Cycles of MCMM conformational search performed on the Jak3 1 complex giving mobility to the remains and the ligand inside a 4 distance permit a possible hydrogen bond between Caspase inhibition the nitrile purpose and Gln988, a relationship that could be lost in Jak2. But, the docking present of just one in Jak2 does retain the key hydrogen bond with Arg980. It’s uncertain how this lone change may possibly affect binding, but given the relative Kd and IC50 values claimed for 1 at both objectives the big difference is presumably negligible. This really is also consistent with the proven fact that, because of the unique conformation of the part of the initial loop located instantly ahead of the APE design, in Jak2 Glu1015 points far from the binding site and wouldn’t take proximity with the nitrile moiety. From the docking comparisons, the related disassociation constants for 1 at Jak3 and Jak2 aren’t surprising. Early results from the clinical use of 1 demonstrate effectiveness, but in addition unwanted anemia and neutropenia. 26 This shows that unwelcome downregulation of Jak2 is occurring to a significant degree. None the less, Dizocilpine 77086-21-6 phase 1 clinical evaluations demonstrated a fair safety profile and numerous phase 2 evaluations are underway. The IC50 values claimed by Changelian et al. Show a small degree of selectivity between Jak3 and Jak2. This data was collected via ELISA and is presumably more accurate compared to the Kd determinations shown here. Nonetheless, whether 1 binds/inhibits Jak2 at 1 nM or 20 nM levels, it’s likely that the physiological ranges of the drug will surpass the amount needed for successful downregulation of Jak2. The more convincing studies, however, are cell based studies such as the assessment of inhibition Inguinal canal of Stat4 phosphorylation by 1 and the prior report that 1 effectively inhibits IL 2 stimulated cell proliferation while having much weaker impact on granulocyte macrophage colony stimulation factor induced proliferation. These results may provide tantalizing hints to the method by which cytokine receptor/Jak sets trigger signaling cascades. Kinases are among the most intriguing therapeutic goals in the human proteome and kinase inhibitors are becoming staples of the pharmacopeia. A major doctrine of drug design is to restrict the number of chiral centers placed into small molecules intended for clinical use for an array of reasons. 1 goes against convention and features not just one, but two chiral centers. Employing a combination of molecular modeling, target profiling and cell based studies we’ve found A 205804 251992-66-2 that the chiral nature of just one is a key element that describes its ability to bind and inhibit its primary target. In addition, discrete stereoisomers of 1 may prove of good use starting points for novel small molecules targeting alternate branches of the kinome.

CP690550, was found to diminish mortality and lower target organ damage in rats put through GVHD by controlling Raf inhibition donor CD4 T cell mediated?? Generation and inhibition of Th1 differentiation. Specic inhibitors of Janus kinase 3 have been tested as cure for GVHD. The usage of the JAK 3 chemical, WHI P131, showed increased mortality rates and reduced liver and skin injury. Still another JAK 3 inhibitor, 4 amino 6,7 dimethoxyquinazoline, increased mortality rates and ameliorated the clinical apparent symptoms of GVHD. A specic Brutons tyrosine kinase inhibitor, was also tested as remedy for GVHD, treated rats had less scientific GVHD and showed enhanced survival rates. The combined treatment of LFM A13 with JANEX 3 was more efficient than treatment with LFM A13 or JANEX 3 alone. Taken together, these results suggest that signaling molecules downstream MK 801 cost of chemokine signaling may be of good use targets for managing GVHD. In the context of the treatment of hematological malignances, such as for instance leukemia, engraftment of donor cells is essential to revive the immune system after ablative therapy. Along with rebuilding the disease fighting capability, the engrafted cells are believed to contribute to chemotherapy by causing an anti tumor effect, an effect that is known as. A few therapies that decrease GVHD may possibly decrease GVL, which is an undesirable outcome of such therapies. Consequently, it’s generally speaking accepted that, in the context of haematopoietic stem cell transplantation, a therapy should reduce Infectious causes of cancer or prevent GVHD but ideally shouldn’t alter the associated GVL. Although a promising system is represented by the chemokine system to a target to produce new GVHD therapies, it’s also vital that you recognize the role of chemokines in GVL answer. Evaluation of GVL hasn’t been the major focus of studies involving chemokines and GVHD. But, we have found several studies Dalcetrapib price showing that, by interfering with the chemokine system, it’s possible to diminish GVHD without interfering with GVL. Our group and Choi et al. Indicated that, inspite of the important activity of CCR1 and its ligands, CCL3, and CCL5, in the GVHD result, neutralization of CCL3, or the absence of CCR1 in donor cells did not interfere with GVL. The ability of T cells to remove tumor cells remained unaltered upon neutralization of CCL3 by evasin 1 in mice put through GVHD. The absence of CCR1 in donor cells also maintained the GVL response in rats put through GVHD. Ueha et al. veried the GVL result in a study investigating the function of fractalkine in GVHD. In this research, CX3CL1 was important for GVHD growth, but not for the GVL reaction, and treatment with anti CX3CL1 lowered GVHD without changing GVL.

