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Background and Objective: The role of ion channels and particularly cationic channels in the pathogenesis of various diseases are being considered carefully. The diabetes mellitus is a common disease which is initiated by ion channel disturbances. This study was done to determine the characteristics of hepatocyte rough endoplasmic reticulum single cationic channel in Streptozocin- induced diabetic rats. Materials and Methods: This experimental study was done on 10 male adult Wistar rats and animals were randomly allocaied into diabetic and control groups. Diabetes induced by STZ (65 mg/kg/bw) intraperitounally. Rough endoplasmic reticulum vesicles were extracted following rat liver excision, homogenization and ultracentrifuging. The bilayer membrane formation was prepared by painting phosphatidylcholine on 250µM aperture in between Cis and Trans sides. The RER vesicles incorporation was performed through gentle and delicate touch of membrane using a dentistry needle. The Pclamp9 software was used for ion channel activity characteristic analysis. Results: The cationic channel current amplitude did not change significantly in voltages more than +3o mV but their open probability (Po) decreased in diabetic group (P

Abstract: Recently, we have reported electropharmacological properties of a charybdotoxin (ChTx)- and ATP-insensitive–iberiotoxin (IbTx)-sensitive large conductance Ca+2-activated potassium (BKCa) channel in almost purified brain mitochondrial inner membrane vesicles. In this work, we report the single-channel characterization of a new BK channel from rat brain mitochondrial inner membrane (mitochondrial large conductance Ca2+-activated potassium channel, mitoBKCa channel) incorporated into a planar lipid bilayer. The channel conductance was 565 pS in 200 mM KCl cis/50 mM KCl trans. The channel open probability appeared voltage-independent at −40 to +40 mV. Adding 10 mM 4-aminopyridine (4-AP) at positive and negative potentials and 2.5 mM ATP at positive voltages inhibited the channel activities. Notably, addition of 70 μM glibenclamide to the cis side had no effect on the channel behavior. Hence, it can be concluded that there are, at least, two different types of mitoBKCa channels in brain mitochondrial membrane, and the IbTx-, ChTx-, and ATP-sensitive mitoBKCa channels may be activated during the decline of cell metabolism. [Copyright &y& Elsevier]

Background: H. pylori are generally considered as extracellular organisms, with exclusive colonization of the gastric milieu. Yet, several extra gastric manifestations are associated with this infection. The aim of the present study was to investigate the feasibility of toxin transfer by extracellular vesicles, from bacterial and epithelial origins., Methods: Tox-positive H. pylori and its two cagA and vacA mutant strains were used to produce bacterial vesicles (BVs) and to infect AGS cells. The produced BVs and the infected cell vesicles (ICVs) were collected by ultracentrifugation and evaluated by western blotting, DLS and electron microscopy. These two sets of vesicles were applied to a second set of recipient AGS cells, in which the acellular transfer of toxins, IL-8 production and downstream morphologic changes were assessed, by western blotting, ELISA and light microscopy, respectively., Results: The BVs were positive for H. pylori membrane markers (BabA and UreB), VacA and CagA toxins, except for from the corresponding mutant strains. The ICVs were larger in size and positive for bacterial markers, as well as epithelial markers of CD9, LGR5, but negative for nuclear (Ki76) or cytoplasmic (β-actin) markers. Bacteria-independent transfer of CagA and VacA into the recipient cells occurred upon treatment of cells with BVs and ICVs, followed by cellular vacuolation and elongation. IL-8 production was induced in recipient AGS cells, treated with BVs (1279.4 ± 19.79 pg/10 6  cells), early (8 h, 1171.4 ± 11.31 pg/10 6  cells) and late (48 h, 965.4 ± 36.77 pg/10 6  cells) ICVs (P < 0.0001)., Conclusion: Our data indicates that ICVs, with mixed bacterial and epithelial constituents, similar to BVs, are capable of transferring bacterial toxins into the recipient cells, inducing IL-8 production and subsequent morphologic changes, in an acellular manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

There is a growing interest in designing a nanocarrier containing an EGFR targeting affibody to direct toward cancer cells. Here, cytolysin A was cloned at the N-terminus of Z EGFR:1907 affibody to guarantee its surface presentation on the OMVs while targeting the epidermal growth factor receptors (EGFRs). A separate construct including a fusogenic peptide (GALA) was also designed for the endosomal escape of the nanocarrier. Binding of the two constructs ClyA-affi EGFR and ClyA-affi EGFR -GALA to domain III of EGFR was investigated using molecular docking and molecular dynamic simulations. The higher stability of the ClyA-affi EGFR -GALA/EGFR as compared to the ClyA-affi EGFR /EGFR complex was evident. The ClyA-affi EGFR -GALA structure showed a higher RMSD during the first half of the simulation time implying a much less stable behavior. Plateau state of the radius of gyration plot of ClyA-affi EGFR -GALA confirmed a well-folded structure in the presence of the GALA sequence. Solvent accessible surface area for both proteins was in the same range. The data obtained from hydrogen bond analysis revealed a more equilibrated and stable form of the ClyA-affi EGFR -GALA structure upon interaction with EGFR. The data provided here was a requisite for our biological evaluation of the synthesized constructs as a component of a novel drug delivery system., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

