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BACKGROUND: Pegunigalsidase alfa is a PEGylated α-galactosidase A enzyme replacement therapy. BALANCE (NCT02795676) assessed non-inferiority of pegunigalsidase alfa versus agalsidase beta in adults with Fabry disease with an annualised estimated glomerular filtration rate (eGFR) slope more negative than -2 mL/min/1.73 m2/year who had received agalsidase beta for ≥1 year. METHODS: Patients were randomly assigned 2:1 to receive 1 mg/kg pegunigalsidase alfa or agalsidase beta every 2 weeks for 2 years. The primary efficacy analysis assessed non-inferiority based on median annualised eGFR slope differences between treatment arms. RESULTS: Seventy-seven patients received either pegunigalsidase alfa (n=52) or agalsidase beta (n=25). At baseline, mean (range) age was 44 (18-60) years, 47 (61%) patients were male, median eGFR was 74.5 mL/min/1.73 m2 and median (range) eGFR slope was -7.3 (-30.5, 6.3) mL/min/1.73 m2/year. At 2 years, the difference between median eGFR slopes was -0.36 mL/min/1.73 m2/year, meeting the prespecified non-inferiority margin. Minimal changes were observed in lyso-Gb3 concentrations in both treatment arms at 2 years. Proportions of patients experiencing treatment-related adverse events and mild or moderate infusion-related reactions were similar in both groups, yet exposure-adjusted rates were 3.6-fold and 7.8-fold higher, respectively, with agalsidase beta than pegunigalsidase alfa. At the end of the study, neutralising antibodies were detected in 7 out of 47 (15%) pegunigalsidase alfa-treated patients and 6 out of 23 (26%) agalsidase beta-treated patients. There were no deaths. CONCLUSIONS: Based on rate of eGFR decline over 2 years, pegunigalsidase alfa was non-inferior to agalsidase beta. Pegunigalsidase alfa had lower rates of treatment-emergent adverse events and mild or moderate infusion-related reactions. TRIAL REGISTRATION NUMBER: NCT02795676.

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs. Gs heterotrimers are considered to be poor providers of free Gβγ subunits. Here, the authors show that—despite this—Gs-derived Gβγ dimers are active transducers of GPCR-initiated Ca2+ signals involving phosphoinositide-based signaling routes. [ABSTRACT FROM AUTHOR]

Laccases are important and valuable enzymes with a great potential for biotechnological applications. In this study, two novel laccases, LacHU1 and LacHU2, from Alternaria sp. HU have been purified and characterized. The molecular mass of each isoenzyme was ~66 kDa. LacHU1 laccases was yellow and had no typical blue oxidase spectra and LacHU2 had a blue color and characteristic absorption spectra. The catalytic efficiency of LacHU1 for most substrates was higher than that of LacHU2 laccase. Both isoenzymes effectively oxidize flavonoids. Alternaria sp. laccases were successfully immobilized on magnetic nanoparticles. The thermostability of immobilized laccases increased and optimal pH shifted to more alkaline compared to the free laccases. Potential applications of laccases from Alternaria sp. HU are in the oxidation of flavonoids in cotton or in water treatment processes. [ABSTRACT FROM AUTHOR]

Cytochrome P450 enzymes are a superfamily of enzymes responsible for the metabolism of a variety of medicines and xenobiotics. Among the Cytochrome P450 family, five isozymes that include 1A2, 2C9, 2C19, 2D6, and 3A4 are most important for the metabolism of xenobiotics. Inhibition of any of these five CYP isozymes causes drug-drug interactions with high pharmacological and toxicological effects. So, the inhibition or non-inhibition prediction of these isozymes is of great importance. Many techniques based on machine learning and deep learning algorithms are currently being used to predict whether these isozymes will be inhibited or not. In this study, three different molecular or substructural properties that include Morgan, MACCS and Morgan (combined) and RDKit of the various molecules are used to train a distinct SVM model against each isozyme (1A2, 2C9, 2C19, 2D6, and 3A4). On the independent dataset, Morgan fingerprints provided the best results, while MACCS and Morgan (combined) achieved comparable results in terms of balanced accuracy (BA), sensitivity (Sn), and Mathews correlation coefficient (MCC). For the Morgan fingerprints, balanced accuracies (BA), Mathews correlation coefficients (MCC), and sensitivities (Sn) against each CYPs isozyme, 1A2, 2C9, 2C19, 2D6, and 3A4 on an independent dataset ranged between 0.81 and 0.85, 0.61 and 0.70, 0.72 and 0.83, respectively. Similarly, on the independent dataset, MACCS and Morgan (combined) fingerprints achieved competitive results in terms of balanced accuracies (BA), Mathews correlation coefficients (MCC), and sensitivities (Sn) against each CYPs isozyme, 1A2, 2C9, 2C19, 2D6, and 3A4, which ranged between 0.79 and 0.85, 0.59 and 0.69, 0.69 and 0.82, respectively. [ABSTRACT FROM AUTHOR]