HGF inhibition somewhat decreased custom peptide price cyst development and growth in both established and minimal infection settings of CCS. We examined the tumors that developed despite anti HGF antibody treatment and found that c Met was strongly stimulated in these tumors. This result, taken together with the xenograft little condition finding, suggests that the antibody most potently inhibits the survival/proliferation of isolated tumor cells or really small tumors. After the tumefaction becomes established, the antibody might be no further effective at inhibiting autocrine signaling. It is possible that the local accessibility to antibody is insufficient to block the HGF produced by a growing tumor or that the microenvironment of a more substantial tumor fosters HGF signaling. Nevertheless, the minimal illness model may possibly imitate the scenario faced by physicians with a top risk cancer. After resection of a big primary cyst in the absence of gross metastatic disease, tiny disease often contributes to regional or distant recurrences and ergo such HGF reduction may possibly demonstrate efficacy in the adjuvant setting. Targeting MITF triggered h Met in melanoma could offer an identical therapeutic purpose. Hit down data suggest that the significance of c Met to CCS may often be independent of HGF creation, though it remains to be determined ATP-competitive ALK inhibitor exactly what portion of CCS cancers present c Met initial. Additionally, other techniques could result in c Met initial. For as seen in other cyst types example, in vivo, activation could be mediated through paracrine mechanisms. Our research suggests the possibility of therapeutically targeting HGF:c Met in CCS. Pathological interrogation of h Met expression and phosphorylation status in human cancers should enable selection of individuals most likely to respond to HGF:c Met directed Immune system therapy. Because the essential role of microorganisms in its etiology was scientifically demonstrated in the mid 60s, the research work was long centered on identifying the pathogenic microorganisms and their virulence Gemcitabine Gemzar factors. This seek out offender organisms was encouraged by the fact that colonization of the presence and mouth of dental biofilm is usually connected with health, much like the colonization of the colon. Different therapeutic strategies aimed at the organisms have been studied over time, including systemic and local delivery of antibiotic and antimicrobial agents. The explanation for these therapeutic approaches is the undeniable fact that some species of bacteria are thought to play prominent roles in periodontal illness based on their increased prevalence in the microbial flora associated diseased states.

Chemotaxis was significantly reduced by inhibition of MET expression. The simultaneous expression of c Met and HGF by CCS292 cells and the basal degree of phospho c Met suggest that c Met could be triggered by an autocrine pathway.

The recent identification of a fully human custom peptide price monoclonal anti HGF antibody, offered an opportunity to study the result of HGF inhibition on CCS. To show the activity of AMG 102 on CCS derived HGF, 501mel cells were treated with CCS conditioned media that have been pretreated with AMG 102. At all concentrations tested, AMG 102 entirely blocked cMet activation. This result confirms that c Met service in this melanoma cell line is mediated entirely by HGF and maybe not by still another secreted aspect in the conditioned medium. We then examined the result of HGF inhibition on CCS by managing CCS292 cells with increasing levels of AMG 102.

As opposed to an isotype matched get a grip on antibody, AMG 102 BI-1356 ic50 resulted in a marked, although partial, decrease in activated h Met. Decreased phospho c Met was associated with an increase as a whole c Met, probably reflecting a reduced rate of receptor turnover in the absence of continuous, autocrine ligand activation. We also examined whether AMG 102 mediated c Met inhibition influenced intracellular signaling in CCS292 cells. Both AKT and MAPK signaling were inhibited by AMG 102 therapy in a dose dependent manner.

