Rajeshwary Ghosh

The necessity of and strategies for improving confidence in the accuracy of western blots

Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts; however, several recent findings suggest that western blots may not be as reliable as previously assumed. This is not surprising since many labs are unaware of the limitations of western blotting. In this manuscript, we review essential strategies for improving confidence in the accuracy of western blots. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein. Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed.

NSAIDs and Cardiovascular Diseases : Role of Reactive Oxygen Species

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs worldwide. NSAIDs are used for a variety of conditions including pain, rheumatoid arthritis, and musculoskeletal disorders. The beneficial effects of NSAIDs in reducing or relieving pain are well established, and other benefits such as reducing inflammation and anticancer effects are also documented. The undesirable side effects of NSAIDs include ulcers, internal bleeding, kidney failure, and increased risk of heart attack and stroke. Some of these side effects may be due to the oxidative stress induced by NSAIDs in different tissues. NSAIDs have been shown to induce reactive oxygen species (ROS) in different cell types including cardiac and cardiovascular related cells. Increases in ROS result in increased levels of oxidized proteins which alters key intracellular signaling pathways. One of these key pathways is apoptosis which causes cell death when significantly activated. This review discusses the relationship between NSAIDs and cardiovascular diseases (CVD) and the role of NSAID-induced ROS in CVD.

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

Different effects of the nonsteroidal anti-inflammatory drugs meclofenamate sodium and naproxen sodium on proteasome activity in cardiac cells.

The use of nonsteroidal anti-inflammatory drugs (NSAIDs) like meclofenamate sodium (MS), used to reduce pain, has been associated with an increased risk of cardiovascular disease (CVD). Naproxen (NAP), another NSAID, is not associated with increased risk of CVD. The molecular mechanism(s) by which NSAIDs induce CVD is unknown. We investigated the effects of MS and NAP on protein homeostasis and cardiotoxicity in rat cardiac H9c2 cells and murine neonatal cardiomyocytes. MS, but not NAP, significantly inhibited proteasome activity and reduced cardiac cell viability at pharmacological levels found in humans. Although proteasome subunit gene and protein expression were unaffected by NSAIDs, MS treated cell lysates showed higher 20S proteasome content, while purified proteasomes from MS treated cells had lower proteasome activity and higher levels of oxidized subunits than proteasomes from control cells. Addition of exogenous proteasome to MS treated cells improved cell viability. Both MS and NAP increased ROS production, but the rate of ROS production was greater in MS than in NAP treated cells. The ROS production is likely from mitochondria, as MS inhibited mitochondrial Complexes I and III, major sources of ROS, while NAP inhibited Complex I. MS also impaired mitochondrial membrane potential while NAP did not. Antioxidants were able to prevent the reduced cell viability caused by MS treatment. These results suggest that NSAIDs induce cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction and may explain why some NSAIDs should not be given to patients for long periods.

The Activation by Glucose of Liver Membrane Nitric Oxide Synthase in the Synthesis and Translocation of Glucose transporter-4 in the Production of Insulin in the Mice Hepatocytes

Introduction Glucose has been reported to have an essential role in the synthesis and secretion of insulin in hepatocytes. As the efflux of glucose is facilitated from the liver cells into the circulation, the mechanism of transportation of glucose into the hepatocytes for the synthesis of insulin was investigated. Methods Grated liver suspension (GLS) was prepared by grating intact liver from adult mice by using a grater. Nitric oxide (NO) was measured by methemoglobin method. Glucose transporter-4 (Glut-4) was measured by immunoblot technique using Glut-4 antibody. Results Incubation of GLS with different amounts of glucose resulted in the uptake of glucose by the suspension with increased NO synthesis due to the stimulation of a glucose activated nitric oxide synthase that was present in the liver membrane. The inhibition of glucose induced NO synthesis resulted in the inhibition of glucose uptake. Glucose at 0.02M that maximally increased NO synthesis in the hepatocytes led to the translocation and increased synthesis of Glut-4 by 3.3 fold over the control that was inhibited by the inhibition of NO synthesis. The glucose induced NO synthesis was also found to result in the synthesis of insulin, in the presence of glucose due to the expression of both proinsulin genes I and II in the liver cells. Conclusion It was concluded that glucose itself facilitated its own transportation in the liver cells both via Glut-4 and by the synthesis of NO which had an essential role for insulin synthesis in the presence of glucose in these cells.

