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Background Increasing intramuscular fat (IMF) is an important strategy to improve meat quality, but the regulation mechanism of IMF deposition needs to be systematically clarified. Results A total of 520 chickens from a selected line with improved IMF content and a control line were used to investigate the biochemical mechanism of IMF deposition in chickens. The results showed that the increased IMF would improve the flavor and tenderness quality of chicken meat. IMF content was mainly determined both by measuring triglyceride (TG) and phospholipid (PLIP) in muscle tissue, but only TG content was found to be decisive for IMF deposition. Furthermore, the increase in major fatty acid (FA) components in IMF is mainly derived from TGs (including C16:0, C16:1, C18:1n9c, and C18:2n6c, etc.), and the inhibition of certain very-long-chain FAs would help to IMF/TG deposition. Conclusions Our study elucidated the underlying biochemical mechanism of IMF deposition in chicken: Prevalent accumulation of long-chain FAs and inhibitions of medium-chain FAs and very long chain FA would jointly result in the increase of TGs with the FA biosynthesis and cellular uptake ways. Our findings will guide the production of high-quality chicken meat.

The current conditions in the progression of the SARS-CoV2 pandemic changed the current scientific paradigm, and we now observe a novel rhythm and way of evaluating the collected information. Previous experiences in epidemics with similar viruses (viz., SARS-CoV1, and MERS-CoV) and collected information about the viral transmission and replication can be used to overcome the SARS-CoV2 pandemic. Although SARS-CoV2 emerged very recently, there are plenty of scientific studies about similar viruses to comment on the current situation. Inhibition of SARS-CoV2 spike protein activation, inhibition of virus endocytosis, using a soluble form of ACE2, peptide or non-peptide analogs of ACE2, and sustaining ACE2/Angiotensin-(1–7)/Mas receptor pathway activation can be proposed for use in therapeutic studies. In this review, the biochemical mechanism of SARS-CoV2 and ACE2 binding, virus-cell membrane fusion, and endocytosis of virus to host cells are discussed according to the currently available literature. The significant contribution of this review may be to provide useful information to researchers into the SARS-CoV2 outbreak.

13 p.-9 fig.-3 tab., Protein tyrosine phosphatase 1B (PTP1B) is a very promising target for the treatment of metabolic disorders such as type II diabetes mellitus. Although it was validated as a promising target for this disease more than 30 years ago, as yet there is no drug in advanced clinical trials, and its biochemical mechanism and functions are still being studied. In the present study, based on our experience generating PTP1B inhibitors, we have developed and implemented a scaffold-hopping approach to vary the pyrrole ring of the pyrrolo[1,2-a]quinoxaline core, supported by extensive computational techniques aimed to explain the molecular interaction with PTP1B. Using a combination of docking, molecular dynamics and end-point free-energy calculations, we have rationally designed a hypothesis for new PTP1B inhibitors, supporting their recognition mechanism at a molecular level. After the design phase, we were able to easily synthesize proposed candidates and their evaluation against PTP1B was found to be in good concordance with our predictions. Moreover, the best candidates exhibited glucose uptake increments in cellulo model, thus confirming their utility for PTP1B inhibition and validating this approach for inhibitors design and molecules thus obtained., We are grateful for funding from the Spanish Ministerio de Economía y Competitividad (CTQ2017-85263-R MINECO/FEDER UE to J.J.V.); Instituto de Salud Carlos III and FEDER funds (RD16/0009/0015, RD16/0009/18, PI17/01513 and PI17/00625 to J.J.V., D.R.-P. and M.R.-P.). J. G−M was the recipient of an FPU fellowship (FPU16/01647) Spanish Ministerio de Educación y Cultura.

Biochemical mechanisms emerged and were integrated into the metabolic plan of cellular life long before molecular oxygen accumulated in the biosphere. When oxygen levels finally rose, they threatened specific types of enzymes: those that use organic radicals as catalysts, and those that depend upon iron centers. Nature has found ways to ensure that such enzymes are still used by contemporary organisms. In some cases they are restricted to microbes that reside in anoxic habitats, but in others they manage to function inside oxygen-filled cells. In the latter case, it is frequently true that the ancestral enzyme has been modified to fend off poisoning. In this review we survey a range of protein adaptations that permit radical-based and low-potential iron chemistry to succeed in oxic environments. In many cases, accessory domains shield the vulnerable radical or metal center from oxygen. In others, the structures of iron cofactors evolved to less oxidizable forms, or alternative metals replaced iron altogether. The overarching view is that some classes of biochemical mechanism are intrinsically incompatible with the presence of oxygen. The structural modification of target enzymes is an under-recognized response to this problem.

Summary High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K+. The K+ dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K+ by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K+ re-addition promptly restored glycolysis-dependent adenosine-5′-triphosphate generation. Moreover, expression and activation of KV1.3, a voltage-gated K+ channel, lowered cellular K+ content and the glycolytic activity of HEK293 cells. Our findings unveil K+ as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K+ channels and cell metabolism., Graphical abstract, Highlights • HKII expression sensitizes cellular metabolism for intracellular K • Intracellular K+ depletion abates the metabolic activity of HKII-positive cells • (Re-)elevations of intracellular K+ restore glycolysis, Here, Bischof et al. report that the activity of hexokinase-II requires a high intracellular K+ concentration. Cells that rely on hexokinase-II activity for augmented aerobic glycolysis are thus vulnerable to intracellular K+ depletion.