Small molecule inhibitors of c Met provide an alternative technique to modulate c Met. SU11274 is definitely an inhibitor of c Met with action in both ligand independent and dependent models. Treatment with SU11274 at levels reported to Eumycetoma inhibit c Met triggered a dosedependent decrease in phospho c Met. The inhibition of phospho h Met was connected with decreased buy MK-2206 downstream MAPK and AKT phosphorylation. We then examined survival and cell growth after SU11274 treatment. 1 cell proliferation was transiently decreased by uM SU11274.

But, 10 uM therapy led to a sustained reduction in cell proliferation and reduced cell viability. The information using both an inhibitor of HGF or the c Met kinase inhibitor advise that c Met plays an essential role in a subset of CCS and that its action plays a dominant role in activation of two pathways central to survival and cell growth. Because HGF ignited c Met activation is apparently a main activator of both survival and proliferation trails in CCS, we examined the effect of HGF inhibition on cyst cell proliferation in culture and in vivo.

CCS cell lines were cultured by us in the current presence of the selective HGF chemical, AMG 102. A significant reduction in growth was noted in two CCS lines.

The immune methods response to antigen will depend on the relative frequencies of responding T and B cells and around the thresholds of binding HSP90 inhibition affinity that their receptors display, the amounts of antigen existing, and the period through which the antigen remains in secondary lymphoid tissue, wherever primary immune responses are initiated. Tolerance induction would be the method by which the immune procedure is able to adapt to exogenous antigens and it is characterized by an antigen specific nonreactivity. T and B cell tolerance can be established or disrupted either centrally, at the web-site of key lymphocyte growth during the thymus or bone marrow, or peripherally during the lymphoid tissue exactly where antigen recognition and processing take place. While in the peripheral immune method the key mechanisms that induce and sustain tolerance include things like clonal deletion, anergy, ignorance, and suppression.

Ignorance describes the problem whereby T cells fail to reply to a particular antigen. This can be on account of reduced levels of AG-1478 structure antigen that happen to be inadequate to activate T cells, antigens which are physically separated from T cells. Antigens that happen to be presented from the absence of co stimulation signaling can induce anergy, characterized by state of T cell unresponsiveness. Deletion of T cells can occur when the cell is activated from the absence of co stimulation, or as a consequence of a lack of growth aspects. Tolerance induction by suppression is surely an active method by which a regulatory subset of T cells exclusively suppresses the activity of T cells.

In an hard work to prevent immune responses through gene transfer, viral gene therapy vectors are built to incorporate few or no viral coding genes and prevent expression of pathogenic genes. Elements influencing the host immune response towards the vector, such as route of vector Plastid administration, dose of vector, option of promoter/ enhancer, alterations to vector genome sequence and/or structure, the status plus the nature of the target tissue, and patient related elements are all vital to your advancement of a clinically related gene based mostly strategy to treat human conditions. For some clinical situations, fetal or neonatal therapy are significant for that treatment method of the disorder and in these tactics the immune responses to your vector and/or transgene might be minimized. Transgene expression limited to your target tissue by using tissue unique promoters has become extensively exploited in order to avoid immune responses towards the transgene.

One particular crucial method to avoid supplier PF 573228 an immune response will be to stop transgene expression within antigen presenting cells, which include dendritic cells, B cells, or macrophages. However, the uptake of exogenous protein by APC and presentation within the context of important histocompatibility complicated class I or class II isn’t going to demand direct transduction of APCs by the recombinant vectors.

The cells have been maintained on ice until the i was measured. The i was measured by using an emission wavelength of 520 nm and alternating excitatory wavelengths of 340 and 380 nm. Employing external calibration, we then calculated i based on the equation i _, exactly where Adrenergic Receptors R could be the uorescence intensity of your Ca2 delicate dye fura 2 at excitation wavelengths of 340 and 380 nm, Rmin may be the minimum uorescence ratio of about 0. 768 and Rmax will be the maximum uorescence ratio of about 35. 1. The coecient Sf2 indicates the free dye measured at wavelength of 380 nm and Sb2 indicates Ca2 bound dye at 380 nm. According to experimental information, Sf2/Sb2 for fura 2 is about 15. 3. Kd is the eective dissociation constant of fura 2, which was about 135 nmol l1.