Neutralization by Insulin of the Hypertensive Effect of Dermcidin Isoform 2: An Environmentally Induced Diabetogenic and Hypertensive Protein

The effect of dermcidin isoform 2 (dermcidin), an environmentally induced stress protein, was investigated on the genesis of diabetes mellitus and hypertension, the two major atherosclerotic risk factors. The role of dermcidin as an atherosclerotic risk factor related to the impaired systemic insulin level was investigated. Dermcidin was prepared by electrophoresis using plasma from the subjects with acute ischemic heart disease. Injection of 0.2 μM dermcidin in mice increased the blood glucose level from 98 ± 2.45 mg/dL to 350  ± 10.2 mg/dL which was normalized by the oral administration of acetyl salicylic acid (aspirin) after 24 h. Hypertensive subjects with systolic and diastolic blood pressure of 165 mm and 95 mm of Hg, respectively, had plasma dermcidin level of 95 nM. Ingestion of acetyl salicylic acid (aspirin) (150 mg/70 kg body weight) decreased the systolic and diastolic pressures to 125 mm and 80 mm of Hg, respectively, with decrease of dermcidin level to 15 nM. Incubation of kidney cortex cells with 0.2 μM dermcidin-inhibited synthesis of (r)-cortexin, an antihypertensive protein, and the basal (r)-cortexin level was reduced from 33 nM to 15 nM. Addition of 25 μunits of insulin/mL was found to reverse the inhibition of cortexin synthesis. The effect of dermcidin as a diabetogenic and a hypertensive agent could be controlled either by aspirin or by insulin.

Diclofenac induces proteasome and mitochondrial dysfunction in murine cardiomyocytes and hearts.

BACKGROUND One of the most common nonsteroidal anti-inflammatory drugs (NSAIDs) used worldwide, diclofenac (DIC), has been linked to increased risk of cardiovascular disease (CVD). The molecular mechanism(s) by which DIC causes CVD is unknown. METHODS Proteasome activities were studied in hearts, livers, and kidneys from male Swiss Webster mice treated with either 100mg/kg DIC for 18h (acute treatment) or 10mg/kg DIC for 28days (chronic treatment). Cultured H9c2 cells and neonatal cardiomyocytes were also treated with different concentrations of DIC and proteasome function, cell death and ROS generation studied. Isolated mouse heart mitochondria were utilized to determine the effect of DIC on various electron transport chain complex activities. RESULTS DIC significantly inhibited the chymotrypsin-like proteasome activity in rat cardiac H9c2 cells, murine neonatal cardiomyocytes, and mouse hearts, but did not affect proteasome subunit expression levels. Proteasome activity was also affected in liver and kidney tissues from DIC treated animals. The levels of polyubiquitinated proteins increased in hearts from DIC treated mice. Importantly, the levels of oxidized proteins increased while the β5i immunoproteasome activity decreased in hearts from DIC treated mice. DIC increased ROS production and cell death in H9c2 cells and neonatal cardiomyocytes while the cardioprotective NSAID, aspirin, had no effect on ROS levels or cell viability. DIC inhibited mitochondrial Complex III, a major source of ROS, and impaired mitochondrial membrane potential suggesting that mitochondria are the major sites of ROS generation. CONCLUSION These results suggest that DIC induces cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction.

Reduction of death rate due to acute myocardial infarction in subjects with cancers through systemic restoration of impaired nitric oxide.