Summary CD40-Ligand (CD40L)-CD40 interaction regulates immune responses against pathogens, autoantigens, and tumor and transplantation antigens. Single amino acid mutations within the 115–155 amino acids stretch, which is responsible for CD40L functions, result in XIgM syndrome. We hypothesize that each of these amino acids of CD40L encodes specific message that, when decoded by CD40 signaling, induces a specific profile of functions. We observed that every single substitution in the XIgM-related amino acids in the 115–155 41-mer peptide in CD40L selectively altered CD40 signaling and effector functions—cytokine productions, HMGCoA reductase, ceramide synthase, inducible nitric oxide synthase and arginase expression, survival of B cells, and control of Leishmania infection and anti-leishmanial T cell response—suggesting residue-specific encoding of a distinct set of messages that collectively define CD40L pleiotropy, serve as a target for engineering the ligand to generate superagonists as immunotherapeutic, and implicate the evolutionary diversification of functions among the ligands in a protein superfamily., Graphical Abstract, Highlights • Contact residues on CD40L, when mutated to Valine, alter signaling specificity • The specificity is dictated by Residue-specific CD40L-CD40 interaction • Cytokine release, apoptosis, antileishmanial activity imply functional specificity • Specific CD40-CD40L interaction for antileishmanial functions in BALB/c mice, Biochemical Mechanism; Biochemistry; Immunology

Cutroneo, Kenneth Robert. Ph.D., University of Rhode Island, September, 1970. Alteration of Proline Hydroxylase Activity by Corticosteroids. Major Professor: Dr. George C. Fuller. iii The biochemical mechanism by which glucocorticoids affect collagen metabolism was investigated . Triamcinolone, hydrocortisone and methylprednisolone significantly decreased liver proline hydroxylase activity in vivo. This was not a manifestation of an antianabolic effect on protein. Triamcinolone inhibited liver proline hydroxylase activity in a dose dependent manner in vivo. An observed elevation of liver hydroxylase activity in adrenalectomized animals was decreased to control level by hydrocortisone treatment. Subdermal implants of three types of sponges (polyurethane , cellulose and polyvinyl) were used to induce granuloma growth as a model system of inflam.~ation. The anti-inflammatory activity of corticosteroids was correlated with inhibition of proline hydroxylase activity in granuloma tissue . . The data indicate that corticosteroids decrease collagen syntre sis by inhibiting the rate limiting enzyme. This inhibitory effect on collagen metabolism is correlated with observed anti-inflamma tory activity.

Marine brown macroalgae contain several bioactive compounds with potent anti-inflammatory properties but undescribed pharmacological properties. This study provided the first description of the biochemical mechanism of anti-inflammation from two bioactive compounds (fucoxanthin and sargachromenol) found in brown macroalgae (Sargassum spp.) based on a functional module-based analysis of a protein-protein interaction (PPI) network. The constructed PPI network of fucoxanthin and sargachromenol with 18 and 5 inflammatory proteins, respectively, have scale-free, small world and modular properties. There were 6 and 1 inflammatory modules found associated with the anti-inflammatory actions of fucoxanthin and sargachromenol, respectively. Of particular interest was that the anti-inflammatory effect of fucoxanthin and sargachromenol may be partly attributable to regulation of the I-kappa B kinase/NF-kappa B cascade and regulation of gene expression, respectively. These can be used to search for potential targets of fucoxanthin and of sargachromenol to treat inflammation. Therefore, functional module-based analysis of a PPI network can be an initial method for elucidating the anti-inflammatory mechanism of active compounds and finding their targets to validate in a wet laboratory clinical application and for further drug development. Keywords: Anti-inflammation, Biochemical mechanism, Functional module-based analysis, Marine-derived bioactive compounds, Protein-protein interaction network

Summary MicroRNAs (miRNAs) are part of a complex regulatory network that modulates cellular lipid metabolism. Here, we identify miR-124 as a regulator of triglyceride (TG) metabolism. This study advances our knowledge of the role of miR-124 in human hepatoma cells. Transcriptional profiling of Huh7.5 cells overexpressing miR-124 reveals enrichment for host factors involved in fatty acid oxidation among repressed miRNA targets. In addition, miR-124 down-regulates arylacetamide deacetylase (AADAC) and adipose triglyceride lipase, lipases proposed to mediate breakdown of hepatic TG stores for lipoprotein assembly and mitochondrial β-oxidation. Consistent with the inhibition of TG and fatty acid catabolism, miR-124 expression promotes cellular TG accumulation. Interestingly, miR-124 inhibits the production of hepatitis C virus, a virus that hijacks lipid pathways during its life cycle. Antiviral activity of miR-124 is consistent with repression of AADAC, a pro-viral host factor. Overall, our data highlight miR-124 as a novel regulator of TG metabolism in human hepatoma cells., Graphical Abstract, Highlights • miR-124 regulates triglyceride and fatty acid metabolism • miR-124 represses genes associated with fatty acid and triglyceride breakdown • miR-124 promotes triglyceride accumulation in hepatoma cells, Biological Sciences; Biochemistry; Biochemical Mechanism