It has been described that using noninvasive exposure to 40-Hz white light LED reduces amyloid-beta, a peptide thought to initiate neurotoxic events in Alzheimer's disease (AD). However, the mechanisms remain to be identified. Since AD impairs mitochondrial potassium channels and respiratory chain activity, the objectives of the current study were to determine the effect of 40-Hz white light LED on structure-function of mitoK ATP channel and brain mitochondrial respiratory chain activity, production of reactive oxygen species (ROS), and ΔΨ m in AD. Single mitoK ATP channel was considered using a channel incorporated into the bilayer lipid membrane and expression of mitoK ATP -Kir6.1 subunit as a pore-forming subunit of the channel was determined using a western blot analysis in Aβ1-42 toxicity and light-treated rats. Our results indicated a severe decrease in mito-K ATP channel permeation and Kir6.1 subunit expression coming from the Aβ1-42-induced neurotoxicity. Furthermore, we found that Aβ1-42-induced neurotoxicity decreased activities of complexes I and IV and increased ROS production and ΔΨ m . Surprisingly, light therapy increased channel permeation and mitoK ATP -Kir6.1 subunit expression. Noninvasive 40-Hz white light LED treatment also increased activities of complexes I and IV and decreased ROS production and ΔΨ m up to ~ 70%. Here, we report that brain mito-K ATP channel and respiratory chain are, at least in part, novel targets of 40-Hz white light LED therapy in AD., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background & Objective: This study examined the potential of human epididymis protein 4 (HE4) as a marker in early diagnosis or as a prognostic factor for breast cancer (BC) patients., Methods: A total of 31 patients diagnosed with BC were enrolled in the study between 2008 and 2018. The mRNA and protein expression levels of HE4 were analyzed by immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) in the BC tissue and the non-tumoral adjacent tissue. Using ELISA technique, HE4 plasma levels were also measured in 43 BC patients compared to 43 healthy individuals. The correlation between HE4 expression and clinicopathological features was then investigated., Results: An increase in HE4 expression was observed at mRNA and protein levels in the BC group compared to the control group ( P <0.01, P <0.0001, respectively). In addition, the relative expression of HE4 mRNA in BC patients showed a significant correlation with the differentiation grade of cancer cells ( P <0.001). Plasma levels of HE4 was also associated with grade ( P <0.0001), stage, and tumor size in BC patients (for both P <0.01). Patients with metastatic BC ( P <0.01), lymphatic invasion, and lymph node involvement (for both P <0.05) showed significantly higher plasma levels of HE4 expression than patients without metastasis., Conclusion: According to our findings, upregulation of HE4 may be related to invasive BC phenotype. Measuring plasma levels of HE4 could be useful as a screening test in early diagnosis of BC.

Objectives: Gaucher disease (GD) is the most common autosomal recessive disorder of glycolipid storage. It results from mutations in the glucocerebrosidase ( GBA ) gene and leads to GBA deficiency. Different mutations are associated with different phenotypes in the three major types of GD., Materials and Methods: The spectrum of mutations in GBA gene in 26 unrelated patients with GD from different Iranian populations was determined by DNA sequencing, polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), and amplification-refractory mutation system (ARMS) methods. An in silico analysis was also performed for novel mutations., Results: Six new mutations were identified in this study. The newly detected mutations that could be theoretically harmful included p.I200T (c.599T>C), p.H312D (c.934C>G), p.L325S (c.974T>C), p.L393V (c.1177C>G), p.S439G (c.1315A>G), and p.M455R (c.1365G>A). Also, p.L483P, p.N409S, p.W420X, p.E379K, p.R398Q, p.N227S, p.R202Q, and p.D448H mutations were identified in the patients. Besides, two new complex mutations, namely, p.S439G/p.S439G+p.E379K/- and p.R202Q/p.R202Q+p.N227S/p.N227S, were detected. The most common GBA mutation in the population was p.L483P with an allele frequency of 32.7%, followed by p.N409S (19.2%)., Conclusion: The present study detected six new mutations of GBA gene among GD patients. Two mutations (p.L483P and p.N409S) were especially common among Iranians; this finding can be used in implementing screening programs and understanding the molecular basis of GD., Competing Interests: All authors of this article have no financial or personal involvement or any conflicts of interest.