Introduction Excessive aggregation of platelets at the site of plaque rupture on the coronary artery led to the formation of thrombus which is reported to precipitate acute myocardial infarction (AMI). Nitric oxide (NO) has been reported to inhibit platelet aggregation and induce thrombolysis through the in situ formation of plasmin. As the plasma NO level in AMI patients from two different ethnic groups was reduced to 0 µM (median) compared to 4.0 µM (median) in normal controls, the effect of restoration of the NO level to normal ranges on the rate of death due to AMI was determined. Methods and Results The restoration of plasma NO level was achieved by a sticking small cotton pad (10×25 mm) containing 0.28 mmol sodium nitroprusside (SNP) in 0.9% NaCl to the abdominal skin of the participants using non-toxic adhesive tape which was reported to normalize the plasma NO level. The participants (8,283) were volunteers in an independent study who had different kinds of cancers and did not wish to use any conventional therapy for their condition but opted to receive SNP “pad” for their condition for 3 years. The use of SNP “pad” which normalized (≈4.0 µM) the plasma NO level that in consequence reduced the death rate due to AMI, among the participants, was found to be significantly reduced compared to the death due to AMI in normal population. Conclusion Our data suggested that the use of SNP “pad” significantly reduced the death due to AMI. Trial Registration www.ctri.nic.in 004236

Extra pancreatic synthesis of insulin

It is currently believed that insulin, an essential hormone for carbohydrate metabolism, is produced only in the pancreas. Many investigators on the other hand had reported that various cells in different organs of the body beside pancreas are also capable of synthesizing insulin. This hormone not only has a critical role in the carbohydrate metabolism, but the protein is also reported to prevent the atherosclerosis and hypertension. The multifunctional synthesis of the protein in different cells in various organs is presented in the review. Insulin was reported to be synthesized in the liver, brain, thymus, adipocytes, gastrointestinal tract, bone marrow and in the leukocytes. Insulin synthesis was confirmed by cDNA analysis, amino acid sequence and by bioassay of the hormone. Liver was found to synthesize insulin, and glucose was found to stimulate NO synthesis in the liver and NO thus produced stimulated insulin and Glut-4 synthesis in the liver. Insulin synthesis occurs not only in human but also all animals. The lymphocytes and the leucocytes in the circulation were found to synthesize insulin. It is possible that synthesis of insulin in leucocytes could be involved for the ready supply of insulin in the prevention of thrombosis in platelets which did not produce any insulin. The synthesis of insulin in different organs in many different animals has also been reported. It was concluded that the synthesis of insulin in different cells was essential to maintain the cellular integrity from carbohydrate derived energy for all living organisms.

In reply: Maspin and it’s evolving role in tumor progression in systemic malignancies

In reference to the letter by Dr. Kapoor [1], the author elegantly described the role of maspin synthesis in the possible contribution both in bladder and colorectal cancer. In the letter, the author claimed that the expression of maspin (gene) indicated a worst prognostic outcome in bladder and colorectal cancer. It should be noted here that cancer is generally believed to be a disease of the gene. The heterogeneous nature of the cancer cells, particularly with different cancers, cannot be equated to uniformity in their expression of genes particularly because the expression of any gene is a multistep process. As cancer cells sometimes do undergo rapid mutation, it will almost be impossible to predict the expression of a particular gene in the pathogenesis of any cancer. For example, although the mutation of maspin genes has been claimed to result in breast cancer, the addition of nitric oxide (NO) to the in vitro reaction mixture containing ER(-) neutrophils from breast cancer subjects, which do not synthesize maspin in the presence of estrogen, resulted in the expression of the maspin gene supposedly that was mutated and resulted in the pathogenesis of the breast cancer [2]. Also it should be noted that the role of estrogen and progesterone in the pathogenesis of breast cancer in the context of maspin synthesis might not be extended to the cases of bladder and colorectal cancer. It should also be noted that the expression of estrogenor progesterone-dependent synthesis of maspin was not ‘‘autonomous’’ in nature in that the effect of these steroids in the synthesis of the anti-breast cancer protein was inhibited in the presence of insulin in the system owing to ‘‘cross talk’’ between the insulin and the steroid receptors on the cell surface. As the systemic presence of insulin would ‘‘downregulate’’ the steroid receptor numbers owing to the existence of type 2 diabetes mellitus [3], the role of systemic increase or decrease of maspin synthesis in predicting the recurrence of cancers needs to be considered with appropriate caution. However, in contrast to the negative role of maspin gene expression in colorectal cancer, as described by the letter’s writer, we have found impressive good prognostic outcomes in colorectal cancer through the systemic increase of NO by using acetyl salicylic acid that increased systemic maspin synthesis [4]. Also it must be noted that the increase of NO by using acetyl salicylic acid was apparently found to reduce the metastasis of breast cancer, a notorious malignant condition with a high tendency to metastasize.