Activating mutations in KRAS are among the most common tumor driver mutations. Until recently, KRAS had been considered undruggable with small molecules; the discovery of the covalent KRASG12C inhibitors ARS-853 and ARS-1620 has demonstrated that it is feasible to inhibit KRAS with high potency in cells and animals. Although the biological activity of these inhibitors has been described, the biochemical mechanism of how the compounds achieve potent inhibition remained incompletely understood. We now show that the activity of ARS-853 and ARS-1620 is primarily driven by KRAS-mediated catalysis of the chemical reaction with Cys12 in human KRASG12C, while the reversible binding affinity is weak, in the hundreds of micromolar or higher range. The mechanism resolves how an induced, shallow and dynamic pocket not expected to support high-affinity binding of small molecules can nevertheless be targeted with potent inhibitors and may be applicable to other targets conventionally considered undruggable.

High mortality is a frequent occurrence during live transport of shrimp species and the biochemical mechanism remains unknown. This study aimed to explore the influence of combined stress of acute cold exposure (AC) and waterless duration (WD) on survivability and biochemical response of shrimp L. vannamei during live transport. The shrimps in NC and AC groups remained the total survivability throughout the experiment while the shrimps exposed to AC + WD stress exhibited significantly higher mortality since 6h afterwards (P 0.05) and the median survival time was calculated at 10.46 h. Moreover, the typical combined stress points at AC + WD3h, AC + WD6h and AC + WD9h were assigned for exploring the immunological and antioxidative responses. For immunity response, the total hemocyte counts (THC) decreased with the prolongation of duration time and the activities of non-specific immunity enzymes such as phenol oxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), aspartate aminotransferase (AST) and alanine transaminase (ALT) were significantly elevated in AC + WD9h groups (P 0.05). Moreover, compared with that in NC group, the significant accumulation of reactive oxygen species (ROS) was observed in AC group and then reduced in combined stress groups (P 0.05), with the highest level of malonaldehyde (MDA) in AC and AC + WD3h groups. Overall, the significant elevation of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) was detected in AC + WD9h group (P 0.05). Furthermore, the accumulative pathological impairment on hepatopancreas tissue revealed the cytoskeleton degradation. In addition, correlation analyses visualized the correlation between oxidative stress and biochemical response. This study not only deepens our understanding on the biochemical mechanism of shrimp mortality induced by combined stress, but also provides a potential strategy for improving the management of L. vannamei during live transport.

o,p’-Dichlorodiphenyldichloroethane (o,p’-DDD, mitotane) is used in the treatment of adrenocortical cancer and Cushing’s disease. This medicine induces numerous biochemical changes in the adrenal cortex, as well as disorder in the mitochondrial structure. Therewith, the level of produced corticosteroid hormones is significantly reduced. One of the possible causes can be a decrease in the NADPH level due to inhibition of the activity of its reduction system and increased NADPH consumption during the glutathione reduction catalyzed by glutathione reductase. o,p’-DDD is partially metabolized in the adrenal glands, and the main metabolite (in terms of quantity) is o,p’-dichlorodiphenylacetic acid. However, attempts to find a physiologically active component among metabolites were unsuccessful. The most pronounced changes caused by o,p’-DDD were found in the mitochondria of the adrenal cortex. The respiration at the level of IV and I complexes is suppressed, the protein content of these complexes decreases. The phospholipid composition of the tissue altered and the concentration of diphosphatidylglycerol, the most important component of mitochondrial membranes, decreased. In our opinion, o,p’-DDD, owing to its high lipophilicity, accumulates in the mitochondria membranes and causes conformational disorder followed by disorder in mitochondrial functions. It was shown that o,p’-DDD acts as an inhibitor of acyl-CoA-cholesterol acyltransferase (ACAT, SOAT1). Therefore, adenocorticocytes accumulate free cholesterol, causing endoplasmic reticulum stress, mitochondrial swelling and caspases activation. Increased apoptosis leads to a decline in adrenal function and to a decrease in weight of adrenal glands.

Mycotoxins are highly dangerous natural compounds produced by various fungi. Enzymatic transformation seems to be the most promising method for detoxification of mycotoxins. This review summarizes current information on enzymes of different classes to convert various mycotoxins. An in-depth analysis of 11 key enzyme mechanisms towards dozens of major mycotoxins was realized. Additionally, molecular docking of mycotoxins to enzymes’ active centers was carried out to clarify some of these catalytic mechanisms. Analyzing protein homologues from various organisms (plants, animals, fungi, and bacteria), the prevalence and availability of natural sources of active biocatalysts with a high practical potential is discussed. The importance of multifunctional enzyme combinations for detoxification of mycotoxins is posed.

