Estrogen receptors as potential therapeutic target in endometrial cancer.

Endometrial cancer (EC) is one of the most common gynecological carcinomas in both developed and developing countries. Majority of the gynecological malignancies are hormonally driven where estrogen signaling acts as an oncogenic signal. Estrogen's effects are mediated via classical nuclear estrogen receptors; estrogen receptor alpha and beta (ERα and ERß) and a trans-membrane G protein-coupled estrogen receptor (GPR30 and GPER). ERs and GPER through ligand binding triggers multiple downstream signaling pathways causing cell cycle regulation, cell differentiation, migration, and apoptosis in various tissues including endometrium. Although the molecular aspect of estrogen function in ER-mediated signaling is now partly understood, the same is not true for GPER-mediated signaling in endometrial malignancies. Understanding the physiological roles of ERα and GPER in EC biology therefore leads to the identification of some novel therapeutic targets. Here we review the effect of estrogen signaling through ERα-and GPER in EC, major types, and some affordable treatment approaches for endometrial tumor patients which has interesting implications in understanding uterine cancer progression.

Characterization of a novel family VIII esterase EstM2 from soil metagenome capable of hydrolyzing estrogenic phthalates.

BACKGROUND: Microbes are rich sources of enzymes and esterases are one of the most important classes of enzymes because of their potential for application in the field of food, agriculture, pharmaceuticals and bioremediation. Due to limitations in their cultivation, only a small fraction of the complex microbial communities can be cultured from natural habitats. Thus to explore the catalytic potential of uncultured organisms, the metagenomic approach has turned out to be an effective alternative method for direct mining of enzymes of interest. Based on activity-based screening method, an esterase-positive clone was obtained from metagenomic libraries. RESULTS: Functional screening of a soil metagenomic fosmid library, followed by transposon mutagenesis led to the identification of a 1179 bp esterase gene, estM2, that encodes a 392 amino acids long protein (EstM2) with a translated molecular weight of 43.12 kDa. Overproduction, purification and biochemical characterization of the recombinant protein demonstrated carboxylesterase activity towards short-chain fatty acyl esters with optimal activity for p-nitrophenyl butyrate at pH 8.0 and 37 °C. Amino acid sequence analysis and subsequent phylogenetic analysis suggested that EstM2 belongs to the family VIII esterases that bear modest similarities to class C ß-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C ß-lactamases but did not exhibit ß-lactamase activity. Guided by molecular docking analysis, EstM2 was shown to hydrolyze a wide range of di- and monoesters of alkyl-, aryl- and benzyl-substituted phthalates. Thus, EstM2 displays an atypical hydrolytic potential of biotechnological significance within family VIII esterases. CONCLUSIONS: This study has led to the discovery of a new member of family VIII esterases. To the best of our knowledge, this is the first phthalate hydrolase (EstM2), isolated from a soil metagenomic library that belongs to a family possessing ß-lactamase like catalytic triad. Based on its catalytic potential towards hydrolysis of both phthalate diesters and phthalate monoesters, this enzyme may find use to counter the growing pollution caused by phthalate-based plasticizers in diverse geological environment and in other aspects of biotechnological applications.

Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1.

UNLABELLED: The gene encoding a nonoxidative decarboxylase capable of catalyzing the transformation of 2-hydroxy-1-naphthoic acid (2H1NA) to 2-naphthol was identified, recombinantly expressed, and purified to homogeneity. The putative gene sequence of the decarboxylase (hndA) encodes a 316-amino-acid protein (HndA) with a predicted molecular mass of 34 kDa. HndA exhibited high identity with uncharacterized amidohydrolase 2 proteins of various Burkholderia species, whereas it showed a modest 27% identity with γ-resorcylate decarboxylase, a well-characterized nonoxidative decarboxylase belonging to the amidohydrolase superfamily. Biochemically characterized HndA demonstrated strict substrate specificity toward 2H1NA, whereas inhibition studies with HndA indicated the presence of zinc as the transition metal center, as confirmed by atomic absorption spectroscopy. A three-dimensional structural model of HndA, followed by docking analysis, identified the conserved metal-coordinating and substrate-binding residues, while their importance in catalysis was validated by site-directed mutagenesis. IMPORTANCE: Microbial nonoxidative decarboxylases play a crucial role in the metabolism of a large array of carboxy aromatic chemicals released into the environment from a variety of natural and anthropogenic sources. Among these, hydroxynaphthoic acids are usually encountered as pathway intermediates in the bacterial degradation of polycyclic aromatic hydrocarbons. The present study reveals biochemical and molecular characterization of a 2-hydroxy-1-naphthoic acid nonoxidative decarboxylase involved in an alternative metabolic pathway which can be classified as a member of the small repertoire of nonoxidative decarboxylases belonging to the amidohydrolase 2 family of proteins. The strict substrate specificity and sequence uniqueness make it a novel member of the metallo-dependent hydrolase superfamily.

