Understanding gene expression in specific tissues and their modulation under environmental stimuli, such as nutritional deficiency, reveals the key physiological regulatory mechanisms of an organism. This study examined the tissue-specific expression of insulin-like peptide (ILP) genes (BmX and BmZ) in Bombyx mori larvae and their responses to hyperglycaemia, food deprivation and hormonal (20-hydroxyecdysone and bovine insulin) treatments. mRNA expression levels of BmX and BmZ were analyzed in the brain, fat body, midgut and ovary. The results revealed that BmX was highly expressed in the fat body, while both genes were abundant in the ovary. Hyperglycaemia increased BmX mRNA expression level in the midgut (3.07-fold) and brain (7.53-fold), while BmZ mRNA expression level was increased in all tissues except the midgut. Nutrient deficiency upregulated BmX mRNA expression level (1.36-fold) in the fat body while reducing it (-0.53-fold) in the midgut. Food deprivation progressively increased (0.77-fold at 24 h and 2.34-fold at 72 h) BmX mRNA expression level in the fat body, while both BmX and BmZ transcripts declined in the midgut. Insulin suppressed BmX (-0.25-fold) and BmZ (-0.91-fold) mRNA expression levels in food-deprived larvae in the fat body, whereas 20E consistently downregulated BmX, BmZ, and BmInR (insulin receptor) mRNA expression levels in all the conditions. These findings revealed the complex interaction of gene expression, tissue specificity, and environmental factors in B. mori larvae and provided insights into adaptive responses to nutritional stress and hormonal regulation in the insect with potential applications in sericulture and agricultural biotechnology.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder and its concurrent presence with chronic kidney disease (CKD) is a significant concern. Glycerol kinase (GK) and glycerol 3-phosphate shuttle enzymes (cGPDH and mGPDH) facilitate the regulation of endogenous glucose production in many cell lines. This research investigates the functions of GK, cGPDH, and mGPDH in HEK293 cells. Standard protocols were employed to assess enzyme activity, mRNA- and protein-expression, glucose uptake, and production. Homology modeling and molecular docking were employed to elucidate interactions of genistein and metformin with these enzymes. The secondary structures of GK, cGPDH and mGPDH and the thermal stability of cGPDH and mGPDH were analyzed by CD spectra. Genistein inhibited GK activity by 40%, while metformin decreased cGPDH and mGPDH activity by 58% and 55%, respectively, in HEK293 cells. Nonetheless, the expression levels of mRNA and protein remained unaltered. Genistein and metformin inhibited HEK293 glucose production by 0.46-fold and 0.63-fold, respectively. Genistein reduced glucose uptake by 0.26-fold, while metformin increased it by 0.51-fold. Genistein allosterically interacted with GK with a CDocker energy of -27.71, while metformin interacted with Gln295 and Lys296 of the catalytic loop of cGPDH and the FAD+ binding domain of mGPDH, yielding CDocker energies of -11.12 and -13.34, respectively. This study indicated the role of genistein and metformin on GK, cGPDH, and mGPDH in HEK293 cells.

Glycerol kinase (GK) catalyzes the transfer of a phosphate group from ATP to glycerol. GK is the key enzyme for utilizing glycerol as a carbon and energy source. The functionality of GK displays distinctive characteristics based on the tissue type, reflecting the distinct glycerol metabolic pathways in different tissues. Enzyme kinetics and western blot analysis were performed using different mouse tissues to determine the activity and protein expression of GK. The highest specific activities of GK were observed in muscle and heart tissues, with 1.433 U/ mg protein and 1.451 U/mg protein, respectively. The enzyme exhibited optimal activity at pH 7.5 and showed consistency in various buffers. The highest Vmax for ATP was in muscle and heart tissues (3.075 U/mg protein and 2.743 U/mg protein), with the highest Km for ATP in muscle and testis (0.529 mM and 0.582 mM). Additionally, for glycerol, the highest Vmax was also in muscle and heart tissues (2.970 U/mg protein and 3.220 U/mg protein), with the highest Km in these tissues being 6.496 mu M and 7.142 mu M, respectively. Western blot analysis indicated that GK is expressed in all tissues analyzed. This study revealed the tissue-specific functionality of GK in various mouse tissues, emphasising its crucial role in glycerol metabolism. This study also provided basic outlines of GK expression patterns in various tissues of mice and may provide important information for the regulation of GK for its critical role in type 2 diabetes mellitus.