The modify of i in response to phenylephrine or KCl was evaluated by using regular physiologic salt solution containing Ca2. Pretreatment reversible Akt inhibitor of tanshinone IIA was carried out to determine its antagonism of Ca2. We administered the K channel blockers, then added tanshinone IIA to find out this inhibition of i by tanshinone IIA that concerned the opening of K channels. for your number of animals in each and every group as indicated inside the tables and gures. Statistical dierences among groups had been established by using two way repeatedmeasure ANOVA. Dunnett variety post hoc comparisons were utilised to find out the source of signicant dierences in which ideal P worth. 05 was thought of statistically signicant. A dosedependent reduce of SBP in SHR obtained an i. p. injection of danshen was shown in Figure 1, the maximal eect was achieved by 60 min treatment with danshen at ten mg kg1.

The eect of danshen within the reduction of SBP was maintained for 150 min. No transform of SBP was observed in WKY obtaining Papillary thyroid cancer the related administration of danshen at ten mg kg1 for 60 min. Just after treatment with tanshinone IIA, SBP was noticeably decreased in SHR, a 60 min therapy with tanshinone IIA with the oral dosage of 60 mg kg1 signicantly lowered SBP in SHR Having said that, administering WKY with tanshinone IIA for 60 min failed to modify the SBP. The SHR aortic ring strips strongly contracted after an preliminary application of phenylephrine or KCl. Although tanshinone IIA didn’t inuence resting vascular tone, it dilated the two phenylephrineand KCl induced contractions within a concentration dependent method.

At the maximal concentration, tanshinone IIA signicantly attenuated the tonic contraction of SHR aortic rings induced by phenylephrine to 24. 9 5. 2% from the maximal supplier Ataluren contraction. Also, the eect of tanshinone IIA on KCl induced tonic vasoconstriction approached 28. 3 5. 4% of the maximal contraction. No dierence is often observed relating to the soothing eect of tanshinone IIA on phenylephrine induced tonic vasoconstriction amongst SHR aortic rings with or with no functional endothelium. manner.

The inducing eects would decrease their intestinal absorption and so increase rst pass clearance of CYP3A4 and/or P gp substrates. In future scientific studies other danshen preparations containing a higher written content of cryptotanshinone and tanshinone IIA ought to be evaluated for their ability to induce in vivo CYP3A4 and P gp. mGluR Conrmation with the outcomes of this review will demand bigger, controlled trials. In conclusion, persistent administration of danshen tablets resulted inside a signicant decline in oral bioavailability of midazolam, which might be the consequence on the induction of intestinal CYP3A4. If an orally administered drug is often a substrate of CYP3A and has minimal oral bioavailabity as a result of in depth pre systemic metabolic process by enteric CYP3A4, then administration of danshen tablets could possess a signicant eect on systemic exposure.

Use of CYP3A substrates with concurrent danshen tablet use may contact for caution, depending on the drugs publicity response connection. Dose adjustment of CYP3A substrates could be essential in individuals obtaining concomitant therapy with danshen preparations containing FGFR2 inhibitor lipophilic elements. Bunge is usually a well known plant utilized in traditional Chinese medicine to deal with many entities, this kind of as cardiovascular disease, angina pectoris, hyperlipidemia, and acute ischemic stroke. Tan shen extracts include a number of constituents which include watersoluble phenolic acids and lipophilic tanshinones. Recently, other research and our very own discovered that extracts of tan shen exhibit signicant antitumor activity by dierent mechanisms in many varieties of tumor cells.

We previously showed that DHTS markedly inhibited the proliferation of breast cancer cells by induction of G1 phase arrest and enhanced reduction with the mitochondrial membrane prospective and cytochrome c release. Furthermore, the inhibitory action was ranked as follows: DHTS tanshinone I cryptotanshinone I. Tanshinone I was also shown to induce cancer cell apoptosis in human Metastatic carcinoma myeloid leukemia cells and human nonsmall cell lung cancer whereas tanshinone IIA induced apoptosis in human HeLa and rat glioma cells. Even though numerous mechanisms had been proposed to describe the antitumor eects with the dierent tan shen constituents, this kind of as inactivation of the PI3K/Akt/survivin signaling pathways, reductions of interleukin 8, Ras mitogen activated protein kinase, Rac1, interference with microtubule assembly, and inhibition of constitutive STAT3 activation, this problem has not been convincingly claried.