Summary SARM1, an NAD-utilizing enzyme, regulates axonal degeneration. We show that CZ-48, a cell-permeant mimetic of NMN, activated SARM1 in vitro and in cellulo to cyclize NAD and produce a Ca2+ messenger, cADPR, with similar efficiency as NMN. Knockout of NMN-adenylyltransferase elevated cellular NMN and activated SARM1 to produce cADPR, confirming NMN was its endogenous activator. Determinants for the activating effects and cell permeability of CZ-48 were identified. CZ-48 activated SARM1 via a conformational change of the auto-inhibitory domain and dimerization of its catalytic domain. SARM1 catalysis was similar to CD38, despite having no sequence similarity. Both catalyzed similar set of reactions, but SARM1 had much higher NAD-cyclizing activity, making it more efficient in elevating cADPR. CZ-48 acted selectively, activating SARM1 but inhibiting CD38. In SARM1-overexpressing cells, CZ-48 elevated cADPR, depleted NAD and ATP, and induced non-apoptotic death. CZ-48 is a specific modulator of SARM1 functions in cells., Graphical Abstract, Highlights • CZ-48, a cell-permeant mimetic of NMN, activates SARM1 but inhibits CD38 enzymatically • SARM1 catalysis is similar to CD38, but with higher cyclase activity • Activation by CZ-48 or NMN elicits conformational changes in SARM1 • Activation of SARM1 causes cADPR production, NAD depletion, and non-apoptotic cell death, Biochemistry; Enzymology; Biochemical Mechanism

It can be deduced from this concise review that bradykinin and polyphenols could have a double role in health. Nevertheless, if their bioavailability is improved by their encapsulation, polyphenols could diminish the binding affinity of bradykinin to its receptors by causing a conformational rearrangement of the peptide. Besides, the interaction between polyphenols and bradykinin should play a role in pain. Indeed, the formation of the polyphenol-bradykinin aggregates might alleviate the pain sensation caused by bradykinin by preventing: the production of nitric oxide, the activation of phospholipase A2, as well as that of nuclear factor-kappa B. To conclude, this work falls within the basic science, given that it seeks to provide a probable biochemical mechanism of the pharmacological action of polyphenols on the pain sensation caused by bradykinin.