Although it is well established that diabetes impairs mitochondrial respiratory chain activity, little is known of the effects of intranasal insulin (INI) on the mitochondrial respiratory chain and structure-function of mitoBK Ca channel in diabetes. We have investigated this mechanism in an STZ-induced early type 2 diabetic model. Single ATP-sensitive mitoBK Ca channel activity was considered in diabetic and INI-treated rats using a channel incorporated into the bilayer lipid membrane. Because mitoBK Ca channels have been involved in mitochondrial respiratory chain activity, a study was undertaken to investigate whether the NADH, complexes I and IV, mitochondrial ROS production, and ΔΨ m are altered in an early diabetic model. In this work, we provide evidence for a significant decrease in channel open probability and conductance in diabetic rats. Evidence has been shown that BK Ca channel β2 subunits induce a left shift in the BK Ca channel voltage dependent curve in low Ca 2+ conditions,; our results indicated a significant decrease in mitoBK Ca β2 subunits using Western blot analysis. Importantly, INI treatment improved mitoBK Ca channel behaviors and β2 subunits expression up to ~70%. We found that early diabetes decreased activities of complex I and IV and increased NADH, ROS production, and ΔΨ m . Surprisingly, INI modified the mitochondrial respiratory chain, ROS production, and ΔΨ m up to ~70%. Our results thus demonstrate an INI improvement in respiratory chain activity and ROS production in brain mitochondrial preparations coming from the STZ early diabetic rat model, an effect potentially linked to INI improvement in mitoBK Ca channel activity and channel β2 subunit expression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We have determined the electropharmacological properties of a new potassium channel from brain mitochondrial membrane using a planar lipid bilayer method. Our results show the presence of a channel with a conductance of 150 pS at potentials between 0 and -60 mV in 200 mM cis /50 mM trans KCl solutions. The channel was voltage independent, with an open probability value of approximately 0.6 at different voltages. ATP did not affect current amplitude or open probability at positive and negative voltages. Notably, adding iberiotoxin, charybdotoxin, lidocaine or margatoxin had no effect on the channel behavior. Similarly, no changes were observed by decreasing the cis pH to 6. Interestingly, the channel was inhibited by adding sodium in a dose-dependent manner. Our results also indicated a significant increase in mitochondrial complex IV activity and membrane potential and a decrease in complex I activity and mitochondrial ROS production in the presence of sodium ions. We propose that inhibition of mitochondrial potassium transport by sodium ions on potassium channel opening could be important for cell protection and ATP synthesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Background: In the human genome, the transcription factors (TFs) and transcription factor-binding sites (TFBSs) network has a great regulatory function in the biological pathways. Such crosstalk might be affected by the single-nucleotide polymorphisms (SNPs), which could create or disrupt a TFBS, leading to either a disease or a phenotypic defect. Many computational resources have been introduced to predict the TFs binding variations due to SNPs inside TFBSs, sTRAP being one of them., Methods: A literature review was performed and the experimental data for 18 TFBSs located in 12 genes was provided. The sequences of TFBS motifs were extracted using two different strategies; in the size similar with synthetic target sites used in the experimental techniques, and with 60 bp upstream and downstream of the SNPs. The sTRAP (http://trap.molgen.mpg.de/cgi-bin/trap&#95;two&#95;seq&#95;form.cgi) was applied to compute the binding affinity scores of their cognate TFs in the context of reference and mutant sequences of TFBSs. The alternative bioinformatics model used in this study was regulatory analysis of variation in enhancers (RAVEN; http://www.cisreg.ca/cgi-bin/RAVEN/a). The bioinformatics outputs of our study were compared with experimental data, electrophoretic mobility shift assay (EMSA)., Results: In 6 out of 18 TFBSs in the following genes COL1A1, Hb ḉᴪ, TF, FIX, MBL2, NOS2A, the outputs of sTRAP were inconsistent with the results of EMSA. Furthermore, no p value of the difference between the two scores of binding affinity under the wild and mutant conditions of TFBSs was presented. Nor, were any criteria for preference or selection of any of the measurements of different matrices used for the same analysis., Conclusion: Our preliminary study indicated some paradoxical results between sTRAP and experimental data. However, to link the data of sTRAP to the biological functions, its optimization via experimental procedures with the integration of expanded data and applying several other bioinformatics tools might be required., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Introduction: Rheumatoid arthritis (RA) is categorized as an autoimmune disease with a frequency of 0.2-1% worldwide. It is reported that various autoantibodies are produced in the RA population, particularly against citrullinated peptides. Among various candidate markers for RA diagnosis, the citrullinated proteins have the highest specificity and sensitivity for both diagnosis and prognosis of RA. Anti-mutated citrullinated vimentin and α-enolase constitute a new class of autoantibodies for early detection of RA., Material and Methods: 45 serum samples and 19 synovial fluid (SF) specimens collected from RA patients were considered for American College of Rheumatology criteria and 20 serum samples and 10 SF specimens were provided from healthy subjects as a control group. To assess the quantity of anti-citrullinated protein antibodies (ACPA), anti-mutated citrullinated vimentin (MCV) and anti-α-enolase in the serum and SF of RA patients were determined by the enzyme-linked immunosorbent assay (ELISA) method. For the evaluation of disease activity and joint destruction, we used the Disease Activity Score of 28 joints based on erythrocyte sedimentation rate (ESR) Disease Activity Score 28 (DAS28). Furthermore, to measure the molecular weight of vimentin and α-enolase, electrophoresis on 10% SDS-PAGE was performed as described before., Results: The anti-α-enolase level among serum samples from RA patients was significantly higher than in healthy subjects (4.49 ±0.20 ng/ml vs. 0.76 ±0.12 ng/ml) ( p < 0.001). There was a direct relation between α-enolase quantity and (rheumatoid factor) RF and C-reactive protein (CRP) levels. The mean ESR value in positive and negative ACPA patients was 38.2 ±22.6 mm/h and 9.2 ±5.8 mm/h respectively ( p < 0.0001). The mean DAS28-ESR was 3.3. The level of anti-MCV in the serum of RA patients (244.6 ±53.3 U/ml) was higher than in serum of the healthy group (148.73 ±71.8) ( p < 0.0001). The level of anti-MCV in the SF of patients was 687.5 ±148.4 U/ml., Conclusions: In conclusion, both autoantibodies against MCV and α-enolase are two important markers that increase in serum and SF of RA patients and are specific for diagnosis of RA disease., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2020 Narodowy Instytut Geriatrii, Reumatologii i Rehabilitacji w Warszawie.)

