Flavonoids derived from medicinal plants as a COVID-19 treatment.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 disease. Through its viral spike (S) protein, the virus enters and infects epithelial cells by utilizing angiotensin-converting enzyme 2 as a host cell's receptor protein. The COVID-19 pandemic had a profound impact on global public health and economies. Although various effective vaccinations and medications are now available to prevent and treat COVID-19, natural compounds derived from medicinal plants, particularly flavonoids, demonstrated therapeutic potential to treat COVID-19 disease. Flavonoids exhibit dual antiviral mechanisms: direct interference with viral invasion and inhibition of replication. Specifically, they target key viral molecules, particularly viral proteases, involved in infection. These compounds showcase significant immunomodulatory and anti-inflammatory properties, effectively inhibiting various inflammatory cytokines. Additionally, emerging evidence supports the potential of flavonoids to mitigate the progression of COVID-19 in individuals with obesity by positively influencing lipid metabolism. This review aims to elucidate the molecular structure of SARS-CoV-2 and the underlying mechanism of action of flavonoids on the virus. This study evaluates the potential anti-SARS-CoV-2 properties exhibited by flavonoid compounds, with a specific interest in their structure and mechanisms of action, as therapeutic applications for the prevention and treatment of COVID-19. Nevertheless, a significant portion of existing knowledge is based on theoretical frameworks and findings derived from in vitro investigations. Further research is required to better assess the effectiveness of flavonoids in combating SARS-CoV-2, with a particular emphasis on in vivo and clinical investigations.

Effects of Vinblastine and Vincristine on the function of chronic myeloid leukemic cells through expression of A20 and CYLD.

Chronic myelogenous leukemia (CML) is characterised by the translocation of regions of the BCR and ABL genes, leading to the fusion gene BCR-ABL forming the Philadelphia (Ph) chromosome. Vinblastine (Vinb) and Vincristine (Vinc) are Vinca alkaloids and frequently used in combination chemotherapy in leukemias and lymphomas. Deubiquitinating enzyme (DUB) genes such as A20, Otubain 1 and CYLD are known as inhibitors of functional activation of immune cells mediated through the NF-κB/STAT pathway. Little is known about the regulatory role of Vinb/Vinc on the function of CML cells and the contribution of the DUBs to those effects. In the end, the gene expression profile was determined by quantitative RT-PCR, physiological properties of CML cells by flow cytometry and cytokine production by ELISA. As a result, inactivated expression of the DUBs A20, CYLD, Otubain 1 and Cezanne and enhanced activation of CD11b+ and CD4T cells were observed in CML patients. Importantly, Vinc enhanced the expression of A20 and CYLD and inhibited the proliferation and survival of CML (K562) cells. The effects were abolished in the presence of A20 siRNA, while cell proliferation only depended on the presence of CYLD. In conclusion, the up-regulation of A20 by Vinc could involve inhibitory effects on the proliferation and survival of K562 cells. The events might contribute to the anticancer effect of Vinc on A20-sensitive CML cells.

The role of interleukin­7 serum level as biological marker in breast cancer: a cross­sectional, observational, and analytical study.

BACKGROUND: The important role that the immune system plays in malignant diseases is well known. The action of interleukin-7 (IL-7) as a cytokine has been observed in many cellular processes, both in normal cells of the immune system and in some cancer cells. The aim of this study has been to explore whether there is any elevation of interleukin-7 serum levels in early invasive breast cancer (EIBC) patients in comparison with healthy controls. In addition, the correlation between the IL-7 serum level and the histopathological characteristics of the tumor has been evaluated. METHODS: This cross-sectional, observational, and analytical study included 213 consecutive patients with EIBC (113 from Croatia and 100 from Kosovo) and 62 healthy participants as the control group (30 from Croatia and 32 from Kosovo). Blood samples have been taken from patients confirmed with breast cancer (BC) by biopsy, prior to surgical intervention and other oncological treatments, as well as from healthy participants. A serum IL-7 level has been measured, using the "Sandwich" ELISA Immunoenzyme test. In addition, after the surgical intervention, histopathological specimen examinations and immunohistochemistry have been performed and analyzed. The differences in the distribution of the numerical variables have been analyzed with the Mann-Whitney U test and Kruskal-Wallis ANOVA test. Correlations have been tested with Pearson coefficients. A P-value < 0.05 has been accepted as statistically significant. RESULTS: The serum level of IL-7 in EIBC patients was significantly higher than in control cases (P 0.001). Patients with invasive lobular carcinoma (ILC) seem to have a lower IL-7 serum level compared to other histological subtypes, and the difference has been significant (P = 0.043). There has been no correlation between IL-7 serum level and histopathological characteristics of the tumor, with neither age nor menopausal status of the patients. CONCLUSIONS: Noting the significant increase in the IL-7 serum level in the EIBC patients as compared to the healthy control group, the use of IL-7 as a potential diagnostic indicator for BC, as well as in the follow-up of the patients after treatment, can be assumed. The lack of correlation with tumor size, lymph node metastasis, and all other histopathological characteristics of the tumor questions its use as a prognostic indicator.