BackgroundThe outcome of a given antibiotic treatment on the growth capacity of bacteria is largely dependent on the initial population size (the Inoculum Effect, IE). For some specific classical antibiotic drugs this phenomenon is well established in both in-vitro and in-vivo studies, and its precise mechanisms, its clinical implications and its mathematical modelling are at the forefront of current research. Traditional view of the IE is that it is mainly attributed to β-lactam antibiotics in relation to β-lactamase producing bacteria, and that some antibiotics do not induce an IE at all. The study of antimicrobial peptides had emerged in the past two decades as a possible additional strategy for combatting infections, and their mechanism of operation and clinical implications are extensively studied. Yet, no previous studies addressed the possible induction of IE under the action of classical cationic antimicrobial peptides (CAMPs).Based on mathematical reasoning regarding bacteria-neutrophils interaction, we hypothesized that CAMPs also induce an IE in bacterial growth, and questioned what are the similarities and differences between the IE induced by CAMPs and that induced by classical antibiotics. To this aim we also needed to better understand the characteristics of the IE induced by classical antibiotics.Principal FindingsWe characterized and built a model of in-vitro IE in E. coli cultures using a large variety of antimicrobials, including 6 conventional antibiotics, and for the first time, 4 cationic antimicrobial peptides (CAMPs). Each combination of bacterial initial load and antimicrobial concentration experiment was done in duplicate, with 48 such combinations in each experiment. Each experiment was repeated 4-6 times, sometimes with some adjustments in the tested concentrations to get better resolution of the IE. Each growth curve was processed independently, to correctly reflect the initial exponential growth that might lead to large deviations even between duplicates. By using Optical Density (OD) to monitor the bacterial density, we were able to gather growth curves from this extensive data set and from these curves extract, by data processing, the corresponding growth functions. We show that this process allows us to clearly differentiate between simple one-dimensional deterministic bacterial growth dynamics and more complex behaviour.In all agents we tested, including all cationic antimicrobial peptides and all conventional antibiotics, independently of their biochemical mechanism of action, an “inoculum effect” was found. At a certain range of concentrations, which is specific for every drug and experimental setting, the system exhibits a bistable behaviour in which large loads survive and small loads are inhibited. Moreover, we characterized three distinct classes of drug-induced bi-stable growth dynamics and demonstrated that in rich medium, CAMPs correspond to the simplest class, bacteriostatic antibiotics to the second class and all other traditional antibiotics to the third, more complex class. In particular, for the first two classes, of cationic antimicrobial peptides and of the commercial bacteriostatic antibiotics, the bacterial growth can be explained by a very simple deterministic one-dimensional mathematical model. These findings provide a unifying universal framework to describe the dynamics of the inoculum effect induced by antimicrobials with inherently different killing mechanisms.Limitations of the results: The IE we detect is in-vitro, in rich medium, and the simple deterministic one dimensional models apply to this setting for the CAMPs and the bacteriostatic antibiotics only. While these findings can be used as a building block to more complex settings, with in-vivo being the most complex of all, it is clear that additional studies are needed in order to address these complexities. Another limitation is the OD methodology which does not clearly differentiate between live, dormant and dead cells and also does not detect small bacterial loads that are below the reader detection level. Nonetheless, since only live bacteria grow, the growth functions that we find experimentally are independent of the dead and dormant bacteria, and the bacterial density axis may be at most shifted by small amount due to this effect. The behaviour at small loads, below the OD detection level, is also irrelevant for the current study as we are concerned with the IE at high inoculum. Finally, this study is conducted at the population level only, with the point of view that IE is induced by deterministic non-linear interactions between the bacteria and the anti-microbial agent, without delving into the details of the particular molecular mechanisms that lead to this particular interaction. Such detailed nonlinear molecular mechanisms that induce IE are known to exist for some of the agents we use. Future studies are needed to better understand the detailed molecular mechanisms in the other cases.Conclusions & SignificanceThe vast increase in bacterial resistance, highlights the need for new approaches to eradicate bacterial infections, by either the development of new antimicrobial agents, or new strategies of treatment. Developing treatment strategies requires a better understanding of the Inoculum Effect (IE). We demonstrate that IE is abundant in the application of both classical antimicrobial peptides and classical antibiotics to bacteria. Furthermore, we show that IE falls into three universality classes of bi-stable behaviours and that classical antimicrobial peptides form a class of their own – the simplest and most predictable class. These findings propose a new exciting viewpoint on the universality features of IE that may serve as building blocks for the design of better treatment strategies for infection.We stress that the detection of IE in CAMPs may have important implications for their mode of operation, and this finding may lead to further explorations of this phenomenon both in terms of mechanistic models and in terms of clinical and biological implications.While bacterial IE was identified in previous studies of particular conventional antibiotic agents and bacteria, previous explanations of its appearance included genetic and/or phenotypic population heterogeneity and additional time-dependent factors. These were modelled, for example, by deterministic multi-dimensional equations of classical reaction kinetics. Here we show that for some cases (the bacteriostatic antibiotics) a one dimensional model can explain the resulting growth curves by density dependant mechanisms alone. By Ockham’s razor principle, we assert that such models are adequate for describing the IE in bacteriostatic antibiotics. On the other hand, we also show that for all other cases (growth with all other classical antibiotics and growth in poor medium) simple one dimensional deterministic models cannot describe the dynamics, and thus multi-dimensional models may be needed to describe IE in these cases. Additionally, contrary to some other studies, we show that IE appears in every antibiotic we tested (in particular antibiotics that are not β-lactams), so additional molecular mechanisms for creating the non-linear bacterial-drug interaction need to be identified.Finally, density dependent phenomena are abundant in biology and may appear in other pathogenesis systems, where densities matter. Here we demonstrated that such phenomena can sometimes be described by very simple growth dynamics. Such simple models may serve as building blocks to more complex models such as in-vivo ones and may also inspire detailed studies aimed at deciphering the specific dominant molecular mechanisms of the detected IE. We propose that the principles and methodologies developed here for studying IE by observing the population level dynamics may be applicable to diverse biological situations.Authors SummaryThe vast increase in bacterial resistance highlights the need for new approaches to eradicate bacterial infections, by either the development of new antimicrobial agents, or new strategies of treatment. Since the outcome of a given antibiotic treatment on the growth capacity of bacteria is largely dependent on the initial population size (Inoculum Effect, IE), developing treatment strategies requires a better understanding of this effect. We characterized and built a model of this effect in E. coli cultures using a large variety of antimicrobials, including conventional antibiotics, and for the first time, cationic antimicrobial peptides (CAMPs). Our results show that all classes of antimicrobial drugs induce an inoculum effect. Moreover, we characterized three distinct classes of drug-induced bi-stable growth dynamics and demonstrated that in rich medium, CAMPs correspond to the simplest class, bacteriostatic antibiotics to the second class and all other traditional antibiotics to the third, more complex class. These findings provide a unifying universal framework to describe the dynamics of the inoculum effect induced by antimicrobials with inherently different killing mechanisms. These findings propose a new exciting viewpoint on the universality features of IE that may serve as building blocks for the design of better treatment strategies for infection.

Summary Aberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (β-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity., Graphical Abstract, Highlights • A class of carboxamide-substituted benzhydryl amine (CBA) Wnt inhibitors • A biological active, biotinylated CBA to identify KDM3A as a direct target • CBA-1 interacted with the Mn2+ ion in the JmjC domains of KDM3A/3B • CBA-1 inhibited Wnt signaling in colon cancer cells and in zebrafish models, Biological Sciences; Biochemistry; Biochemical Mechanism

Summary Inhibition of the ATR kinase has emerged as a therapeutically attractive means to target cancer since the development of potent inhibitors, which are now in clinical testing. We investigated a potential link between ATR inhibition and the autophagy process in esophageal cancer cells using four ATR inhibitors including two in clinical testing. The response to pharmacological ATR inhibitors was compared with genetic systems to investigate the ATR dependence of the effects observed. The ATR inhibitor, VX-970, was found to lead to an accumulation of p62 and LC3-II indicative of a blocked autophagy. This increase in p62 occurred post-transcriptionally and in all the cell lines tested. However, our data indicate that the accumulation of p62 occurred in an ATR-independent manner and was instead an off-target response to the ATR inhibitor. This study has important implications for the clinical response to pharmacological ATR inhibition, which in some cases includes the blockage of autophagy., Graphical Abstract, Highlights • Inhibition of ATR using VX-970 leads to an accumulation of p62 • VX-970-mediated accumulation of p62 occurs independently of ATR • VX-970-mediated accumulation of p62 is consistent with blocked autophagy, Biological Sciences; Biochemistry; Biochemical Mechanism; Cancer

