RETRACTED ARTICLE: Sodium nitroprusside enhances callus induction and shoot regeneration in high value medicinal plant Canscora decussata

Plant Cell, Tissue and Organ Culture (PCTOC) -     

Variation in the male pheromones and mating success of wild caught Drosophila melanogaster

Drosophila melanogaster males express two primary cuticular hydrocarbons (male-predominant hydrocarbons). These act as sex pheromones by influencing female receptivity to mating. The relative quantities of these hydrocarbons vary widely among natural populations and can contribute to variation in mating success. We tested four isofemale lines collected from a wild population to assess the effect of intrapopulation variation in male-predominant hydrocarbons on mating success. The receptivity of laboratory females to males of the four wild-caught lines varied significantly, but not consistently in the direction predicted by variation in male-predominant hydrocarbons. Receptivity of the wild-caught females to laboratory males also varied significantly, but females from lines with male-predominant hydrocarbon profiles closer to a more cosmopolitan one did not show a correspondingly strong mating bias toward a cosmopolitan male. Among wild-caught lines, the male-specific ejaculatory bulb lipid, cis-vaccenyl acetate, varied more than two-fold, but was not associated with variation in male mating success. We observed a strong inverse relationship between the receptivity of wild-caught females and the mating success of males from their own lines, when tested with laboratory flies of the opposite sex.     

LCN2 and TIMP1 as Potential Serum Markers for the Early Detection of Familial Pancreatic Cancer

High-risk individuals of familial pancreatic cancer (FPC) families are considered to be good candidates for screening programs to detect early PC or its high-grade precursor lesions, especially pancreatic intraepithelial neoplasia (PanIN) 2/3 lesions. There is a definite need for diagnostic markers as neither reliable imaging methods nor biomarkers are available to detect these lesions. On the basis of a literature search, the potential serum markers neutrophil gelatinase-associated lipocalin (LCN2), metallopeptidase inhibitor 1 (TIMP1), chemokine (C-X-C motif) ligand 16 (CXCL16), IGFBP4, and iC3a, which were first tested in transgenic KrasLSL.^G12D/+;p53^R172H/+;Pdx1-Cre mice, were identified. ELISA analyses of LCN2, TIMP1, and CXCL16 revealed significantly higher levels in mice with PanIN2/3 lesions or PC compared to mice with normal pancreata or PanIN1 lesions. Analysis of preoperative human serum samples from patients with sporadic PC (n = 61), hereditary PC (n = 24), chronic pancreatitis (n = 28), pancreatic neuroendocrine tumors (n = 11), and FPC patients with histologically proven multifocal PanIN2/3 lesions (n = 3), as well as healthy control subjects (n = 20), confirmed significantly higher serum levels of LCN2 and TIMP1 in patients with PC and multifocal PanIN2/3 lesions. The combination of LCN2 and TIMP1 as a diagnostic test for the detection of PC had a sensitivity, specificity, and positive predictive value of 100% each. Although this preliminary finding needs to be validated in a large series of individuals at high risk for FPC, serum measurement of LCN2 and TIMP1 might be a promising screening tool.     

Absence of Nitric-oxide Synthase in Sequentially Purified Rat Liver Mitochondria

Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[¹⁴C]arginine to l-[¹⁴C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.Nitric oxide (NO^·)² is a highly diffusible, hydrophobic, and gaseous free radical (1) that is responsible for autocrine and paracrine signaling activities (2). NO^· can readily partition into and through membranes (3–5) to influence biological functions such as blood pressure regulation, platelet aggregation and adhesion, neurotransmission, and cellular defense (4, 6–11). The mechanism by which NO^· influences biological functions is by binding to target proteins that contain heme and/or thiol(s). Alternatively, NO^· can combine with to produce the highly reactive species peroxynitrite.Mitochondria are highly compartmentalized, membranous organelles that contain abundant amounts of reactive hemoproteins and thiols (12, 13), to which NO^· may bind reversibly (14, 15) or irreversibly (16–18). Mitochondria also generate various amounts of during the process of cellular respiration (19, 20). Studies conducted during the past decade have suggested that NO^· can diffuse into mitochondria and cause mitochondrial dysfunction by reversibly inhibiting cytochrome c oxidase (14, 21, 22) and NADH dehydrogenase (23).In the mid-90s, a putative variant of NOS was proposed to reside within mitochondria. Initially, Kobzik et al. (24) and Hellsten and co-workers (25) observed an apparent endothelial NOS (eNOS) immunoreactivity in skeletal muscle mitochondria. Simultaneously, Bates et al. (26, 27) observed an apparent eNOS histochemical reactivity in inner mitochondrial membrane preparations, isolated from rat liver, brain, heart, skeletal muscle, and kidney. Tatoyan and Giulivi (28), acting on these initial observations, performed experiments in an attempt to confirm the identity of this putative mtNOS. Relying on immunochemical analysis, Tatoyan and Giulivi (28) claimed that inducible NOS (iNOS) was the NOS isoform present in rat liver mitochondria. This same group using mass spectrometry later presented data in support of the putative mtNOS being a variant of nNOS (29). Ghafourifar and Richter (30) had reported previously that the putative mtNOS was calcium-sensitive and constitutive in nature. Since these reports, different groups have reported the presence of each of the three main isoforms of NOS within mitochondria (29, 31, 32). Also, biochemical characterization of the putative mtNOS performed by Giulivi and co-workers (29) revealed certain post-translational modifications (myristoylation and phosphorylation of the protein) that are thought to be unique to eNOS. During the last decade, various reports have supported the presence of at least one of the three main isoforms of NOS residing in mitochondria. However, the more recent reports tend to question this claim (33–36). Because of the contradictory reports regarding the existence of a putative mtNOS, Brookes (33) compiled a critical and thorough review of the literature published up to 2003 dealing with the putative mtNOS. This review brought to light the diverse technical issues involved in the aforementioned studies. Major issues were the degree of purity of mitochondrial preparations (37, 38), shortcomings of measurement methodology (29, 39–41), use of inappropriate, or total lack of, experimental controls and confusing technical practices. Lacza et al. (42) has reviewed the more recent developments in the area of mitochondrial NO^· production and discussed some of the shortcomings of certain techniques still being used.In light of this ongoing controversy regarding the presence or absence of a mtNOS, we designed and carefully conducted properly controlled studies to either confirm or refute the existence of any NOS isoform within mitochondria. Ultrapure rat liver mitochondria were isolated using repeated differential centrifugation followed by Percoll gradient purification. Proteomic analyses were then performed using a nano-HPLC-coupled nanospray LTQ-MS. To avoid the interfering factors that are rampant in NO^· trapping assays (43), the NOS-catalyzed conversion of l-[¹⁴C]arginine to l-[¹⁴C]citrulline was used to probe for NOS activity in mitochondria. Appropriate controls were employed and, for inhibition studies, high concentrations of l-thiocitrulline (TC) (44) were used. Additionally, immunochemical analyses were performed with ultrapure mitochondria using nNOS, eNOS, and CaM antibodies. The problems faced with the commonly used techniques in mtNOS studies are discussed.     

Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.