The mechanisms underlying the role of Vitex agnus-castus in mastalgia.

Medicinal plants exert therapeutic effects or have beneficial healing functions on the human or animal body. Medicinal plants are widely used in traditional medicine as an interesting alternative and/or complementary to science-based medicine. Compared to chemical drugs, medicinal plants have a lower risk of side effects, are eco-friendly, and have cost-effective production. This encouraged researchers to extensively exploit them for their therapeutic use. One of the most well-known medicinal plants is Vitex agnus-castus L., which belongs to the Verbenaceae family. This shrub tree is mainly grown in tropical and sub-tropical regions. The parts of VAC, especially berries and leaves, contain essential oils, flavonoids, and diterpenes. Many medical benefits of VAC have already been reported, including mastalgia, regulating menstrual cycles and premenstrual complaints, and infertility. Respiratory and cardiovascular effects are also reported. In this review, we will analyze and characterize the known roles of VAC in mastalgia, as well as the mechanism of action reported in in vitro and/or in vivo studies, and show the potential for alternative therapeutic uses in mastalgia, also known as breast pain (Fig. 2, Ref. 40). Keywords: mastalgia, Vitex agnus-castus, therapy, traditional medicine.

Regulation of cell activation by A20 through STAT signaling in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the hematologic malignancy characterized by the aberrant proliferation of immature lymphoid cells. A20 is a deubiquitinase gene that inhibits functional activation of immune cells mediated through NF-κB/STAT pathways and frequently found inactivated in lymphoma. IL-6 is a pro-inflammatory cytokine secreted by immune cells under the pathogenic conditions and regulated by STAT signaling. Little is known about the role of A20 in regulating the function of ALL blasts and underlying molecular mechanisms. The present study, therefore, explored whether A20 expression contributes to IL-6 induced cell migration and activation of myeloid cells in ALL. To this end, blood samples of thirty-five adult ALL patients were examined. Gene expression profile was determined by quantitative RT-PCR, immunophenotype by flow cytometry, secretion of inflammatory cytokines by ELISA, and cell migration by a transwell migration assay. As a result, the expression of A20 was inactivated in ALL. Immunophenotypic analysis indicated that percent of CD11b+CD40+ expressing cells present in ALL was significantly reduced when transfected with PEM-T easy A20. Importantly, IL6-induced CXCL12-mediated migration of ALL blasts was dependent on the presence of A20. The inhibitory effects of A20 on activated myeloid cells and migration of ALL blasts were mediated through the STAT pathway upon IL-6 challenge. In addition, the CA-125 level was much higher in elderly females than either young female or male ALL patients or healthy donors. In conclusion, the inhibitory effects of A20 on activation of ALL blasts are expected to affect the immune response to treatment for adult ALL patients.

Klotho-Dependent Role of 1,25(OH)2D3 in the Brain.

The antiaging protein Klotho is encoded by the Klotho gene first identified as an 'aging suppressor', in mice. Klotho deficiency is involved in premature aging and early death, while its overexpression is related to longevity. Klotho is mostly expressed in the kidney, but also in the brain, and in other organs. Two forms of Klotho, the cell membrane and secreted form, have pleiotropic activities that include regulation of general metabolism, oxidative stress, and mineral metabolism that correlates with its effect on accelerating aging. Membrane Klotho serves as an obligate co-receptor for the fibroblast growth factor (FGF), while secreted Klotho plays its role as a humoral factor. Klotho protein participates in the regulation of several biological activities, including regulation of calcium-phosphate homeostasis and PTH as well as vitamin D metabolism. The active form of vitamin D, 1,25(OH)2D3 (1,25-dihydroxy-vitamin D3 = calcitriol), acts as a neurosteroid that participates in the regulation of multiple brain functions. It provides neuroprotection and suppresses oxidative stress, inhibits inflammation and inflammatory mediators, and stimulates various neurotrophins. Calcitriol is involved in many brain-related diseases, including multiple sclerosis, Alzheimer´s disease, Parkinson´s disease, and schizophrenia. This review covers the most recent advances in Klotho research and discusses Klotho-dependent roles of calcitriol in neuro-psycho-pathophysiology.