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by -45 %, while metformin reduced cGPDH and mGPDH activities by -32 % and -43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 mu M), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 mu M) and mGPDH (Ki 54.70 mu M) activities. siRNAs transfection showed -50 % and -35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

Abstract Background: Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in Type 2 diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown. Methods: The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics. Results: The overexpressed bioactive His-GK was purified apparently to homogeneity (∼295-fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of ∼55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis–Menten kinetics with Km value of 5.022 µM (R2=0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2=0.928) and 0.223 mM (R2=0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined. Conclusion: The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.

Glycerol 3-phosphate (G3P) shuttle is composed of mGPDH and cGPDH and serves as the interface between carbohydrate- and lipid-metabolism. Recently, these metabolic enzymes have been implicated in type II diabetes mellitus but the detailed kinetic parameters and crystal structure of human mGPDH is unknown, though fewer studies on cGPDH are available. To characterize these enzymes, the human mGPDH and cGPDH genes were optimized and cloned into the pET-SUMO vector and pET-24a(+) vector, respectively, and over-expressed in Escherichia coli BL21 (DE3). However, SUMO-mGPDH was expressed as inclusion bodies. Hence, various culture parameters, solubilizing agents and expression vectors were used to solubilize the protein but they did not produce functional SUMO-mGPDH. Over-expression of SUMO-mGPDH along with molecular chaperone (pG-KJE8) produced a functional SUMO-mGPDH. The functional SUMO-mGPDH was purified and characterized using NAD+/NADH redox method. cGPDH was also over-expressed and purified for its characterization. DLS analysis and CD spectra of the purified proteins were performed. The mGPDH was a monomeric enzyme with MW of ∼74 kDa and displayed optimal activity in the Tris-HCl buffer (pH 7.4); while, cGPDH was a homodimer with a monomeric MW of ∼37 kDa and showed optimal activity in imidazole buffer (pH 8.0). The Kmapp was 0.475 mM for G3P, and 0.734 mM for DHAP. These methods may be used to characterize these enzymes to understand their role in metabolic disorders.

Background: Cancer metastasis is one of the major clinical challenges worldwide and is highly associated with patient's deaths. Therefore, targeting metastatic related proteins is the key to the development of anticancer therapy. Osteopontin (OPN) overexpression is associated with breast cancer progression and metastasis; thus, it is considered a promising biomarker. Many in vitro and in vivo studies have been reported that the use of isoflavones (particularly genistein) for the treatment of different types of cancer, including breast cancer, be promising chemopreventive agents. However, their precise mode of action in breast cancer treatment remains unclear till today. Methods: Literature survey was performed for screening out the genistein with therapeutic potentials and their mode of action using published articles available at the web databases in the public domains (like PubMed, SCI Finder, Science Direct, and Google Scholar) till June 2021. Results: In this review article, we have reviewed the development of novel anti-tumour strategies like inhibition of OPN at the transcriptional- or translational- level, development of monoclonal antibodies against OPN, and its downstream signalling pathways, etc. The inhibitory effect of the isoflavone(s) (e.g. genistein) on breast cancer proliferation, migration and invasion has also been reviewed. A newly elucidated anti-carcinogenic mode of action of the isoflavone including epigenetic modifications, topoisomerase inhibition and modulation of miRNA expression has also been discussed. Reports on the regulation of OPN expression by the isoflavone and the signalling pathways involved therein have been reviewed. Conclusion: Taken together, the isoflavone (genistein) could be used as a promising agent for cancer chemoprevention and/or treatment, though more clinical trials are needed for its validation.