During the current examine, we demonstrate that DHTS is capable of potently induce ER pressure in prostate carcinoma cells, as indicated by elevated amounts of GRP78/Bip and CHOP/GADD153, leading to apoptosis. In addition, DHTS induced the accumulation of polyubiquitinated proteins and HIF 1, indicating that DHTS may well be a proteasome inhibitor which produces ER strain or enhanced ATP-competitive Aurora Kinase inhibitor apoptosis caused by the traditional ER anxiety dependent mechanism. DHTS was obtained from Xian Honson Biotechnology. The purity was about 95% according to a higher effectiveness liquid chromatographic evaluation.

Hop extract is employed as herbal medication for your therapy mGluR of the selection of disorders, such as anxiety, insomnia, and restlessness. Additionally, it has estrogenic exercise. Because of this, hop extract has been investigated as being a probable therapy to the management of postmenopausal signs and symptoms. Chemicals existing in hops include terpenes, bitter acids, chalcones, avonol glycosides, and catechins. The bitter acids comprise of acids and B acids. It has been shown that an ethanolic extract of hops of unknown chemical composition increases PXR mediated transcriptional activity, as assessed in an in vitro cellbased luciferase reporter gene assay. Comparative examination signifies the extent of PXR activation from the ethanolic extract of hops is related to that obtained with St. Johns wort and Gugulipid.

Constant together with the nding that hop extract increases PXR activity, treatment of key cultures of human hepatocytes using the extract increases CYP3A4 mRNA expression. Experiments with colupulone present that deacetylase inhibitor this compound increases PXR action. Nevertheless, it stays to get demonstrated conclusively that colupulone is responsible for that human PXR activating result of hop extract. It’s probably that colupulone can be an activator of rodent PXR because of former ndings displaying that this B acid is an inducer of hepatic CYP3A gene expression in mice and rats. H. perforatum is frequently identified as St. Johns wort. This plant has a extended history of use as herbal medication in Europe and it is recognized as an anti depressant. The antidepressant action of St.

Johns wort has become linked to its inhibition of synaptosomal reuptake of serotonin, Skin infection norepinephrine, and dopamine. The chemical constituents in St. Johns wort include naphthodianthrones which include hypericin and pseudohypericin, phlorolucins like hyperforin, avonoids like hyperoside, quercetin, and rutin, carbolic acids, xanthones, proanthocyanidins, anthraquinones, carotenoids, cumarine, and volatile oils. Hyperforin is shown to possess inhibitory impact on neurotransmitter reuptake. As pointed out over, St. Johns wort was the rst herbal medication reported to activate PXR. The mechanism of human PXR activation by St. Johns wort entails direct ligand binding for the receptor. Constant with all the nding that St. Johns wort activates PXR, this herbal medication is regarded to induce PXR regulated genes, including CYP3A4, in major cultures of human hepatocytes.

Many of the clinical herb?drug interactions with St. Johns wort can now be explained about the basis of PXR activation by this herbal medication. Chemical examination identied hyperforin like a constituent Ataluren ic50 in St. Johns wort that activates human PXR. This compound activates human PXR transcriptional activity with an EC50 value in reduced nanomolar concentrations, and it’s 1 of your most potent activators of human PXR identied to date.

An alcoholic extract of C. forkohlii of undened chemical composition has been reported Raf inhibition to activate mouse PXR dependant on the experimental nding indicating the extract increases Cyp3a11 messenger RNA expression in major hepatocytes isolated from wild variety mice, whereas it’s tiny or no result on Cyp3a11 mRNA expression in hepatocytes isolated from PXR knockout mice. As pointed out previously, Cyp3a11 is usually a gene subject to regulation by PXR. It’s not recognized which person chemical constituent is straight accountable for or contributes towards the activation of mouse PXR by C. forkohlii extract. On the other hand, candidate compounds contain forskolin and 1,9 dideoxyforskolin, that’s a different diterpene existing during the roots of C. forkohlii.