Aim: Trace elements play a key role in human metabolism. The aim of the present study was to measure essential trace elements in the serum of patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML)., Materials and Methods: 32 patients with ALL and 16 patients with AML were studied. The control group consisted of 36 subjects. Serum levels of the trace elements selenium, copper and zinc were measured by spectrophotometry., Results: The mean of copper concentrations in the groups of patients with AML and ALL was significantly higher than in the control group (p < 0.0001), whereas serum levels of selenium and zinc were significantly lower in AML patients (p < 0.0001). Also in ALL patients the levels of selenium and zinc were significantly decreased compared with the control group (p < 0.0001; p = 0.0003)., Conclusion: Our results indicate that the levels of zinc and selenium are significantly decreased and copper levels are significantly increased in the serum of patients with acute leukemia (AML, ALL).

Background: Rheumatoid Arthritis (RA) is a chronic multi systemic disorder with the unclarified ethiopathology. Although several markers have been presented for recognition of RA, but none of them has been specific. New markers such as HLA typing and activity of Adenosine Deaminase (ADA) isoenzymes could be useful and specific., Objective: The aim of this study is to evaluate the pattern of ADA isoenzymes activity and HLA typing in both RA patients and healthy cases., Methods: Blood samples were collected from 55 RA patients and 60 healthy subjects, over a period of 6 months. Levels of C-reactive Protein (CRP), Rheumatoid Factor (RF) and ADA (ADA1, ADA2, total ADA) were measured using AVITEX kit and HITACHI Auto Analyzer. In addition, HLA-DRB1*01,*04 and *10 was detected using PCR-SSP., Results: ADA activity, particularly ADA2 level, was significantly higher among RA group (Pv <0.05). The concentrations of tADA in patients with RF and CRP positive were significantly higher (Pv <0.05). The allele prevalence of DRB1*01 was significantly higher in RA patients (13.1%) compared with control group (5.5%, respectively) (P <0.05, Bonferroni adjustment P<0.003). Calculated sensitivity and specificity for diagnostic tests in this study are listed as: CRP (75%), RF (80%), ADA (84%) and RF (90%), ADA (83%), CRP (72%), respectively., Conclusion: Increased tADA level and the frequency of DRB1*10 and *01 caused susceptibility to RA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Acute lymphoblastic leukemia (ALL) is known as the most prevalent pediatric malignancy all around the world. Identification of specific biomarker is necessary for early diagnosis and effective therapy. It is believed that Adenosine deaminase (ADA) as an enzyme involved in the purine salvage pathway increases in ALL patients. Herein, the quantity and pattern of ADA isoenzymes were surveyed among ALL patients in comparison to healthy subjects., Methods: Serum and RBC samples of three different groups of ALL patients, including newly diagnosed cases without any drugs administration, subjects with the relapsed disease, patients in the remission stage after therapy, and the healthy subjects were enrolled in the study. Then, the activity and pattern of ADA1, ADA2 and ADA1+cp were determined using ADA kit and electrophoresis on SDS-PAGE, respectively. To confirm the presence of ADA enzyme, the fresh serums, extractions from erythrocytes, JM cell line as a human T lymphocyte line and J774 A.1 as mouse monocyte line were electrophoresed on 1.2% agarose gel and stained with the specific dye., Results: The activities of ADA1 isoenzyme and total ADA in new cases and subjects with the relapsed disease were significantly higher than their activities in the patients in the remission stage and healthy controls (p<0.001). The unbounded ADA1 isoenzyme was found to exist in the erythrocyte, lymphocyte and monocyte. But in serum, all the ADA1 was bounded to the cp protein., Conclusions: ADA1 is the key isoenzyme elevating in ALL patients, therefore this isoenzyme could be a useful biomarker to diagnose ALL patients and monitor their therapies., Competing Interests: Conflict of interest statement:The authors stated that they have no conflicts of interest regarding the publication of this article.