Glucosinolates, synthesized by the glucosinolate biosynthesis pathway, are the secondary metabolites used as a defence mechanism in the Brassicaceae plants, including Arabidopsis thaliana. The first committed step in the pathway, catalysed by methylthioalkylmalate (MAM) synthase (EC: 2.3.3.17), is to produce different variants of glucosinolates. Phylogenetic analyses suggest that possibly MAM synthases have been evolved from isopropylmalate synthase (IPMS) by the substitutions of five amino acid residues (L143I, H167L, S216G, N250G and P252G) in the active site of IPMS due to point mutations. Considering the importance of MAM synthase in Brassicaceae plants, Petersen et al. (2019) made an effort to characterise the MAM synthase (15 MAM1 variants) in vitro by single substitution or double substitutions. In their study, the authors have expressed the variants in Escherichia coli and analysed the amino acids in the cultures of E. coli in vivo. Since modifying the MAM synthases by transgenic approaches could increase the resistance of Brassicaceae plants for enhancing the defence effect of glucosinolates and their degraded products; hence, MAM synthases should be characterized in detail in vivo in A. thaliana along with the structural analysis of the enzyme for meaningful impact and for its imminent use in vivo.

Artemisinin (ART) has been in use against different cancer cells and its derivatives and conjugates are more cytotoxic to iron-rich cancer cells. It is desirable to develop easily achievable synthetic 1,2,4-trioxanes having the same pharmacophore as that of ART. To explore more efficient compounds, a 1,2,3-triazole tethered 1,2,4‑trioxane trimer (4T) was synthesized and the anti-cancer effects of ART and 4T on MDA-MB-435 and MDA-MB-231 cells were investigated concerning regulation of osteopontin (OPN) expression, which is associated with cancer progression and malignancy. 1H NMR and 13C NMR, oxidative stress analysis, flow cytometry, western blot, Real-Time PCR, transfections, luciferase assay, cell viability, proliferation, migration and chemotactic invasion assays were used in this study. It was observed that the 4T induced apoptosis by inhibiting Bcl-2 (~0.6-fold) and cleavage of caspase-3 (intrinsic pathway) in these metastatic cancer cells, and also reduced colony formation, migration and invasion of these cancer cells. The treatment of 4T decreased the reduced glutathione level and increased the activities of glucose-6-phosphate dehydrogenase and glutathione reductase in the 4T treated cancer cells as compared to their respective controls. Further, the expression of OPN was diminished (~0.5-fold) by the 4T in these cell lines. It was also observed that the key mitogen-activated protein kinase pathway proteins, mitogen-activated protein kinase kinase1/2 (~1.8-fold) and extracellular signal-regulated kinase1/2 (~16-fold), were also activated following the treatment of the 4T. However, the phosphorylated c-Jun level, a component of activator protein-1, was significantly reduced in these cancer cells upon 4T treatment. Taken together, we hypothesize that 4T may be useful for controlling cancer progression and malignancy.

Heavy metal contaminations may have devastating effects on the ecological balance of the environment and diversity of aquatic organisms. Furthermore, they cause great threat to the Indian aquatic ecosystem. In this study, we analyzed the effect of arsenic (As) and lead (Pb) on the blood glucose level and their possible involvement in the mitogen-activated protein kinase (MAPK) pathway in liver and muscle tissues of Heteropneustes fossilis. The catfish, H. fossilis, were exposed to an acute (35.09 ppm As2O3 and 66.20 ppm PbCl2 for ~96 h) and chronic (LC50/20th ppm of both As2O3 and PbCl2 for ~30 d) concentration of As and Pb. Thereafter, the blood glucose level and the extracellular signal-regulated protein kinase (ERK) expression level in liver and muscle tissues of the fish were analyzed. It was found that As and Pb caused hyperglycemia in H. fossilis. Both on acute and chronic treatment with As and Pb, no significant change in p-ERK1/2 expression level was found in the muscle tissue of H. fossilis, whereas, in the liver tissue, the p-ERK1/2 expression level showed a significant increase in both acute (96 h) and chronic treatment (10 d and 30 d) of As and Pb. Therefore, it can be concluded that As and Pb could be highly toxic to the aquatic fauna, which could be a potential threat to human health as well.