Degradation Pathways of 2- and 4-Nitrobenzoates in Cupriavidus sp. Strain ST-14 and Construction of a Recombinant Strain, ST-14::3NBA, Capable of Degrading 3-Nitrobenzoate.

UNLABELLED: Strain ST-14, characterized as a member of the genus Cupriavidus, was capable of utilizing 2- and 4-nitrobenzoates individually as sole sources of carbon and energy. Biochemical studies revealed the assimilation of 2- and 4-nitrobenzoates via 3-hydroxyanthranilate and protocatechuate, respectively. Screening of a genomic fosmid library of strain ST-14 constructed in Escherichia coli identified two gene clusters, onb and pob-pca, to be responsible for the complete degradation of 2-nitrobenzoate and protocatechuate, respectively. Additionally, a gene segment (pnb) harboring the genes for the conversion of 4-nitrobenzoate to protocatechuate was unveiled by transposome mutagenesis. Reverse transcription-PCR analysis showed the polycistronic nature of the gene clusters, and their importance in the degradation of 2- and 4-nitrobenzoates was ascertained by gene knockout analysis. Cloning and expression of the relevant pathway genes revealed the transformation of 2-nitrobenzoate to 3-hydroxyanthranilate and of 4-nitrobenzoate to protocatechuate. Finally, incorporation of functional 3-nitrobenzoate dioxygenase into strain ST-14 allowed the recombinant strain to utilize 3-nitrobenzoate via the existing protocatechuate metabolic pathway, thereby allowing the degradation of all three isomers of mononitrobenzoate by a single bacterial strain. IMPORTANCE: Mononitrobenzoates are toxic chemicals largely used for the production of various value-added products and enter the ecosystem through industrial wastes. Bacteria capable of degrading mononitrobenzoates are relatively limited. Unlike other contaminants, these man-made chemicals have entered the environment since the last century, and it is believed that bacteria in nature evolved not quite efficiently to assimilate these compounds; as a consequence, to date, there are only a few reports on the bacterial degradation of one or more isomers of mononitrobenzoate. In the present study, fortunately, we have been able to isolate a Cupriavidus sp. strain capable of assimilating both 2- and 4-nitrobenzoates as the sole carbon source. Results of the biochemical and molecular characterization of catabolic genes responsible for the degradation of mononitrobenzoates led us to manipulate a single enzymatic step, allowing the recombinant host organism to expand its catabolic potential to assimilate 3-nitrobenzoate.

Metabolism of 2-hydroxy-1-naphthoic acid and naphthalene via gentisic acid by distinctly different sets of enzymes in Burkholderia sp. strain BC1.

Burkholderia sp. strain BC1, a soil bacterium, isolated from a naphthalene balls manufacturing waste disposal site, is capable of utilizing 2-hydroxy-1-naphthoic acid (2H1NA) and naphthalene individually as the sole source of carbon and energy. To deduce the pathway for degradation of 2H1NA, metabolites isolated from resting cell culture were identified by a combination of chromatographic and spectrometric analyses. Characterization of metabolic intermediates, oxygen uptake studies and enzyme activities revealed that strain BC1 degrades 2H1NA via 2-naphthol, 1,2,6-trihydroxy-1,2-dihydronaphthalene and gentisic acid. In addition, naphthalene was found to be degraded via 1,2-dihydroxy-1,2-dihydronaphthalene, salicylic acid and gentisic acid, with the putative involvement of the classical nag pathway. Unlike most other Gram-negative bacteria, metabolism of salicylic acid in strain BC1 involves a dual pathway, via gentisic acid and catechol, with the latter being metabolized by catechol 1,2-dioxygenase. Involvement of a non-oxidative decarboxylase in the enzymic transformation of 2H1NA to 2-naphthol indicates an alternative catabolic pathway for the bacterial degradation of hydroxynaphthoic acid. Furthermore, the biochemical observations on the metabolism of structurally similar compounds, naphthalene and 2-naphthol, by similar but different sets of enzymes in strain BC1 were validated by real-time PCR analyses.