We have characterized the conduction and blocking properties of two different chloride channels from brain mitochondrial inner membranes after incorporation into planar lipid bilayers. Our experiments revealed the existence of channels with a mean conductance of 158 ± 7 and 301 ± 8 pS in asymmetrical 200 mM cis/50 mM trans KCl solutions. We determined that the channels were ten times more permeable for Cl - than for K + , calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channels were bell-shaped voltage dependent, with maximum open probability 0.9 at ± 20 mV. Two mitochondrial chloride channels were blocked after the addition of 10 µM DIDS. In addition, 158 pS chloride channel was blocked by 300 nM NPPB, acidic pH and 2.5 mM ATP, whereas the 301 pS chloride channel was blocked by 600 µM NPPB but not by acidic pH or ATP. Gating and conducting behaviors of these channels were unaffected by Ca 2+ . These results demonstrate that the 158 pS anion channel present in brain mitochondrial inner membrane, is probably identical to IMAC and 301 pS Cl channel displays different properties than those classically described for mitochondrial anion channels.

Finding new compounds with antifungal properties is an important task due to the side effects of common antifungal drugs and emerging antifungal resistance in fungal strains. ∆ 24 -sterol methyltransferase (24-SMT) is a crucial enzyme that plays important roles in fungal ergosterol biosynthesis pathway and is not found in humans. In the present study, the effects of α-bisabolol on Aspergillus fumigatus Af239 growth and ergosterol synthesis on the base of 24-SMT enzyme activity were studied; in addition, the expression of erg6, the gene encoded 24-SMT, was considered. To our knowledge, this is the first report demonstrating that α-bisabolol inhibits A. fumigatus growth specifically via suppressing fungal 24-SMT. Since this enzyme is a specific fungal enzyme not reported to exist in mammalian cells, α-bisabolol may serve as a lead compound in the development of new antifungal drugs. Fungi were cultured in presence of serial concentrations of α-bisabolol (0.281-9 mM) for 3 days at 35 °C. Mycelia dry weight was determined as an index of fungal growth and ergosterol content was assessed. Microsomal 24-SMT activity was assayed in presence of α-bisabolol as an inhibitor, lanosterol as a substrate and [methyl-H 3 ] AdoMet (S-Adenosyl methionin). In addition, the expression of erg 6 gene (24-SMT encoding gene) was determined after treatments with various concentrations of α-bisabolol. Our results demonstrated that α-bisabolol strongly inhibited A. fumigatus growth (35.53-77.17%) and ergosterol synthesis (26.31-73.77%) dose-dependently and suppressed the expression of erg 6 gene by 76.14% at the highest concentration of 9 mM. α-bisabolol inhibited the activity of 24-SMT by 99% at the concentration of 5 mM. Taken together, these results provides an evidence for the first time that α-bisabolol inhibits A. fumigatus Af239 growth via affecting microsomal ∆ 24 -sterol methyltransferase as a crucial enzyme in ergosterol biosynthetic pathway.

Background and Aims . In the present study, we have investigated the activity of adenosine deaminase (ADA) as a diagnostic marker in type 2 (or II) diabetes mellitus (T2DM). Design and Methods . The deaminase activity of ADA1 and ADA2 was determined in serum from 33 patients with type 2 (or II) diabetes mellitus and 35 healthy controls. We also determined the proportion of glycated hemoglobin (HbA1c). Results . Our results showed significant differences between total serum ADA (tADA) and ADA2 activities in the diabetic groups with HbA1c < 8 (%) and HbA1c ≥ 8 (%) with respect to the values in healthy individuals ( p < 0.001). ADA2 activity in patients with high HbA1c was found to be much higher than that in patients with low HbA1c ( p = 0.0001). In addition, total ADA activity showed a significant correlation with HbA1c ( r = 0.6, p < 0.0001). Conclusions . Total serum ADA activity, specially that due to ADA2, could be useful test for the diagnosis of type 2 (or II) diabetes mellitus., Competing Interests: The authors declare no conflict of interests.

We report in a previous study the presence of a large conductance K+ channel in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity in this case was found to decrease in presence of ATP 100 µM on the cytoplasmic side and was totally inhibited at ATP concentrations greater than 0.25 mM. Although such features would be compatible with the presence of a KATP channel in the RER, recent data obtained from a brain mitochondrial inner membrane preparation have provided evidence for a Maxi-K channel which could also be blocked by ATP within the mM concentration range. A series of channel incorporation experiments was thus undertaken to determine if the ATP-sensitive channel originally observed in the RER corresponds to KATP channel. Our results indicate that the gating and permeation properties of this channel are unaffected by the addition of 800 nM charybdotoxin and 1 µM iberiotoxin, but appeared sensitive to 10 mM TEA and 2.5 mM ATP. Furthermore, adding 100 µM glibenclamide at positive potentials and 400 µM tolbutamide at negative or positive voltages caused a strong inhibition of channel activity. Finally Western blot analyses provided evidence for Kir6.2, SUR1 and/or SUR2B, and SUR2A expression in our RER fractions. It was concluded on the basis of these observations that the channel previously characterized in RER membranes corresponds to KATP, suggesting that opening of this channel may enhance Ca2+ releases, alter the dynamics of the Ca2+ transient and prevent accumulation of Ca2+ in the ER during Ca2+ overload.