Every of those chemical compounds is proven to act as an agonist of mouse PXR, as judged by their ability to bind towards the ligandbinding domain of PXR, recruit coactivator to PXR, and dissociate corepressor from PXR. The two forskolin and 1,9 dideoxyforskolin also activate human PXR exercise in vitro. Depending on the reported in vitro EC50 of 0. 4?twelve ?M in human PXR activation order AG-1478 by forskolin and plasma forskolin concentration of 4 ?M, this compound is predicted to get capable of activating PXR in vivo. Commiphora mukul, which is often known as Commiphora wightii or guggul tree, is indigenous to India, Pakistan, and Bangladesh. It’s medicinal value in standard Ayurvedic medicine. Extracts of guggul, which can be the gum resin from the bark in the C. mukul tree, is accessible as an in excess of thecounter dietary supplement in different Western countries, like the USA.

It really is used by shoppers being a naturally Lymphatic system happening cholesterol decreasing agent. Chemical examination signifies that guggul consists of a mixture of diterpenes, sterols, steroids, esters, and increased alcohols. Guggulsterone and guggulsterone would be the energetic compounds with cholesterol reducing action. Caspase-9 inhibitor Mechanistic scientific studies have proposed that these two pregnane derivatives act by antagonizing the farnesoid X receptor and up regulating the expression on the bile acid export pump. Gugulipid extract is capable of activating human and mouse PXR, as assessed in an in vitro cell primarily based luciferase reporter gene assay. In the highest concentration investigated, the extent of human PXR activation by Gugulipid is roughly 80% of that by rifampicin, and that is a prototypic agonist of human PXR. By comparison, the extent of mouse PXR activation through the exact same concentration of Gugulipid is just like that by PCN, a prototypic agonist of mouse PXR. The mechanism by which Gugulipid activates PXR remains for being elucidated. The effect of guggulsterone and guggulsterone on PXR action has also been studied. The two of those compounds activate PXR in in vitro cell primarily based reporter gene assays.

Intraperitoneal glucose tolerance check was carried out in 1618 h fasted mice injected intraperitoneally with 2 g glucose/kg entire body wt, and insulin sensitivity exams were performed in mice within the random fed state injected IP with 0. 75 units bovine insulin/kg body VEGFR inhibition wt. Insulin articles in islets or pancreas, and glucose stimulated insulin secretion in isolated islets were measured as reported. Numerous very low dose streptozotocin induced diabetes.

Male mice aged ten12 weeks had been injected IP for 5 consecutive days with streptozotocin, order Hesperidin beginning at day 0, and nonfasting blood glucose was measured from snipped tails at distinctive time factors. Immunohistochemistry and insulitis. Parafn embedded pancreatic sections have been immunostained for insulin, glucagon, somatostatin, c Met, and 5bromo 2 deoxyuridine as described.

b Cell mass and islet amount have been measured in 3 insulin stained pancreas sections from every single mouse working with ImageJ.

BrdU incorporation in b and ductal cells Mitochondrion was measured in pancreas sections of mice injected IP with BrdU, killed 6 h later, and stained for insulin and BrdU. b Cell death was established in pancreas sections stained for insulin and employing the terminal deoxynucleotidyl transferasemediated dUTP nick finish labeling system. Sections have been also stained with hematoxylineosin and anti CD3 for pathologic evaluation of islet insulitis. Islet isolation and culture of pancreatic islets and bTC 3 cells.

Mouse islets had been isolated after injection of collagenase P with the pancreatic duct, as previously reported. Human islets were provided through the ICR and JDRF Primary Science Islet Distribution Programs.

Personal mouse and human islets have been hand picked underneath a stereomicroscope, and 100200 islets/mL have been cultured in Roswell Park Memorial Institute medium from the presence or absence of recombinant mouse or human cytokines: interleukin 1b, interferon g, and tumor necrosis element a, respectively.

Evaluation of c Met, HGF, inducible nitric oxide synthase, and A20 mRNA expression in isolated islets was performed by genuine time PCR employing specic primers. In a distinct set of real time PCR experiments, mouse insulinoma bTC 3 cells had been plated in Dulbeccos modied Eagles medium with 10% fetal bovine serum.

Twenty 4 hours later on, cells have been serum depleted and taken care of with 1 mmol/L STZ or 50 units/mL IL 1b, 1,000 units/mL IFN g, and 1,000 units/mL TNF a for 16 h in advance of harvesting and RNA isolation. Medium nitric oxide, monocyte chemoattractant protein 1, and monokine induced by g IFN concentration measurements. Medium from islet cultures containing one hundred islets/mL was analyzed for nitric 850649-62-6 Alogliptin oxide by adding an equal volume of Greiss reagent.