AMP-activated protein kinase α1 regulates cardiac gap junction protein connexin 43 and electrical remodeling following pressure overload.

BACKGROUND/AIMS: Adenosine 5'-monophosphate (AMP)-activated protein kinase (Ampk) modulates a wide array of cellular functions and regulates various ion channels and transporters. In failing human hearts an increased Ampkα1 activity was observed. The present study aimed to uncover the impact of Ampkα1 on cardiac electrical remodeling. METHODS: Gene-targeted mice lacking functional Ampkα1 (Ampkα1-/-) and corresponding wild-type mice were exposed to pressure overload by "transverse aortic constriction" (TAC). In vivo electrophysiology was performed with a single catheter technique, myocardial conduction velocities and conduction characteristics investigated in isolated hearts, transcript levels quantified by RT-PCR and protein abundance determined by Western blotting. Moreover, connexin 43 (Cx43) was expressed in Xenopus oocytes with or without coexpression of wild-type or mutant AMPK and Cx43 protein abundance quantified utilizing confocal microscopy. RESULTS: TAC treatment increased Ampkα1 protein expression in cardiac tissue from wild-type mice. TAC further increased left ventricular conduction inhomogeneity and triggered conduction blocks, effects blunted in the Ampkα1(-/-) mice. TAC treatment decreased Cx43 protein abundance in cardiac tissue, an effect significantly blunted in the Ampkα1(-/-) mice. TAC treatment did not modify Cx43 mRNA levels but increased ubiquitination of Cx43 protein, an effect mitigated by Ampkα1 deficiency. As shown in Xenopus oocytes, Cx43 cell membrane protein abundance was significantly downregulated by wild-type AMPK(WT) and constitutively active AMPK(γR70Q), but not by catalytically inactive AMPK(αK45R). CONCLUSION: Ampkα1 stimulates ubiquitination of the gap junction protein Cx43, thereby contributing to gap junction remodeling following pressure overload.

Energy-sensitive regulation of Na+/K+-ATPase by Janus kinase 2.

Janus kinase 2 (JAK2) contributes to intracellular signaling of leptin and erythropoietin, hormones protecting cells during energy depletion. The present study explores whether JAK2 is activated by energy depletion and regulates Na(+)/K(+)-ATPase, the major energy-consuming pump. In Jurkat cells, JAK2 activity was determined by radioactive kinase assay, phosphorylated JAK2 detected by Western blotting, ATP levels measured by luciferase assay, as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance determined by real-time PCR and Western blotting, respectively. Ouabain-sensitive K(+)-induced currents (Ipump) were measured by whole cell patch clamp. Ipump was further determined by dual-electrode voltage clamp in Xenopus oocytes injected with cRNA-encoding JAK2, active (V617F)JAK2, or inactive (K882E)JAK2. As a result, in Jurkat T cells, JAK2 activity significantly increased following energy depletion by sodium azide (NaN3) or 2,4- dinitro phenol (DNP). DNP- and NaN3-induced decrease of cellular ATP was significantly augmented by JAK2 inhibitor AG490 and blunted by Na(+)/K(+)-ATPase inhibitor ouabain. DNP decreased and AG490 enhanced Ipump as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance. The α1-subunit transcript levels were also enhanced by signal transducer and activator of transcription-5 inhibitor CAS 285986-31-4. In Xenopus oocytes, Ipump was significantly decreased by expression of JAK2 and (V617F)JAK2 but not of (K882E)JAK2, effects again reversed by AG490. In (V617F)JAK2-expressing Xenopus oocytes, neither DNP nor NaN3 resulted in further decline of Ipump. In Xenopus oocytes, the effect of (V617F)JAK2 on Ipump was not prevented by inhibition of transcription with actinomycin. In conclusion, JAK2 is a novel energy-sensing kinase that curtails energy consumption by downregulating Na(+)/K(+)-ATPase expression and activity.