In a previous study we reported the presence of a large conductance K(+) channel in the membrane of endoplasmic reticulum (ER) from rat hepatocytes. The channel open probability (Po) appeared voltage dependent and reached to a minimum 0.2 at +50 mV. Channel activity in this case was found to be totally inhibited at ATP concentration 2.5 mM, glibenclamide 100 µM and tolbutamide 400 µM. Existing evidence indicates an impairment of endoplasmic reticulum functions in ER stress condition. Because ER potassium channels have been involved in several ER functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the ER potassium channel function is altered in a high fat diet model of ER stress. Male Wistar rats were made ER stress for 2 weeks with a high fat diet. Ion channel incorporation of ER stress model into the bilayer lipid membrane allowed the characterization of K(+) channel. Our results indicate that the channel Po was significantly increased at voltages above +30 mV. Interestingly, addition of ATP 7.5 mM, glibenclamide 400 µM and tolbutamide 2400 µM totally inhibited the channel activities, 3-fold, 4-fold and 6-fold higher than that in the control groups, respectively. Our results thus demonstrate a modification in the ER K(+) channel gating properties and decreased sensitivity to drugs in membrane preparations coming from ER high fat model of ER stress, an effect potentially linked to a change in ER K(+) channel subunits in ER stress condition. Our results may provide new insights into the cellular mechanisms underlying ER dysfunctions in ER stress.

Background: Rabies is a major zoonotic viral disease and is detected using the World Health Organization standard diagnostic techniques. Rabies detection is preferably done using the fluorescent antibody technique (FAT) that provides reliable diagnosis with almost 100% accuracy for all variant strains, if a proper conjugate is used. Rabies virus nucleoprotein (NP) is the most important protein used in production of a specific diagnostic conjugate., Objectives: The aim of this study was to extract the cell-associated rabies virus NP from infected Baby Hamster Kidney cell clone (BSR) with rabies virus (Pasteur vaccine strain/PV) and purify for a future project to produce an anti-NP conjugate., Materials and Methods: Pasteur vaccine strain (PV) as the standard rabies vaccine strain with a focus-forming dose (FFD) of 105 was inoculated in to the BSR cell culture at a concentration of 10(6) cells per milliliter. Infected cells were harvested 72 hours after infection and the rabies NP was extracted from these cells by low-speed centrifugation and purification by ultracentrifugation in cesium chloride (CsCl) gradient. For analysis, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)., Results: The volume of the lysate was 15 mL and it became 2.5 mL after purification, with a concentration of 3.25 mg/mL. The corresponding band to the cell lysate protein on the SDS-PAGE had a molecular weight of 50 KDa, similar to the molecular weight of NP in rabies virus., Conclusions: The rabies virus NP could be extracted and purified in an appropriate amount from infected cell culture. The results of SDS-PAGE analysis showed that the intact rabies virus NP had been purified properly and thus could be used for further steps to produce the specific diagnostic rabies conjugate.

Background: Defects in endoplasmic reticulum homeostasis are common occurrences in different diseases, such as diabetes, in which the function of endoplasmic reticulum is disrupted. It is now well established that ion channels of endoplasmic reticulum membrane have a critical role in endoplasmic reticulum luminal homeostasis. Our previous studies showed the presence of an ATP-sensitive cationic channel in endoplasmic reticulum. Therefore, in this study, we examined and compared the activities of this channel in control and diabetic rats using single-channel recording techniques., Method: Male Wistar rats were made diabetic for 2 weeks with a single dose injection of streptozotocin (45 mg/kg). Ion channel incorporation of rough endoplasmic reticulum of diabetic hepatocytes into the bilayer lipid membrane allowed the characterization of K+ channel., Results: Ion channel incorporation of rough endoplasmic reticulum vesicles into the bilayer lipid revealed that the channel current-voltage (I-V) relation with a mean slope conductance of 520 ± 19 pS was unaffected in diabetes. Interestingly, the channel Po-voltage relation was significantly lower in diabetic rats at voltages above +30 mV., Conclusion: We concluded that the endoplasmic reticulum cationic channel is involved in diabetes. Also, this finding could be considered as a goal for further therapeutic plans.

Borrelia persica is a strain seen only in the Middle East and responsible for relapsing fever. These spirochetes are notable for multiphasic antigenic variation of polymorphic outer membrane lipoproteins, a phenomenon responsible for immune evasion. Diagnosis of the disease is a problem and requires a fixed antigen like the flagellar antigen. In vitro culture of B. persica was carried out for the first time and flagellar antigen was purified from culture. 10% SDS was added to the mixture to dissolve the cell wall and then the solution was sheared in an Omni mixer. Electron microscopy confirmed the purity of a 42 KDa periplasmic antigen as revealed by SDS-PAGE. Indirect haemagglutination kits were designed using the pure flagella and tested for cross reactivity with another relapsing fever spirochaete Borrelia microtii positive serum. The kit showed 98% sensitivity and 95% specificity.

