RhoA signaling are critical for TGF-beta-induced VSMC development. To identify downstream targets, we performed Affymetrix cDNA array analysis of Monc-1 cells and identified a gene named response gene to complement 32 (RGC-32) to be important for the VSMC differentiation. RGC-32 expression was increased 5-fold after 2 h and 50-fold after 24 h of TGF-beta induction. Knockdown of RGC-32 expression in Monc-1 cells by small interfering RNA significantly inhibited the expression of multiple smooth muscle marker genes, including SM alpha-actin (alpha-SMA), SM22alpha, and calponin. Of importance, the inhibition of RGC-32 expression correlated with the reduction of alpha-SMA while not inhibiting smooth muscle-unrelated c-fos gene expression, suggesting that RGC-32 is an important protein factor for VSMC differentiation from neural crest cells. Moreover, RGC-32 overexpression significantly enhanced TGF-beta-induced alpha-SMA, SM22alpha, and SM myosin heavy chain promoter activities in both Monc-1 and C3H10T1/2 cells. The induction of VSMC gene promoters by RGC-32 appears to be CArG-dependent. These data suggest that RGC-32 controls VSMC differentiation by regulating marker gene transcription in a CArG-dependent manner. Further studies revealed that both Smad and RhoA signaling are important for RGC-32 activation.

The elevated blood pressure of human GRK4gamma A142V transgenic mice is not associated with increased ROS production.

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the sensitivity of GPCRs, including dopamine receptors. The GRK4 locus is linked to, and some of its polymorphisms are associated with, human essential hypertension. Transgenic mice overexpressing human (h) GRK4gamma A142V on a mixed genetic background (C57BL/6J and SJL/J) have impaired renal D(1)-dopamine receptor (D(1)R) function and increased blood pressure. We now report that hGRK4gamma A142V transgenic mice, in C57BL/6J background, are hypertensive and have higher blood pressures than hGRK4gamma wild-type transgenic and nontransgenic mice. The hypertensive phenotype is stable because blood pressures in transgenic founders and F6 offspring are similarly increased. To determine whether the hypertension is associated with increased production of reactive oxygen species (ROS), we measured renal NADPH oxidase (Nox2 and Nox4) and heme oxygenase (HO-1 and HO-2) protein expressions and urinary excretion of 8-isoprostane and compared the effect of Tempol on blood pressure in hGRK4gamma A142V transgenic mice and D(5)R knockout (D(5)(-/-)) mice in which hypertension is mediated by increased ROS. The expressions of Nox isoforms and HO-2 and the urinary excretion of 8-isoprostane were similar in hGRK4gamma A142V transgenic mice and their controls. HO-1 expression was increased in hGRK4gamma A142V relative to hGRK4gamma wild-type transgenic mice. In contrast with the hypotensive effect of Tempol in D(5)(-/-) mice, it had no effect in hGRK4gamma A142V transgenic mice. We conclude that the elevated blood pressure of hGRK4gamma A142V transgenic mice is due mainly to the effect of hGRK4gamma A142V transgene acting via D(1)R and increased ROS production is not a contributor.