Down-regulation of the epithelial Na⁺ channel ENaC by Janus kinase 2.

Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na(+) transport, effects at least partially involving regulation of the epithelial Na(+) channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na(+) channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function (V617F)JAK2 or inactive (K882E)JAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, (V617F)JAK2 or (K882E)JAK2. ENaC was determined from amiloride (50 µM)-sensitive current (I(amil)) in dual electrode voltage clamp. Moreover, I(amil) was determined in colonic tissue utilizing Ussing chambers. As a result, the I(amil) in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Coexpression of JAK2 and Nedd4-2 decreased I(amil) in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 µM) significantly increased I(amil). In colonic epithelium, I(amil) was significantly enhanced by AG490 pretreatment (40 µM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC.

Regulation of the voltage gated K channel Kv1.3 by recombinant human klotho protein.

BACKGROUND/AIMS: Klotho, a protein mainly produced in the kidney and released into circulating blood, contributes to the negative regulation of 1,25(OH)2D3 formation and is thus a powerful regulator of mineral metabolism. As ß-glucuronidase, alpha Klotho protein further regulates the stability of several carriers and channels in the plasma membrane and thus regulates channel and transporter activity. Accordingly, alpha Klotho protein participates in the regulation of diverse functions seemingly unrelated to mineral metabolism including lymphocyte function. The present study explored the impact of alpha Klotho protein on the voltage gated K+ channel Kv1.3. METHODS: cRNA encoding Kv1.3 (KCNA3) was injected into Xenopus oocytes and depolarization induced outward current in Kv1.3 expressing Xenopus oocytes determined utilizing dual electrode voltage clamp. Experiments were performed without or with prior treatment with recombinant human Klotho protein (50 ng/ml, 24 hours) in the absence or presence of a ß-glucuronidase inhibitor D-saccharic acid-1,4-lactone (DSAL, 10 µM). Moreover, the voltage gated K+ current was determined in Jcam lymphoma cells by whole cell patch clamp following 24 hours incubation without or with recombinant human Klotho protein (50 ng/ml, 24 hours). Kv1.3 protein abundance in Jcam cells was determined utilising fluorescent antibodies in flow cytometry. RESULTS: In Kv1.3 expressing Xenopus oocytes the Kv1.3 currents and the protein abundance of Kv1.3 were both significantly enhanced after treatment with recombinant human Klotho protein (50 ng/ml, 24 hours), an effect reversed by presence of DSAL. Moreover, treatment with recombinant human Klotho protein increased Kv currents and Kv1.3 protein abundance in Jcam cells. CONCLUSION: Alpha Klotho protein enhances Kv1.3 channel abundance and Kv1.3 currents in the plasma membrane, an effect depending on its ß-glucuronidase activity.

Upregulation of the creatine transporter Slc6A8 by Klotho.

BACKGROUND/AIMS: The transmembrane Klotho protein contributes to inhibition of 1,25(OH)2D3 formation. The extracellular domain of Klotho protein could function as an enzyme with e.g. ß-glucuronidase activity, be cleaved off and be released into blood and cerebrospinal fluid. Klotho regulates several cellular transporters. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The main site of Klotho protein expression is the kidney. Klotho protein is also appreciably expressed in other tissues including chorioid plexus. The present study explored the effect of Klotho protein on the creatine transporter CreaT (Slc6A8), which participates in the maintenance of neuronal function and survival. METHODS: To this end cRNA encoding Slc6A8 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho protein. Creatine transporter CreaT (Slc6A8) activity was estimated from creatine induced current determined by two-electrode voltage-clamp. RESULTS: Coexpression of Klotho protein significantly increased creatine-induced current in Slc6A8 expressing Xenopus oocytes. Coexpression of Klotho protein delayed the decline of creatine induced current following inhibition of carrier insertion into the cell membrane by brefeldin A (5 µM). The increase of creatine induced current by coexpression of Klotho protein in Slc6A8 expressing Xenopus oocytes was reversed by ß-glucuronidase inhibitor (DSAL). Similarly, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein significantly increased creatine induced current. CONCLUSION: Klotho protein up-regulates the activity of creatine transporter CreaT (Slc6A8) by stabilizing the carrier protein in the cell membrane, an effect requiring ß-glucuronidase activity of Klotho protein.