Climate change and coastal morphodynamics: Interactions on regional scales.

Climate change and its impacts, combined with unchecked human activities, intensify pressures on coastal environments, resulting in modification of the coastal morphodynamics. Coastal zones are intricate and constantly changing areas, making the monitoring and interpretation of data a challenging task, especially in remote beaches and regions with limited historical data. Traditionally, remote sensing and numerical methods have played a vital role in analysing earth observation data and supporting the monitoring and modelling of complex coastal ecosystems. However, the emergence of artificial intelligence-based techniques has shown promising results, offering the additional advantage of filling data gaps, predicting data in data-scarce regions, and analysing multidimensional datasets collected over extended periods of time and larger spatial scales. The main objective of this study is to provide a comprehensive review of the existing literature, discussing both traditional methods and various emerging artificial intelligence-based approaches used in studying the coastal dynamics, shoreline change analysis, and coastal monitoring. Ultimately, the study proposes a climate resilience framework to enhance coastal zone management practices and policies, fostering resilience among coastal communities. The outcome of this study aligns with and supports particularly SDG 13 of the UN (Climate Action) and advances it by identifying relevant methods in coastal erosion studies and proposing integrated management plans informed by real-time data collection and analysis/modelling using physics-based models.

Interaction of climate change and marine pollution in Southern India: Implications for coastal zone management practices and policies.

Climate change and marine litter are inextricably linked, and their interaction manifests differently depending on the specific environmental and biological characteristics, and other human activities taking place. The negative impacts resulting from those synergistic interactions are threatening coastal and marine ecosystems and the many goods and services they provide. This is particularly pervasive in the coastal zone of the Indian subcontinent. India is already experiencing severe climate change impacts, which are projected to worsen in the future. At the same time, the country is gripped by a litter crisis that is overwhelming authorities and communities and hindering the country's sustainable development goals. The coastal environment and communities of the southern states of Kerala and Tamil Nadu are particularly vulnerable to the impacts of climate change. While these state governments and authorities are stepping up efforts to improve the management of their coastal zones, the scale and severity of these issues are mounting. Here we review the combined effects of climate change and marine litter pollution in Southern India, focusing on the Gulf of Mannar Reserve in Tamil Nadu and the Malabar Coast in Kerala. Finally, we discuss effective management options that could help improve resilience and sustainability.

AMP-activated protein kinase connects cellular energy metabolism to KATP channel function.

AMPK is an important sensor of cellular energy levels. The aim of these studies was to investigate whether cardiac K(ATP) channels, which couple cellular energy metabolism to membrane excitability, are regulated by AMPK activity. We investigated effects of AMPK on rat ventricular K(ATP) channels using electrophysiological and biochemical approaches. Whole-cell K(ATP) channel current was activated by metabolic inhibition; this occurred more rapidly in the presence of AICAR (an AMPK activator). AICAR had no effects on K(ATP) channel activity recorded in the inside-out patch clamp configuration, but ZMP (the intracellular intermediate of AICAR) strongly activated K(ATP) channels. An AMPK-mediated effect is demonstrated by the finding that ZMP had no effect on K(ATP) channels in the presence of Compound C (an AMPK inhibitor). Recombinant AMPK activated Kir6.2/SUR2A channels in a manner that was dependent on the AMP concentration, whereas heat-inactivated AMPK was without effect. Using mass-spectrometry and co-immunoprecipitation approaches, we demonstrate that the AMPK α-subunit physically associates with K(ATP) channel subunits. Our data demonstrate that the cardiac K(ATP) channel function is directly regulated by AMPK activation. During metabolic stress, a small change in cellular AMP that activates AMPK can be a potential trigger for K(ATP) channel opening. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Marine litter and climate change: Inextricably connected threats to the world's oceans.