Background Methylmercury (CH3Hg+) toxicity is characterized by challenging conundrums: 1) “selenium (Se)-protective” effects, 2) undefined biochemical mechanism/s of toxicity, 3) brain-specific oxidative damage, 4) fetal vulnerability, and 5) its latency effect. The “protective effects of Se” against CH3Hg+ toxicity were first recognized >50 years ago, but awareness of Se's vital functions in the brain has transformed understanding of CH3Hg+ biochemical mechanisms. Mercury's affinity for Se is ~1 million times greater than its affinity for sulfur, revealing it as the primary target of CH3Hg+ toxicity. Scope of review This focused review examined research literature regarding distinctive characteristics of CH3Hg+ toxicity to identify Se-dependent aspects of its biochemical mechanisms and effects. Conclusions Research indicates that CH3Hg+ irreversibly inhibits the selenoenzymes that normally prevent/reverse oxidative damage in the brain. Unless supplemental Se is provided, consequences increase as CH3Hg+ approaches/exceeds equimolar stoichiometries with Se, thus forming HgSe and inducing a conditioned Se deficiency. As the biochemical target of CH3Hg+ toxicity, Se-physiology provides perspectives on the brain specificity of its oxidative damage, accentuated fetal vulnerability, and latency. This review reconsiders the concept that Se is a “tonic” that protects against CH3Hg+ toxicity and recognizes Se's role as Hg's molecular “target”. As the most potent intracellular nucleophile, the selenoenzyme inhibition paradigm has broad implications in toxicology, including resolution of conundrums of CH3Hg+ toxicity. General significance Mercury-dependent sequestration of selenium and the irreversible inhibition of selenoenzymes, especially those required to prevent and reverse oxidative damage in the brain, are primarily responsible for the characteristic effects of mercury toxicity.

Razor clam is a major cultivated shellfish of great economic importance and high nutritional value. Due to high corruptible potential, razor clam is generally preserved by controlled freezing-point storage (CFPS). Here, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling to investigate the biochemical mechanism of cold stress adaption in razor clam during CFPS. In total, 369 proteins were quantified, and 27 of them were identified as differentially expressed proteins during CFPS, mostly involved in energy metabolism process, DNA duplication and protein synthesis, and stress response, specifically, MAPK is the predominant pathway. Further qPCR results revealed H2A and S6K 2 alpha to be the critical post-transcriptionally regulated genes. Our results provided proteomics information with respect to the biochemical mechanism of cold stress adaption in razor clam, shed light on the further elongation of razor clams storage period, and help clarify the novel mechanisms of cold tolerance.

Oilseeds crops provide edible oil for humans’ diet or oil for industrial purpose. Triacylglycerols (TAG) are the main components in seed oil which is formed by the esterification of fatty acids to glycerol back bone. In plants, fatty acid biosynthesis process initiated in plastid and ended in the endoplasmic reticulum. A series of biochemical steps are involved in TAG formation and many of the genes involved in this process have been identified. The quality of seed oil depends on the type of fatty acids esterified to glycerol. Finally, the TAGs are stored as lipid in seeds. The biosynthesis process of fatty acids and oil formation in seeds is reviewed in this manuscript.

We propose a tripartite biochemical mechanism for memory. Three physiologic components are involved, namely, the neuron (individual and circuit), the surrounding neural extracellular matrix, and the various trace metals distributed within the matrix. The binding of a metal cation affects a corresponding nanostructure (shrinking, twisting, expansion) and dielectric sensibility of the chelating node (address) within the matrix lattice, sensed by the neuron. The neural extracellular matrix serves as an electro-elastic lattice, wherein neurons manipulate multiple trace metals (n > 10) to encode, store, and decode coginive information. The proposed mechanism explains brains low energy requirements and high rates of storage capacity described in multiples of Avogadro number (N(A) = 6 × 10(23)). Supportive evidence correlates memory loss to trace metal toxicity or deficiency, or breakdown in the delivery/transport of metals to the matrix, or its degradation. Inherited diseases revolving around dysfunctional trace metal metabolism and memory dysfunction, include Alzheimer's disease (Al, Zn, Fe), Wilson's disease (Cu), thalassemia (Fe), and autism (metallothionein). The tripartite mechanism points to the electro-elastic interactions of neurons with trace metals distributed within the neural extracellular matrix, as the molecular underpinning of "synaptic plasticity" affecting short-term memory, long-term memory, and forgetting.