Significance of the anti-aging protein Klotho.

The Klotho gene was identified as an 'aging suppressor' in mice. Overexpression of the Klotho gene extends lifespan and defective Klotho results in rapid aging and early death. Both the membrane and secreted forms of Klotho have biological activity that include regulatory effects on general metabolism and a more specific effect on mineral metabolism that correlates with its effect on aging. Klotho serves as a co-receptor for fibroblast growth factor (FGF), but it also functions as a humoral factor that regulates cell survival and proliferation, vitamin D metabolism, and calcium and phosphate homeostasis and may serve as a potential tumor suppressor. Moreover, Klotho protects against several pathogenic processes in a FGF23-independent manner. These processes include cancer metastasis, vascular calcification, and renal fibrosis. This review covers the recent advances in Klotho research and discusses novel Klotho-dependent mechanisms that are clinically relevant in aging and age-related diseases.

Hematological, Biochemical, and Serum Levels of Allergic Mediators in Individuals with and without Allergic Rhinitis.

Background: Allergic rhinitis (AR) is the most prevalent form of non-infectious rhinitis and is characterized by an immune response mediated by immunoglobulin E (IgE). Aim: This study aims to compare the levels of biochemical markers and other parameters in individuals with AR, non-allergic rhinitis(n-AR), allergic rhinitis accompanied by symptoms of the lower respiratory tract(AR-SLRT), and healthy controls. Study Design: Case control study. Methods: Blood samples from the three study groups, AR (n = 22), n-AR (n=20), AR-SLRT group (n = 21), and the control group (n = 18), were analyzed to ascertain the levels of total IgE, specific IgE, periostin, pendrin, vitamin D, thyroid-stimulating hormone (TSH), free triiodothyronine (Ft3), free thyroxine (Ft4), anti-thyroid peroxidase (TPO), and eosinophilic cationic protein (ECP), as well as the leukocyte formula and hemogram. Results: The AR and n-AR groups had significantly higher hematocrit values in comparison to the control group(p<0.05). Further, eosinophil counts were significantly higher in the AR and AR-SLRT groups than in the control group(p<0.05). Total IgE levels were significantly higher in the AR-SLRT group than in the AR, n-AR, and control groups (p<0.05). The AR group had higher total IgE values compared to the control group and the n-AR group(p<0.05). The values of ECP, periostin, pendrin, Ft3, Ft4, TSH, anti-TPO, and vitamin D did not differ significantly between the groups(p>0.05). Conclusion: All the investigated groups did not differ in ECP, periostin, pendrin, Ft3, Ft4, TSH, anti-TPO, or vitamin D parameters. The groups with positive AR and AR-SLRT had higher eosinophil counts than the control group. The group with AR-SLRT had higher total IgE concentrations than the other groups.

Downregulation of the renal outer medullary K(+) channel ROMK by the AMP-activated protein kinase.

The 5'-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca(2+) activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na(+) channel ENaC and the voltage-gated K(+) channel KCNE1/KCNQ1. It is partially effective by decreasing PIP(2) formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K(+) channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK(γR70Q) (AMPK(α1)-HA+AMPK(ß1)-Flag+AMPKγ1(R70Q)), or of inactive AMPK(αK45R) (AMPK(α1K45R)+AMPK(ß1)-Flag+AMPK(γ1)-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence in Xenopus oocytes and western blot in whole kidney tissue. Moreover, renal Na(+) and K(+) excretion were determined in AMPK(α1)-deficient mice (ampk ( -/- )) and wild-type mice (ampk ( +/+ )) prior to and following an acute K(+) load (111 mM KCl, 30 mM NaHCO(3), 4.7 mM NaCl, and 2.25 g/dl BSA) at a rate of 500 µl/h. As a result, coexpression of AMPK(γR70Q) but not of AMPK(αK45R) significantly decreased the current in ROMK1-expressing Xenopus oocytes. Injection of phosphatidylinositol PI((4,5))P(2) significantly increased the current in ROMK1-expressing Xenopus oocytes, an effect reversed in the presence of AMPK(γR70Q). Under control conditions, no significant differences between ampk ( -/- ) and ampk ( +/+ ) mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentrations as well as absolute and fractional Na(+) and K(+) excretion. Following an acute K(+) load, GFR, urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentration were again similar in both genotypes, but renal absolute and fractional Na(+) and K(+) excretion were higher in ampk ( -/- ) than in ampk ( +/+ ) mice. According to micropuncture following a K(+) load, delivery of Na(+) to the early distal tubule but not delivery of K(+) to late proximal and early distal tubules was increased in ampk (-/-) mice. The upregulation of renal ROMK1 protein expression by acute K(+) load was more pronounced in ampk (-/-) than in ampk ( +/+ ) mice. In conclusion, AMPK downregulates ROMK, an effect compromising the ability of the kidney to excrete K(+) following an acute K(+) load.