The global issues of climate change and marine litter are interlinked and understanding these connections is key to managing their combined risks to marine biodiversity and ultimately society. For example, fossil fuel-based plastics cause direct emissions of greenhouse gases and therefore are an important contributing factor to climate change, while other impacts of plastics can manifest as alterations in key species and habitats in coastal and marine environments. Marine litter is acknowledged as a threat multiplier that acts with other stressors such as climate change to cause far greater damage than if they occurred in isolation. On the other hand, while climate change can lead to increased inputs of litter into the marine environment, the presence of marine litter can also undermine the climate resilience of marine ecosystems. There is increasing evidence that that climate change and marine litter are inextricably linked, although these interactions and the resulting effects vary widely across oceanic regions and depend on the particular characteristics of specific marine environments. Ecosystem resilience approaches, that integrate climate change with other local stressors, offer a suitable framework to incorporate the consideration of marine litter where that is deemed to be a risk, and to steer, coordinate and prioritise research and monitoring, as well as management, policy, planning and action to effectively tackle the combined risks and impacts from climate change and marine litter.

The neuronal apoptotic death in global cerebral ischemia in gerbil: important role for sodium channel modulator.

Global ischemia was induced in gerbil by bilateral occlusion of the common carotid arteries for 5 min. Sodium ionophore monensin or sodium channel blocker tetrodotoxin (TTX) was administered at doses of 10 micorg/kg, i.p., 30 min before ischemia induction; the dose was repeated after 22 hr. Subsequently, brain infarct occurred, determined at 24 hr after occlusion. Large, well-demarcated infarcts were observed in both hemispheres, an important observation because it critically influences the interpretation of the data. Because nitric oxide (NO) production is thought to be related to ischemic neuronal damage, we examined increases in Ca(2+) influx, which lead to the activation of nitric oxide synthase (NOS). Then we evaluated the contributions of neuronal NOS, endothelial NOS, and inducible NOS to NO production in brain cryosections. The cytosolic release of apoptogenic molecules like cytochrome c and p53 were confirmed after 24 hr of reflow. TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) labeling detected the apoptotic cells, which were confirmed in neuron-rich cell populations. After 24 hr, all the ischemic changes were amplified by monensin and significantly attenuated by TTX treatment. Additionally, the nesting behavior and histological outcomes were examined after 7 day of reflow. The neuronal damage in the hippocampal area and significant decrease in nesting scores were observed with monensin treatment and reduced by TTX pretreatment after day 7 of reflow. To our knowledge, this report is the first to highlight the involvement of the voltage-sensitive Na(+) channel in possibly regulating in part NO system and apoptosis in a cytochrome c-dependent manner in global ischemia in the gerbil, and thus warrants further investigation.

Effects of bisphenol A (BPA) on brain-specific expression of cyp19a1b gene in swim-up fry of Labeo rohita.

Estrogen regulates numerous developmental and physiological processes and effects are mediated mainly by estrogenic receptors (ERs), which function as ligand-regulated transcription factor. ERs can be activated by many different types endocrine disrupting chemicals (EDCs) and interfere with behaviour and reproductive potential of living organism. Estrogenic regulation of membrane associated G protein-coupled estrogen receptor, GPER activity has also been reported. Bisphenol A (BPA), a ubiquitous endocrine disruptor is present in many household products, has been linked to many adverse effect on sexual development and reproductive potential of wild life species. The present work is aimed to elucidate how an environmentally pervasive chemical BPA affects in vivo expression of a known estrogen target gene, cyp19a1b in the brain, and a known estrogenic biomarker, vitellogenin (Vg) in the whole body homogenate of 30 days post fertilization (dpf) swim-up fry of Labeo rohita. We confirm that, like estrogen, the xenoestrogen BPA exposure for 5-15 days induces strong overexpression of cyp19a1b, but not cyp19a1a mRNA in the brain and increase concentration of vitellogenin in swim-up fry. BPA also induces strong overexpression of aromatase B protein and aromatase activity in brain. Experiments using selective modulators of classical ERs and GPER argue that this induction is largely through nuclear ERs, not through GPER. Thus, BPA has the potential to elevate the levels of aromatase and thereby, levels of endogenous estrogen in developing brain. These results indicate that L. rohita swim-up fry can be used to detect environmental endocrine disruptors either using cyp19a1b gene expression or vitellogenin induction.

Immunolocalization of KATP channel subunits in mouse and rat cardiac myocytes and the coronary vasculature.