AIM:The regulation of secreted osteopontin (OPN) expression by genistein and its functional sequel in the metastatic cancer cells (MDA-MB-435 and MDA-MB-231) was ascertained.MAIN METHODS:Western blot and Real-Time PCR were used to analyse the proteins and mRNA transcripts, respectively. Possible transcriptional regulation of secreted OPN was analyzed by chromatin immunoprecipitation assay, bioinformatics analysis, transfection and luciferase reporter assay. The specific siRNAs and constitutive p-ERKs were used to evaluate the role of the MAPK pathway. The functional sequel of genistein in these cells was analyzed by colony formation-, migration- and invasion- assay.KEY FINDINGS:Secreted OPN expression was inhibited (up to ~0.7-fold) by genistein in these cells. Genistein (50 μM) displayed a reduction in the aggressiveness of these cells concerning colony formation rate, migration, and invasion. The p-ERK½ was increased by ~2.5-fold and ~1.5-fold upon 50 μM genistein and 15 μM resveratrol treatments at 24 h, respectively. Knockdown of ERK½ and PD98059, the inhibitor of MEK, promoted secreted OPN expression in vitro in these cells; while, the transfection of the constitutive active ERK2 (L73P and S151D) decreased the secreted OPN expression. Further, silent mating type information regulation 2 homolog 1 (SIRT1) expression in the cells was increased (~1.6-fold) upon genistein treatment (50 μM) likewise with resveratrol (~1.5-fold), an activator for SIRT1. Knockdown of SIRT1 increased OPN mRNA transcripts expression level and secreted OPN protein level in these cells.SIGNIFICANCE:MAPK pathway and SIRT1 activation are involved in the regulation of secreted OPN by genistein in these cells.

Artemisinin and its analogs have shown potent anticancer activity in primary cancer cultures and cell lines by inhibiting cancer proliferation, metastasis, and angiogenesis. Despite its apparent compatibility to normal cells and low IC50 values in comparison to the commonly used anticancer drugs, the underlying mechanisms behind their cytotoxic effects are not yet fully understood. Surprisingly, the efficacy of synthetic 1,2,4-trioxanes against cancer has not been explored yet. Given the high antitumor activity of artemisinin dimers in comparison to their monomers, we report here the synthesis of simple 1,2,3-triazole conjugated 1,2,4-trioxanes and their potential antitumor activity by studying their inhibitory effect on osteopontin (OPN) expression in MDA-MB-435 breast cancer cells. It may be noted that despite being a strong marker to identify human tumor metastasis, no study on effect of artemisinin and its synthetic and semisynthetic derivatives on OPN expression has ever been studied. Although our initial studies did not notice any straight-line relationship between the number of trioxane units in a molecule to the extent of inhibition of OPN protein expression, we could observe better results in some cases in comparison to artemisinin. We have observed that artemisinin did not show appreciable OPN downregulation in MDA-MB-435 cancer cells, but dihydroartemisinin (DHA) and some synthetic 1,2,4-trioxane monomers and dimers showed downregulation of OPN expression. Therefore, these compounds may act as an anti-metastatic agent in controlling breast cancer cells metastasis.

Heavy metals show a wide range of effect on fishes, out of which arsenic (As) and lead (Pb) are among the leading heavy metal toxicants. These heavy metals are known to alter different biochemical parameters, including glycogen level, in different tissues of fishes. Glycogen level in fish serves as the main source of energy; hence, in this study, the acute toxicity test of As and Pb and their effect on the glycogen content and the enzymes involved therein (glycogen phosphorylase, glycogen synthase, hexokinase, phosphofructokinase and pyruvate kinase) were studied in the liver and muscle tissues of Heteropneustes fossilis. The 96 h LC50 values of As2O3 and PbCl2 on H. fossilis were found to be 35.09 ppm and 66.20 ppm, respectively. On acute exposure to 96 h LC50 values of As2O3 and PbCl2, the glycogen concentration showed a gradual decrease in both liver and muscle tissues of H. fossilis. However, on chronic exposure (LC50/20th ppm), the glycogen content in liver and muscle of H. fossilis was depleted till 20 days; whereas after 30 days, the glycogen level was recovered in both the tissues. The activities of glycogen metabolic enzymes (glycogen phosphorylase and glycogen synthase) and few selected glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) were also altered in H. fossilis when exposed to acute and chronic concentration of As2O3 and PbCl2. Our present results showed that As and Pb induced toxicity stress on the catfish, H. fossilis, which might have caused to alter the carbohydrate metabolism in the fish.