Downregulation of KCNQ4 by Janus kinase 2.

Janus kinase-2 (JAK2) participates in the signaling of several hormones, growth factors and cytokines. Further stimulators of JAK2 include osmotic cell shrinkage, and the kinase activates the cell volume regulatory Na(+)/H(+) exchanger. The kinase may thus participate in cell volume regulation. Cell shrinkage is known to inhibit K(+) channels. Volume-regulatory K(+) channels include the voltage-gated K(+) channel KCNQ4. The present study explored the effect of JAK2 on KCNQ4 channel activity. KCNQ4 was expressed in Xenopus oocytes with or without wild-type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2; and cell membrane conductance was determined by dual-electrode voltage clamp. Expression of KCNQ4 was followed by the appearance of voltage-gated K(+) conductance. Coexpression of JAK2 or of (V617F)JAK2, but not of (K882E)JAK2, resulted in a significant decrease in conductance. Treatment of KCNQ4 and JAK2 coexpressing oocytes with the JAK2 inhibitor AG490 (40 µM) was followed by an increase in conductance. Treatment of KCNQ4 expressing oocytes with brefeldin A (5 µM) was followed by a decrease in conductance, which was similar in oocytes expressing KCNQ4 together with JAK2 as in oocytes expressing KCNQ4 alone. Thus, JAK2 apparently does not accelerate channel protein retrieval from the cell membrane. In conclusion, JAK2 downregulates KCNQ4 activity and thus counteracts K(+) exit, an effect which may contribute to cell volume regulation.

AMP-activated protein kinase in BK-channel regulation and protection against hearing loss following acoustic overstimulation.

The energy-sensing AMP-activated serine/threonine protein kinase (AMPK) confers cell survival in part by stimulation of cellular energy production and limitation of cellular energy utilization. AMPK-sensitive functions further include activities of epithelial Na+ channel ENaC and voltage-gated K+ channel KCNE1/KCNQ1. AMPK is activated by an increased cytosolic Ca2+ concentration. The present study explored whether AMPK regulates the Ca2+-sensitive large conductance and voltage-gated potassium (BK) channel. cRNA encoding BK channel was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (AMPKα1+AMPKß1+AMPKγ1), constitutively active AMPKγR70Q, or inactive AMPKαK45R. BK-channel activity was determined utilizing the 2-electrode voltage-clamp. Moreover, BK-channel protein abundance in the cell membrane was determined by confocal immunomicroscopy. As BK channels are expressed in outer hair cells (OHC) of the inner ear and lack of BK channels increases noise vulnerability, OHC BK-channel expression was examined by immunohistochemistry and hearing function analyzed by auditory brain stem response measurements in AMPKα1-deficient mice (ampk-/-) and in wild-type mice (ampk+/+). As a result, coexpression of AMPK or AMPKγR70Q but not of AMPKαK45R significantly enhanced BK-channel-mediated currents and BK-channel protein abundance in the oocyte cell membrane. BK-channel expression in the inner ear was lower in ampk-/- mice than in ampk+/+ mice. The hearing thresholds prior to and immediately after an acoustic overexposure were similar in ampk-/- and ampk+/+ mice. However, the recovery from the acoustic trauma was significantly impaired in ampk-/- mice compared to ampk+/+ mice. In summary, AMPK is a potent regulator of BK channels. It may thus participate in the signaling cascades that protect the inner ear from damage following acoustic overstimulation.

Down-regulation of the Na+-coupled phosphate transporter NaPi-IIa by AMP-activated protein kinase.