Hypoxia causes left ventricular dysfunction in the human heart, but the biochemical mechanism is poorly understood. Here, we tested whether short-term normobaric hypoxia leads to changes in cardiac energetics and early cardiac dysfunction. Healthy male volunteers (n=12, age 24±2 yr) were exposed to normobaric hypoxia in a purpose-built hypoxic chamber. The partial pressure of oxygen during end-tidal expiration (P ETO 2) was kept between 50 and 60 mmHg, and peripheral oxygen saturation (SaO 2) was kept above 80%. Cardiac morphology and function were assessed using magnetic resonance imaging and echocardiography, both before and after 20 h of hypoxic exposure, and high-energy phosphate metabolism [measured as the phosphocreatine (PCr)/ATP ratio] was measured using 31P magnetic resonance spectroscopy. During hypoxia, P ETO 2 and SaO 2averaged 55 ± 1 mmHg and 83.6 ± 0.4%, respectively. Hypoxia caused a 15% reduction in cardiac PCr/ATP (from 2.0±0.1 to 1.7

The group of Howard Petty has claimed exotic metabolic wave phenomena together with mutually phase-coupled NAD(P)H- and calcium-oscillations in human neutrophils. At least parts of these phenomena are highly doubtful due to extensive failure of reproducibility by several other groups and hints that unreliable data from the Petty lab are involved in publications concerning circular calcium waves. The aim of our theoretical spatiotemporal modeling approach is to propose a possible and plausible biochemical mechanism which would, in principle, be able to explain metabolic oscillations and wave phenomena in neutrophils. Our modeling suggests the possibility of a calcium-controlled glucose influx as a driving force of metabolic oscillations and a potential role of polarized cell geometry and differential enzyme distribution for various NAD(P)H wave phenomena. The modeling results are supposed to stimulate further controversial discussions of such phenomena and potential mechanisms and experimental efforts to finally clarify the existence and biochemical basis of any kind of temporal and spatiotemporal patterns of calcium signals and metabolic dynamics in human neutrophils. Independent of Petty's observations, they present a general feasibility study of such phenomena in cells.

Citric acid is regarded as a metabolite of energy metabolism, of which the concentration will rise to appreciable amounts only under conditions of substantive metabolic imbalances. Citric acid fermentation conditions were established during the 1930s and 1940s, when the effects of various medium components were evaluated. The biochemical mechanism by which Aspergillus niger accumulates citric acid has continued to attract interest even though its commercial production by fermentation has been established for decades. Although extensive basic biochemical research has been carried out with A. niger, the understanding of the events relevant for citric acid accumulation is not completely understood. This review is focused on citric acid fermentation by A. niger. Emphasis is given to aspects of fermentation biochemistry, membrane transport in A. niger and modeling of the production process.

A subgroup of caspase family of inflammatory caspases (-1, -4, -5, -11, and -12) play important role during cytokine maturation and inflammation but their regulation is not well understood as much as the initiator and effector caspases. Here, the biochemical mechanism of caspase-4 activation is elucidated. With citrate, a well-known kosmotrope to enhance the monomer-dimer transition, caspase-4 was activated approximately 40 times that was comparable with that of caspase-9 ( approximately 75-fold increments). The activation reaction was mainly bimolecular (n=1.67+/-0.04) for monomeric caspase-4. In addition, the interdomain cleavage was also responsible to activate caspase-4 more than 100-fold, again comparable with that of effector caspases where the proteolytic processing is considered as the sole activation mechanism. Thus, caspase-4 shows a novel activation mechanism of the synergism between dimerization and proteolysis that sharply differs from the established activation mechanism of dimerization for initiators and interdomain cleavage for effector caspases.

The Gompertz law of mortality quantitatively describes the mortality rate of humans and almost all multicellular animals. However, its underlying kinetic mechanism is unclear. The Gompertz law cannot explain the mortality plateau at advanced ages and cannot give an explicit relationship between temperature and mortality. In this study a reaction kinetics model with a time dependent rate coefficient is proposed to describe the survival and senescence processes. A temperature-dependent mortality function was derived. The new mortality function becomes the Gompertz mortality function with the same relationship of parameters prescribed by the Strehler–Mildvan correlation when age is smaller than a characteristic value δ, and reaches the mortality plateau when age is greater than δ. A closed-form analytical expression for describing the relationship of average lifespan with temperature and other equations are derived from the new mortality function. The derived equations can be used to estimate the limit of average lifespan, predict the maximal longevity, calculate the temperature coefficient of lifespan, and explain the tendency of the survival curve. This prediction is consistent with the most recently reported mortality trajectories for single-year birth cohorts. This study suggests that the senescence process results from the imbalance between damaging energy and protecting energy for the critical chemical substance in the body. The rate of senescence of the organism increases while the protecting energy decreases. The mortality plateau is reached when the protecting energy decreases to its minimal levels. The decreasing rate of the protecting energy is temperature-dependent. This study is exploring the connection between the biochemical mechanism and demography., Highlights • A temperature-dependent mortality function is derived. • The function reaches a mortality plateau as age>δ (a life characteristic value). • An expression for the temperature-dependent average lifespan is derived. • The equation for estimating the limit of average lifespan is derived.

Following a period of intensive and illuminating genetic analysis, the biochemical mechanism of mismatch repair was deciphered by Paul Modrich and his colleagues. The establishment of an in vitro assay that specifically supports strand-specific excision repair of mismatched bases was a seminal contribution to this body of work.

Drug discovery is extremely difficult. There are many unanticipated scientific, medical and business challenges to every drug discovery programme. It is important to increase our understanding of the fundamental properties of effective drugs so that we can anticipate potential problems in developing new agents. This article addresses potential drug discovery and development risks associated with the biochemical mechanism of drug action, and proposes simple rules to minimize these risks.