Genistein (4′,5,7-trihydroxyisoflavone) is naturally present in plants of the soy family and is known to have various pharmacological activities, such as anti-cancer, anti-diabetic, anti-oxidant, etc. The phytoestrogen is one of the major isoflavones found in some medicinal plants having anthelmintic properties. This review describes the putative role of genistein as an anthelmintic, which has been tested on some helminth parasites in vitro. Genistein has been shown to cause paralysis and alterations in the tegument and tegumental enzymes (acid phosphatase, alkaline phosphatase, adenosine triphosphatase, and 5′-nucleotidase) of helminth parasites. Alterations in the activities of several enzymes associated with the coordination system (specifically non-specific esterases, acetylcholine esterase, and nitric oxide synthase), and changes in the concentration of nitric oxide, cGMP, free amino acid pool, and tissue ammonia are observed in helminth parasites treated with genistein. The phytoestrogen also affects the carbohydrate metabolism by altering the activities of key enzymes involved in glycogen- and glucose-metabolism of a cestode parasite. Considering the significance of phosphoenolpyruvate carboxykinase (PEPCK) in glycolysis of the cestode parasite, Ki of the phytoestrogen for PEPCK in the parasite has been determined, and molecular docking of genistein into the active site of the enzyme has also been described. The potential beneficial role of genistein as a natural alternative in management of helminth parasites needs to be further explored, particularly considering its in vivo efficacy and pharmacokinetics.

Breast cancer is the most common cause of cancer‐related mortality in women worldwide and metastatic spread is the leading cause of breast cancer deaths. Osteopontin (OPN) plays a functional role in normal mammary gland development, progression of breast cancer and in malignancy of breast cancer. High expression of OPN is observed in high‐grade metastatic malignant human gliomas and in the highly invasive and metastatic human breast cancer cell lines (e.g. MDA‐MB‐435). Soy isoflavone genistein, a tyrosine kinase inhibitor and agonist of estrogen receptor‐β (ERβ), is known to have antitumoural properties and also induce apoptotic cell death of ERα‐negative breast cancer cells via p53‐dependent pathway. The isoflavone has been reported to exert antiproliferative activity in some breast cancer cell lines; however, its role with respect to OPN expression is not well understood. Therefore, to address this, the expression level of OPN was investigated upon genistein treatment in MDA‐MB‐435. The cells were treated with different concentrations of genistein for different time intervals; the expression of OPN, the signaling pathways and the promoter regions involved in the regulation of OPN were studied. Genistein displayed an inhibitory effect on OPN protein and mRNA levels in a dose dependent manner and treatment with 50 μM genistein for 24 h showed to be promising. Analysis of the signaling pathways showed no activation of Akt/PKB pathway, no alteration in p‐MEK½ expression whilst an increase in the expression of p‐ERK½ was observed in genistein treated group. These results indicate that genistein acts as an anti‐metastatic agent by inhibiting OPN expression through the activation of p‐ERK½ in MDA‐MB‐435 cells. The transcription factors responsible for this event are being studied in details.Support or Funding InformationDBT (GoI) No. BT/253/NE/TBP/2011 dated May 07, 2012This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

To study the various aspects of FESS and Caldwell-Luc approach in non neoplastic maxillary sinus pathologies. Forty patients were selected for the study. The age group ranged from 10-80 years. A male predominance had been seen in the study. In this study,all cases (100%) treated by FESS required General Anaesthesia, whereas, in Caldwell Luc Approach, 13 patients (65%) were operated under General Anaesthesia and 7 patients(35%) were operated under Local Anaesthesia. Subjective Improvement after 6 months of surgery was satisfactory in 90% cases of FESS and 60% cases of Caldwell-Luc Approach.