BACKGROUND/AIMS: The Na(+)-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na(+) gradient across the apical cell membrane, which is maintained by Na(+) extrusion across the basolateral cell membrane through the Na(+)/K(+) ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na(+) accumulation and K(+) loss with eventual decrease of cell membrane potential, Cl(-) entry and cell swelling. Upon energy depletion, early inhibition of Na(+)-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK), a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. METHODS: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPK(α1)-HA+AMPK(ß1)-Flag+AMPK(γ1)-HA), of inactive AMPK(αK45R) (AMPK(α1K45R)+AMPK(ß1)-Flag+AMPK(γ1)-HA) or of constitutively active AMPK(γR70Q) (AMPK(α1)-HA+AMPK(ß1)-Flag+AMPKγ1(R70Q)). NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. RESULTS: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM) to the extracellular bath solution generated a current (Ip), which was significantly decreased by coexpression of wild-type AMPK and of AMPK(γR70Q) but not of AMPK(αK45R). The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM). Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. CONCLUSION: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport. © 2013 S. Karger AG, Basel.

Regulation of KCNQ1/KCNE1 by ß-catenin.

ß-catenin, a multifunctional protein expressed in all tissues including the heart stimulates the expression of several genes important for cell proliferation. Signaling involving ß-catenin participates in directing cardiac development and in the pathophysiology of cardiac hypertrophy. Nothing is known, however, on the role of ß-catenin in the regulation of cardiac ion channels. The present study explored the functional interaction of ß-catenin and KCNE1/KCNQ1, the K⁺ channel complex underlying the slowly activating outwardly rectifying K⁺ current. To this end, KCNE1/KCNQ1 was expressed in Xenopus oocytes with and without ß-catenin and the depolarization (up to + 80 mV) induced current (I(Ks)) was determined using the two-electrode voltage clamp. As a result, ß-catenin enhanced I(Ks) by 30%. The effect of ß-catenin on I(Ks) was not affected by actinomycin D (10 µM), an inhibitor of transcription, indicating that ß-catenin was not effective as transcription factor. Confocal microscopy revealed that ß-catenin enhanced the KCNE1/KCNQ1 protein abundance in the cell membrane. Exposure of the oocytes to brefeldin A (5 µM), an inhibitor of vesicle insertion, was followed by a decline of I(Ks), which was then similar in oocytes expressing KCNE1/KCNQ1 together with ß-catenin and in oocytes expressing KCNE1/KCNQ1 alone. In conclusion, ß-catenin enhances I(Ks) by increasing the KCNE1/KCNQ1 protein abundance in the cell membrane, an effect requiring vesicle insertion into the cell membrane.

Reproductive risk of the silent carrier of Robertsonian translocation.

AIM: The aim of this study was the evaluation of risk among the couples various types of Robertsonian translocations. METHODS: Cytogenetic diagnosis has been carried out according to the Moorhead and Seabright method. RESULTS: Cytogenetics diagnosis was performed in 17 couples having Robertsonian translocations. Among our examined cases, the most frequent (82.3%) cases were with Robertsonian translocations formed by aberrant fusion between heterologous chromosomes 13q and 14q. Three out of seventeen couples affected with Robertsonian translocation 13q;14q suffered from primary infertility. The total number of pregnancy among the couples with Robertsonian translocation has been 45. Of these 80% of pregnancies resulted in spontaneous miscarriages, while 20% of others have gave birth to alive or dead children. In one couple a Robertsonian translocation was caused as a result of fusion of two homologous chromosomes 15q;15q. A patient with this translocation has had 7 pregnancies and all of them ended with abortions. CONCLUSION: Robertsonian translocation caused the primary infertility in three couples and lowering reproductive abilities in 14 others. Robertsonian translocation between 15q;15q caused intrauterine death and spontaneous failures of all pregnancies of the carrier with this translocation.

Malignant Melanoma of the Middle Ear: Case Report.

We present a case of malignant melanoma in the middle ear in a 63-year-old woman who presented with severe pain on the right side of her face and head, tinnitus, photophobia, and hemorrhagic discharge from the right ear, as well as right facial nerve paralysis. After more than two years of conservative treatment for otitis media and sclerotic mastoiditis, a pathologically enlarged lymph node on the right side of the neck appears. It is removed, and the histopathological examination reveals that it is metastatic malignant melanoma. Imaging examinations reveal a tumoral lesion in the middle ear with bone infiltration. The right middle ear is operated on, and the tissue sent for testing reveals melanoma. The patient was treated with Nivolumab after the operation and is now in sustainably good condition.