BACKGROUND: Electrophysiological data suggest that cardiac KATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other KATP channel subunits) is poorly defined. We examined the localization of each of the KATP channel subunits in the mouse and rat heart. RESULTS: Immunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular KATP channels. CONCLUSIONS: Collectively, our data demonstrate unique cellular and subcellular KATP channel subunit expression patterns in the heart. These results suggest distinct roles for KATP channel subunits in diverse cardiac structures.

Complete degradation of di-n-octyl phthalate by Gordonia sp. strain Dop5.

The present study describes the assimilation of di-n-octyl phthalate by an aerobic bacterium, isolated from municipal waste-contaminated soil sample utilizing di-n-octyl phthalate as the sole source of carbon and energy. The isolate was identified as Gordonia sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic and spectrometric analyses revealed a complete di-n-octyl assimilation pathway. In the degradation process, mono-n-octyl phthalate, phthalic acid, protocatechuic acid and 1-octanol were identified as the degradation products of di-n-octyl phthalate. Furthermore, phthalic acid was metabolized via protocatechuic acid involving protocatechuate 3,4-dioxygenase while 1-octanol was metabolized by NAD(+)-dependent dehydrogenases to 1-octanoic acid, which was subsequently degraded via ß-oxidation, ultimately, leading to tricarboxylic acid cycle intermediates. Apart from phthalic acid and 1-octanol metabolizing pathway enzymes, two esterases, di-n-octyl phthalate hydrolase and mono-n-octyl phthalate hydrolase involved in di-n-octyl phthalate degradation were found to be inducible in nature. This is the first report on the metabolic pathway involved in the complete degradation of di-n-octyl phthalate by a single bacterial isolate, which is also capable of efficiently degrading other phthalate esters of environmental concern having either shorter or longer alkyl chains.

Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits.

Cardiac ATP-sensitive K+ (K(ATP)) channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice that express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression with the alpha-myosin heavy chain promoter. Weight gain and development after birth of these mice were similar to nontransgenic mice, but an increased mortality was noted after the age of 4-5 mo. Transgenic mice lacked cardiac K(ATP) channel activity as assessed with patch clamp techniques. Consistent with a decreased current density observed at positive voltages, the action potential duration was increased in these mice. Some myocytes developed EADs after isoproterenol treatment. Hemodynamic measurements revealed no significant effects on ventricular function (apart from a slightly elevated heart rate), whereas in vivo electrophysiological recordings revealed a prolonged ventricular effective refractory period in transgenic mice. The transgenic mice tolerated stress less well as evident from treadmill stress tests. The proarrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggest that these features are intrinsic to the myocardium and that K(ATP) channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations.

Expression of ATP-sensitive K+ channel subunits during perinatal maturation in the mouse heart.

Prevailing data suggest that sarcolemmal ATP-sensitive (K(ATP)) channels in the adult heart consist of Kir6.2 and SUR2A subunits, but the expression of other K(ATP) channel subunits (including SUR1, SUR2B, and Kir6.1) is poorly defined. The situation is even less clear for the immature heart, which shows a remarkable resistance to hypoxia and metabolic stress. The hypoxia-induced action potential shortening and opening of sarcolemmal K(ATP) channels that occurs in adults is less prominent in the immature heart. This might be due in part to the different biophysical and pharmacological properties of K(ATP) channels of immature and adult K(ATP) channels. Because these properties are largely conferred by subunit composition, it is important to examine the relative expression levels of the various K(ATP) channel subunits during maturation. We therefore used RNAse protection assays, reverse transcription-PCR approaches, and Western blotting to characterize the mRNA and protein expression profiles of K(ATP) channel subunits in fetal, neonatal, and adult mouse heart. Our data indicate that each of the K(ATP) channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B) is expressed in the mouse heart at all of the developmental time points studied. However, the expression level of each of the subunits is low in the fetal heart and progressively increases with maturation. Each of the subunits seems to be expressed in ventricular myocytes with a subcellular expression pattern matching that found in the adult. Our data suggest that the K(ATP) channel composition may change during maturation, which has important implications for K(ATP) channel function in the developing heart.

The glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, and pyruvate kinase are components of the K(ATP) channel macromolecular complex and regulate its function.

The regulation of ATP-sensitive potassium (K(ATP)) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K(ATP) channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K(ATP) channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K(ATP) channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K(ATP) channel complex) causes channel closure.