A unified model for initiator caspase activation has previously been proposed based on the biochemical analysis of caspase-8 and -9. Caspase-2 is structurally related to caspase-9, but its mechanism of activation is not known. Using an uncleavable mutant of caspase-2, we show that dimerization (and not processing) is the key event that drives initial procaspase-2 activation. Following dimerization, caspase-2 undergoes autocatalytic cleavage that promotes its stable dimerization and further enhances the catalytic activity of caspase-2. Although the caspase-2 zymogen does not require cleavage for the initial acquisition of activity, intersubunit cleavage is required to generate levels of activity required to induce cell death by overexpression. We also provide evidence that the reported disulfide bond linkage between two caspase-2 monomers is dispensable for caspase-2 dimerization. As caspase-2 does not require cleavage for its initial activation, our findings confirm caspase-2 to be a bona fide initiator caspase.

Certain phytopathogenic fungi differenti- ate by forming sclerotia by an unclear biochemical mechanism. We have proposed that sclerotial differ- entiation might be regulated by fungal antioxidant defense. Part of this defense might be ascorbic acid, which in its reduced form is a well-known antioxi- dant. This natural antioxidant was studied in Sclero- tium rolfsii in relation to oxidative-growth conditions, developmental stages and strain-differentiating abili- ty. The transition of a sclerotial strain from the un- differentiated to the differentiated stage was accom- panied by a sharp shift in the ratio of reduced/oxi- dized ascorbate toward the oxidized form. Ascorbate profiles and lipid peroxidation levels were different between the sclerotial strain grown under high- and low-oxidative stress conditions, as well as between a nonsclerotial S. rolfsii strain grown under high-oxi- dative stress conditions. In addition, the ratio of re- duced/oxidized ascorbate in the nonsclerotial strain remained unchanged throughout growth. Lipid per- oxidation under high-oxidative stress conditions in sclerotial S. rolfsii colonies one day before differen- tiation was 3.6-fold higher than in same-day colonies of this strain grown under low-oxidative stress con- ditions and 2.5-fold higher than in similar-day colo- nies of the nonsclerotial strain grown under high- oxidative stress conditions. Exogenous ascorbate caused a concentration-dependent reduction of lipid peroxidation and a proportional inhibition of the de- gree of sclerotial differentiation in the sclerotial strain grown under high-oxidative stress conditions by lowering its lipid peroxidation before differentia- tion to levels similar to the strain grown under low- oxidative stress conditions and to the nonsclerotial strain. Ascorbic acid might be produced by the scle- rotial strain to reduce oxidative stress, although less efficiently than the nondifferenting strain. The data

Wax esters are unique surface lipids found on the surface of terrestrial organisms. After a brief review of the composition, the mechanisms used in their biosynthesis by sebaceous glands are summarized. The molecular biology of biosynthesis of methyl-branched, short chain and very long chain acids, and the biochemical mechanism of the loss of the carboxyl carbon of the elongated very long chain acids to generate alkanes are reviewed. The molecular biology of biosynthesis of the major types of molecules contains the hydroxyl group(s) to which the acids are esterified and the molecular biology of the esterification process are reviewed. The molecular bases of seasonal and hormonal regulation of biosynthesis of wax esters that implicate some functions of the wax esters are reviewed.

Alkylphosphonates Io were expected to be effective plant PDHc E1 inhibitors because they were obtained by the chemical modification of sodium O-methyl acetylphosphonate as a hit compound, which is a potent plant PDHc E1 inhibitor. Therefore, it is interesting to examine inhibitory activity against plant PDHc E1. The biochemical mechanism was explored through the method of theoretical calculations and biochemical experiments. Several representative alkylphosphonates Io were found to be powerful inhibitors against plant PDHc, but show no significant inhibition against PDHc E1 from pig heart. The degree of herbicidal activity of these compounds positively correlated with their enzyme inhibition.

In a recent issue of Molecular Cell, Shiotani and Zou (2009) elucidate the biochemical mechanism underlying sequential ATM and ATR activation at DNA double-strand breaks, demonstrating that resection transforms ATM substrates into ATR substrates.

During the last 10 years, much progress has been made in understanding signal transduction. However, the function of many newly identified proteins remains unknown. The protein/protein interactions have emerged as a major biochemical mechanism of signal transduction. They are of major interest to elucidate the role of a protein in one or another cellular process. The two-hybrid system is especially well designed for such investigation. Here we show that the contribution of this technique already is and will be essential in dissecting the molecular mechanism of transduction pathways in many cell types.

The effect of intraperitoneal administration of lethal dose (50 mg/kg) of paraquat on the microsomal cysteine levels in the plasma, liver and lung of adult male Wistar rats has been investigated using Rank Chromaspek amino acid analyzer. The microsomal alanine levels were also determined to help in assessing the extent of paraquat interference with cellular protein. DL–Buthionine–[S,R]–Sulfoximine (BSO) and Diethyl maleate (DEM) were used to potentiate the toxic effect of the bipyridyl. The microsomal cysteine levels were significantly (P ≤ 0.05) depressed in the plasma, liver and lung of the paraquat-treated rats compared with the saline-injected group but the alanine levels were not similarly affected. Probably, paraquat poisoning interferes specifically with the cellular cysteine content in the rat. These findings could provide a valuable information on the biochemical mechanism of paraquat intoxication.