Influenza virosome is one of the commercially available vaccines that have been used for a number of years. Like other influenza vaccines, the efficacy of the virosomal vaccine is significantly compromised when circulating viruses do not have a good match with vaccine strains due to antigenic drift or less frequent emergence of a pandemic virus. A major advantage of virosome over other influenza vaccine platforms is its intrinsic adjuvant activity and potential carrier capability which have been exploited in this study to broaden vaccine protectivity by incorporating a conserved component of influenza virus in seasonal vaccine formulation. Influenza nucleoprotein (NP)-encoding plasmid was adsorbed onto surface of influenza virosomes as a virosome/DNA vaccine complex. Mice were immunized with a single dose of the influenza virosome attached with the NP plasmid or NP plasmid alone where both influenza virosomes and NP gene were derived from influenza A virus H1N1 New/Caledonia strain. Analysis of the cellular immune responses showed that 5μg (10-fold reduced dose) of the NP plasmid attached to the virosomes induced T cell responses equivalent to those elicited by 50μg of NP plasmid alone as assessed by IFN-γ and granzyme B ELISPOT. Furthermore, the influenza virosome/NP plasmid complex protected mice against intra-subtypic challenge with the mouse adapted H1N1 PR8 virus, while mice immunized with the virosome alone did not survive. Results of hemagglutination inhibition test showed that the observed intra-subtypic cross-protection could not be attributed to neutralizing antibodies. These findings suggest that influenza virosomes could be equipped with an NP-encoding plasmid in a dose-sparing fashion to elicit anti-influenza cytotoxic immune responses and broaden the vaccine coverage against antigenic drift., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Adenosine deaminase (ADA) plays a crucial role in the development and maintenance of normal immune system. So, any immunological imbalances could associate with its altered activity in serum. This study evaluated the activity of total ADA and its isoenzymes in serum of 45 patients with systemic lupus erythematosus (SLE). Included were 23 patients with active SLE, 22 during the inactive phase of the disease, and 45 healthy subjects. Our results provided evidence that the significantly elevated total ADA activity in serum of SLE patients is correlated mainly with the increased ADA2 level. The highest mean ADA2 activity during the relapse phase of the disease could be an indication to the macrophages, the main source of ADA2. It might be concluded that ADA and its isoenzymes analysis in serum of patients could be used as a useful and non-invasive diagnostic tool in evaluation of SLE active phase and the disease severity.

The purpose in this study was to investigate poly(ethylene glycol)-modified poly (d,l-lactide-co-glycolide) nanoparticles (PLGA-PEG-NPs) loading 9-nitrocamptothecin (9-NC) as a potent anticancer drug. 9-NC is an analog of the natural plant alkaloid camptothecin that has shown high antitumor activity and is currently in the end stage of clinical trial. Unfortunately, at physiological pH, these potent agents undergo a rapid and reversible hydrolysis with the loss of antitumor activity. Previous researchers have shown that the encapsulation of this drug in PLGA nanoparticles could increase its stability and release profile. In this research we investigated PLGA-PEG nanoparticles and their effect on in vitro characteristics of this labile drug. 9-NC-PLGA-PEG nanoparticles with particle size within the range of 148.5 ± 30 nm were prepared by a nanoprecipitation method. The influence of four different independent variables (amount of polymer, percent of emulsifier, internal phase volume, and external phase volume) on nanoparticle drug-loading was studied. Differential scanning calorimetry and X-ray diffractometry were also evaluated for physical characterizing. The results of optimized formulation showed a narrow size distribution, suitable zeta potential (+1.84), and a drug loading of more than 45%. The in vitro drug release from PLGA-PEG NPs showed a sustained release pattern of up to 120 hours and comparing with PLGA-NPs had a significant decrease in initial burst effect. These experimental results indicate that PLGA-PEG-NPs (versus PLGA-NPs) have a better physicochemical characterization and can be developed as a drug carrier in order to treat different malignancies.

Objectives: We aimed to investigate the activity of ADA and its isoenzymes in serum of patients with various primary immunodeficiency (PID) syndromes., Design and Methods: Total ADA (tADA) and its isoenzymes were measured in 76 children with PID syndromes and 30 healthy controls using the Ellis method., Results: Our results indicated that tADA and ADA2 levels were higher in patients with Chronic Granulomatous Disease (CGD), Leukocyte Adhesion Deficiency (LAD), hyper IgM (HIM) and Wiskott-Aldrich Syndrome (WAS) than those of corresponding controls (P<0.01). There was a significant elevation of tADA and ADA1 activities in IgA deficiency patients as compared to healthy individuals (P<0.01)., Conclusions: Our results hypothesized that altered ADA activity may be associated with altered immunity. Therefore, serum ADA level could be used as an indicator along with other parameters in follow up of patients with CGD, LAD, IgA deficiency, HIM and WAS.

