An insight to the toxic effect of Sulfamerazine on Porcine Pancreatic Amylase and Lactate Dehydrogenase Activity: An in vitro study

2021 ◽  
Vol 15 ◽  
Author(s):  
Avirup Malla ◽  
Koel Mukherjee ◽  
Mukulika Mandal ◽  
Aishwarya Mukherjee ◽  
Runa Sur ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Lactic Acidosis ◽  
Conformational Changes ◽  
Bacterial Infections ◽  
Titration Calorimetry ◽  
Dependent Manner ◽  
Pancreatic Amylase ◽  
Lactate Dehydrogenase Activity ◽  
Tract Infections

Background: Sulfamerazine, a sulfonamide has been routinely used to treat various bacterial infections namely Pneumonia, Urinary tract infections, Shigellosis, Bronchitis, Prostatitis, and many more. It interferes with the bacterial folic acid biosynthesis albeit higher eukaryotes are not susceptible to its action due to the inherent absence of this specific pathway. Objective: In spite of its constant use, Sulfamerazine administration evokes serious issues like development of antibacterial resistance through environmental contamination although how it affects eukaryotic system, specifically its target identification has not been addressed in detail. Methods: Hela Cells are cultured as per standard method, amylase and lactate dehydrogenase assay are conducted using standard procedure with spectrophotometer. Binding thermodynamics and conformational study has been estimated with isothermal titration calorimetry as well as with docking. Results: Experimental observations reveal that Sulfamerazine inhibits porcine pancreatic amylase in a noncompetitive mode (IC50 of 0.96 mM). Binding of the drug to porcine pancreatic amylase is entropy driven with conformational changes of the protein as indicated by concomitant red shift. It enhances the inhibitory effects of acarbose and cetapin on their in vitro pancreatic amylase activity. It augments lipid peroxidation and promotes lactic acidosis in a dose dependent manner. Docking studies ensure effective interactions between Sulfamerazine and proteins like lactic dehydrogenase and porcine pancreatic amylase. Conclusion: Detailed study is to be conducted to confirm whether molecular scaffold of Sulfamerazine might serve as an effective repurposed drug acting as a lead molecule for the design of antidiabetic drug of future use. Alternatively, it should be prescribed with caution under specific medical situations like diabetes, cancer, hepatic disorders manifesting lactic acidosis to avoid crisis.

Lactate Dehydrogenase Activity is Inhibited by Methylmalonate in vitro

2006 ◽  
Vol 31 (4) ◽  
pp. 541-548 ◽  
Author(s):  
Laura O. Saad ◽  
Sandra R. Mirandola ◽  
Evelise N. Maciel ◽  
Roger F. Castilho
Keyword(s):  
Lactate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Lactate Dehydrogenase Activity

An update on cefepime and its future role in combination with novel β-lactamase inhibitors for MDR Enterobacterales and Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Burcu Isler ◽  
Patrick Harris ◽  
Adam G Stewart ◽  
David L Paterson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Studies ◽  
Bacterial Infections ◽  
Animal Studies ◽  
Wide Spectrum ◽  
Clinical Development ◽  
Phase Iii ◽  
Lactam Antibiotic ◽  
Tract Infections

Abstract Cefepime, a wide-spectrum β-lactam antibiotic, has been in use for the treatment of serious bacterial infections for almost 25 years. Since its clinical development, there has been a dramatic shift in its dosing, with 2 g every 8 hours being preferred for serious infections to optimize pharmacokinetic/pharmacodynamic considerations. The advent of ESBLs has become a threat to its ongoing use, although future coadministration with β-lactamase inhibitors (BLIs) under development is an area of intense study. There are currently four new cefepime/BLI combinations in clinical development. Cefepime/zidebactam is generally active against MBL-producing Enterobacterales and Pseudomonas aeruginosa, in vitro and in animal studies, and cefepime/taniborbactam has activity against KPC and OXA-48 producers. Cefepime/enmetazobactam and cefepime/tazobactam are potential carbapenem-sparing agents with activity against ESBLs. Cefepime/enmetazobactam has completed Phase III and cefepime/taniborbactam is in Phase III clinical studies, where they are being tested against carbapenems or piperacillin/tazobactam for the treatment of complicated urinary tract infections. While these combinations are promising, their role in the treatment of MDR Gram-negative infections can only be determined with further clinical studies.

Hepatic stem cells and their enzymatic markers- A review

2018 ◽  
Author(s):  
Amit Kumar ◽  
Sourabh Sulabh
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Lactate Dehydrogenase ◽  
Progenitor Cells ◽  
Ethical Issues ◽  
Fetal Liver ◽  
Lactate Dehydrogenase Activity ◽  
Liver Stem Cells ◽  
Enzymatic Markers

Stem cells are those cells which show capacity for self-renewal and ability to give rise to multiple differentiated cellular populations. Enzymatic activity, as a marker for cell proliferation and cell viability, is used by metabolic activity assays. Liver stem cells/progenitor cells can be a useful source of liver treatment. They can repopulate and restore injured liver. Fetal liver stem/ progenitor cells have been found to be more capable in this, but are subjected to ethical issues. Adult liver stem cells and stem cells from animals can be used. Alkaline phosphatase and lactate dehydrogenase are enzymatic markers of in vitro hepatocyte culture. During in vitro cell culture, in the culture medium, secreted alkaline phosphatase activity increases during exponential growth of cells, whereas low extracellular lactate dehydrogenase activity indicates increased number of viable cells. Alkaline phosphatase and lactate dehydrogenase activities can be used to assess hepatocytes proliferation in vitro.

scholarly journals ACTIVATION LOOP PHOSPHORYLATION OF A NON-RD RECEPTOR KINASE INITIATES PLANT INNATE IMMUNE SIGNALING

2021 ◽  
Author(s):  
Kyle W Bender ◽  
Daniel Couto ◽  
Yasuhiro Kadota ◽  
Alberto P Macho ◽  
Jan Sklenar ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Stress Responses ◽  
Elongation Factor ◽  
Cytoplasmic Domain ◽  
Dependent Manner ◽  
Immune Signaling ◽  
Receptor Kinases

Receptor kinases (RKs) play fundamental roles in extracellular sensing to regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) function primarily as peptide receptors that regulate myriad aspects of plant development and response to external stimuli. Extensive phosphorylation of LRR-RK cytoplasmic domains is among the earliest detectable responses following ligand perception, and reciprocal transphosphorylation between a receptor and its co-receptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model RK, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in anti-bacterial immunity. These results reveal a non-catalytic role for the EFR cytoplasmic domain in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RK complexes with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor which could initiate signaling in a feed-forward fashion either allosterically or through driving the dissociation of negative regulators of the complex.

scholarly journals Enhanced Expression of miR-34a Enhances Escherichia coli Lipopolysaccharide-Mediated Endometritis by Targeting LGR4 to Activate the NF-κB Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Xiaofei Ma ◽  
Baoyi Yin ◽  
Shuai Guo ◽  
Talha Umar ◽  
Junfeng Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Luciferase Reporter ◽  
Blue Staining ◽  
Histopathological Analysis ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Gram Staining

Background. Persistent endometritis caused by bacterial infections has lethal effects on the reproductive performance of dairy cattle, which compromises animal welfare and delays or prevents pregnancy. The microRNA (miRNA) miR-34 family plays a pivotal role in the inflammatory process; however, the precise mechanism of miR-34a in endometritis has not been thoroughly elucidated to date. Methods. In this study, the endometrium of cows diagnosed with endometritis was harvested for bacterial culture and Gram staining to evaluate bacterial contamination of the uterus. Based on this, a bovine endometrial epithelial cell (BEND) inflammation model and a mouse model stimulated with lipopolysaccharide (LPS) in vitro and in vivo were constructed. Cell viability was assessed by CCK-8, trypan blue staining, and flow cytometry. H&E was applied to histopathological analysis. Immunohistochemical, immunofluorescence, qRT-PCR, and western blot assays were performed to measure the mRNA and protein expression of relevant genes. Online databases, plasmid construction, and dual-luciferase reporter gene assays were used to predict and validate the interaction between miR-34a and its target gene LGR4. Finally, mice were injected vaginally with a local antagomir to validate the role of miR-34a in murine uterine inflammation. Results. In this study, we observed that Gram-negative bacteria, represented by Escherichia coli, are the predominant pathogenic agents responsible for the recurrent occurrence of endometritis in dairy cows. Further, miR-34a was found to repress the expression of LGR4 by targeting the 3 ′ untranslated region (3 ′ UTR) of LGR4. miR-34a was upregulated in bovine uterine tissues and bovine endometrial epithelial cells stimulated with LPS. miR-34a induced the release of the proinflammatory cytokines IL-1β, IL-6, and TNF-α by activating the phosphorylation of NF-κB p65. Furthermore, IL-1β upregulated miR-34a transcription and downregulated LGR4 expression in an IL-1β-dependent manner. Conclusions. Taken together, our study confirmed that miR-34a is regulated by IL-1β and suppresses the level of the LGR4 3 ′ UTR, which in turn exacerbates the inflammatory response. Thus, the knockdown of miR-34a might be a new direction for the treatment of endometritis.

scholarly journals Structural and Functional study of Mevalonate Diphosphate Decarboxylase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1636-C1636
Author(s):  
Chun-Liang Chen ◽  
Cynthia Stauffacher
Keyword(s):  
Crystal Structure ◽  
Enterococcus Faecalis ◽  
Conformational Changes ◽  
Active Site ◽  
Bacterial Infections ◽  
Mevalonate Pathway ◽  
Functional Study ◽  
Titration Calorimetry ◽  
Mevalonate Diphosphate Decarboxylase ◽  
Near Future

Mevalonate diphosphate decarboxylases (MDD) (EC 4.1.1.33) catalyze the Mg2+-dependent decarboxylation of mevalonate 5-diphosphate (MVAPP) by hydrolyzing adenosine triphosphate (ATP) and producing isopentenyl diphosphate (IPP) in the final step of mevalonate pathway. This enzyme is essential in Enterococcus faecalis and other Gram (+) bacteria; therefore, MDD protein is an ideal drug target for the treatment of bacterial infections. We have studied the enzyme kinetics and structures of MDD from Enterococcus faecalis (MDDEF) which causes clinical enterorococcal infections. In the crystal structure of the MDDEF bound with ATP, the catalytically unfavored orientation of the γ-phosphate of ATP implies that conformational changes of MDDEF might occur in order to accommodate the binding of ATP when the MVAPP binds to the active site in advance. A 10-fold decrease of the dissociation constrant (Kd) value of ATPγS has been observed using isothermal titration calorimetry (ITC) when MDDEF is pre-bound with MVAPP. The increase of binding affinity of ATPγS suggests that cooperative binding of ATP to MDDEF can be achieved by the prerequisite binding of MVAPP. Indeed, the crystal structure of MDDEF soaked with the MVAPP shows that one flexible loop that eventually should bind ATP becomes non-flexible and bends toward the active site of MDDEF. Thus, we hypothesize that the binding of the MVAPP to the active site triggers conformational changes of MDDEF which induces the binding of the other substrate, ATP, in its catalytically favored position. Further experiments will be performed for investigating a substrate-binding mechanism for MDDEF and these will serve as platforms for specific drug development in the near future.

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