Background: The purpose of this study was to evaluate the levels of zinc (Zn), copper (Cu), iron (Fe) and zinc/ copper ratio in the serum of patients with cutaneous leishmaniasis in Qom Province, center of Iran., Methods: Serum levels of zinc and copper were determined by flame atomic absorption spectrophotometer and serum iron concentration was measured by using an Auto Analyzer. The study group consisted of 60 patients with cutaneous leishmaniasis and the control group of 100 healthy volunteers from the same area who were not exposed to cutaneous leishmaniasis., Result: There were no statistically significant differences in age and body mass index between the two groups. Serum Zn (P< 0.001) and Fe (P< 0.05) levels were lower in patients with cutaneous leishmaniasis than the control group. We also found serum Cu concentration (P< 0.05) in the patient group was significantly higher than that of the control group. However, zinc/ copper ratio (P< 0.001) was lower in patients with cutaneous leishmaniasis than in the control group., Conclusion: Our data indicated that Zn/Cu ratio was significantly lower in patients with CL as compared to the controls. Earlier reports suggest that, this ratio imbalance could be a useful marker for immune dysfunction in leishmaniasis. There was also strong association of Zn, Cu and Fe with CL. It suggests the use of blood zinc, copper, iron concentration and the copper/zinc ratio (Zn/Cu), as a means for estimating the prognosis of CL.

ATP-sensitive K+ channels play an important role in regulating membrane potential during metabolic stress. In this work we report the effect of ATP and ADP-Mg on a K+ channel present in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity was found to decrease in presence of ATP 100 microM on the cytoplasmic side and was totally inhibited at ATP concentrations greater than 0.25mM. The effect appeared voltage dependent, suggesting that the ATP binding site was becoming available upon channel opening. Channel activity was suppressed by the nonhydrolyzable ATP analog (ATPgammaS), ruling out a phosphorylation-based mechanism. Notably addition of 2.5mM ADP-Mg to the cytosolic side increased the channel open probability at negative potentials. We conclude that the large conductance voltage-gated cation channel in RER of rat hepatocytes is an ATP and ADP sensitive channel likely to be involved in cellular processes such as Ca(2+) signaling or control of membrane potential across the endoplasmic reticulum membrane.

Background: Amino acid dehydrogenases (L-amino acid: oxidoreductase deaminating; EC 1.4.1.X) are members of the wider superfamily of oxidoreductases that catalyze the reversible oxidative deamination of an amino acid to its keto acid and ammonia with the concomitant reduction of either NAD+, NADP+ or FAD. These enzymes have been received much attention as biocatalysts for use in biosensors or diagnostic kits to screen amino acid metabolism disorders such as phenylketonuria (PKU), maple syrup urine disease (MSUD), homocystinuria (HCY) and hyperprolinemia. This study was aimed to isolation and screening of novel amino acid dehydrogenases from soil bacteriadehydrogenases from soil bacteria., Methods: The enzyme producing bacteria were selected among L-methionine and L-phenylalanine utilizers isolated from soil by thin layer chromatography, activity staining and confirmed by enzyme assay. Bacterial strains were identified by phenotypic and biochemical characteristics. The steady-state kinetic studies of enzymes were also performed., Results: In total of 230 tested strains, four of them were recognized as amino acid dehydrogenase producers that belong to species of Pseudomonas, Citrobacter and Proteus. They exhibited the desired NAD+-dependent dehydrogenase activities toward L-isoleucine, L-methionine, L-cysteine, L-serine and L-glutamine in oxidative deamination reaction. The specific activity of L-isoleucine dehydrogenase, L-methionine dehydrogenase and L-glutamine dehydrogenase for oxidative deamination of L-isoleucine, L-methionine and L-glutamine were 1.59, 1.2 and 0.73 U/mg, respectively. The Kcat/Km (s(-1).mM(-1)) values in these strains were as follows: L-isoleucine, 113.6, L-methionine, 62.05 and L-glutamine, 95.83., Conclusion: This is the first report of occurrence a specific isoleucine dehydrogenase, glutamine dehydrogenase and methionine dehydrogenase in bacteria.

In this work, we report the single channel characterization of a voltage gated cationic channel from rough endoplasmic reticulum (RER) membranes of rat hepatocytes incorporated into a planar lipid bilayer. The channel was found to be cation selective with a main conductance of 598+/-20 pS in 200 mM KCl cis/50 mM KCl trans. The channel open probability appeared voltage dependent with a voltage for half activation (V(1/2)) of 38 mV and an effective gating charge z of -6.66. Adding either 4-AP (5 mM) or ATP (2.5 mM) to the side corresponding to the cell internal medium caused a strong inhibition of the channel activity. This channel is likely to be involved in maintaining proper cation homeostasis in the RER of hepatocytes.

Borrelia microtti and Borrelia persica are 2 Iranian strains of Borrelia found in western Asia and responsible for relapsing fever. The outer surface antigens of Borrelia undergo variations which are responsible for the relapsing phenomenon. The fixed flagellar antigen is required for diagnosis as the variant antigens cannot be used in serological methods of diagnosis. The fixed flagellar antigen was purified for the first time from the Iranian strain of Borrelia microtti using detergent treatment and shearing in an omnimixer. Periplasmic flagella were extracted, as confirmed by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a band corresponding to 42 kDa. Indirect haemagglutination kits were designed using the pure flagella and the complete sonicate of Borrelia and showed 98% sensitivity and 95% specificity.