Bicyclol is a hepatoprotective agent widely used for treating chronic hepatitis and drug-induced liver injuries in clinics. The purpose of the study was to elucidate the contribution of CYP450 enzymes to the metabolism of bicyclol using the relative activity factor approach. After incubation with human liver microsomes and recombinant human liver CYP450 enzymes, the calculated contribution of CYP3A4 and 2C19 to the metabolism of bicyclol was 85.6–90.3% and 9.2–9.7%, respectively. The metabolism was interrupted in the presence of CYP3A4 and 2C19 selective inhibitors. These findings help to predict or avoid metabolic drug–drug interactions or toxicity in clinical applications of bicyclol. [ABSTRACT FROM AUTHOR]

Thiosemicarbazone derivative was obtained by the addition of thiosemicarbazide to 5‐nitro‐thiophene‐2‐carbaldehyde. The addition–cyclization of the 2‐bromoacetophenone derivative to thiosemicarbazone derivative gave the new thiazole derivatives (2 a‐k). Acetylcholinesterase (AChE), butyrylcholinesterase (BChE), antioxidant, and human carbonic anhydrase (hCA) I and II isoform inhibitory activities of the thiazole derivatives 2 a‐k were investigated. The effects of thiazole derivatives on carbonic anhydrase I and II (hCA I–II) isoenzymes were examined in vitro using the esterase method. IC50 values for enzyme inhibition were found to be 2.661–22.712 μM for hCA I and 5.372–26.813 μM for hCA II. All derivatives reduced the activities of carbonic anhydrase I and II isoenzymes and were new potential inhibitor molecule candidates. These compounds were found to have minimal effects on AChE and BChE. The antioxidant properties of the target compounds were determined using DPPH and ferric ion‐chelating assays. In particular, compounds 2 k and 2 h had the highest antioxidant effect in the series with IC50 values of 30.11±0.008 μM and 30.21±0.006 μM, respectively. ADMET properties of the compounds found to be effective were evaluated and their interactions with the enzyme were determined by molecular docking. [ABSTRACT FROM AUTHOR]

Fourteen novel Chalcone‐Cu complexes were effectively synthesized in this work. The newly synthesized Chalcone‐Cu complexes were assessed for their effects on human carbonic anhydrase isoenzymes I and II, acetylcholinesterase enzymes, and antioxidant activity. The intricate compounds exhibited Ki values ranging from 41.65–190.42 nM against hCA I, 15.79–259.07 nM against hCA II, and 14.36–175.73 nM against AChE enzymes. These complexes demonstrated potent inhibitory profiles against the specified metabolic enzymes, surpassing the inhibitory effects of acetazolamide (for hCA I and II) and tacrine (for AChE). The antioxidant properties of the compounds were assessed using DPPH and ABTS radical scavenging assays, revealing that the complexes had moderate to high efficacy in neutralizing free radicals. All complexes underwent molecular docking experiments. Compounds 14, 22, and 23 yielded the highest docking scores. Compound 14 demonstrated a docking score of −6.414 kcal/mol against hCAI, whereas compound 23 attained a docking score of −6.697 kcal/mol against hCA II. Compound 22 exhibited the most favorable docking score of −9.645 kcal/mol against AChE. The acquired results have the potential to help towards the development of new drugs containing Cu complex structures for the treatment of prevalent ailments such as glaucoma and Alzheimer's diseases. [ABSTRACT FROM AUTHOR]

Capitella spp. are found in marine and estuarine benthos world‐wide. They are often treated as environmental sentinels because they occur in high densities in areas of anthropogenic disturbance and organic pollution. However, there are many cryptic species around the world, often grouped into the Capitella capitata complex. Historically, these have been differentiated using allozymes and developmental studies. Recently, single‐gene sequencing, especially cytochrome c oxidase subunit I, and phylogenetic methods have been used to characterise populations around the world. We present molecular support and provide descriptions for five new Capitella spp. from the Gulf of Mexico: Capitella gomexa sp. n., Capitella mandingensis sp. n., Capitella tampe sp. n., Capitella maculosa sp. n. and Capitella loucindae sp. n. Two were recently differentiated on the basis of larval development and morphology. Additionally, we present an updated look at global Capitella phylogeny. While no apparent patterns of phylogeography were recovered, we did find support for a single origin of thoracic acicular spines. [ABSTRACT FROM AUTHOR]

Background: As drug-metabolizing enzyme activities are affected by a variety of factors, such as drug-drug interactions, a method to evaluate drug-metabolizing enzyme activities in real time is needed. In this study, we developed a novel SPECT imaging probe for evaluation of hepatic CYP2D activity. Methods: Iodine-123- and 125-labeled 4-iodobenzylmequitazine (123/125I-BMQ) was synthesized with high labeling and purity. CYP isozymes involved in the metabolism of 125I-BMQ in mouse liver microsomes were evaluated, and the utility of 123/125I-was assessed from biological distribution and SPECT imaging evaluation in normal and CYP2D-inhibited mice. Results: In vitro metabolite analysis using mouse liver microsomes showed that 125I-BMQ is specifically metabolized by CYP2D. Biological distribution and SPECT imaging of 123/125I-BMQ in normal mice showed that injection 123/125I-BMQ accumulated early in the liver and was excreted into the gallbladder and intestines. In CYP2D-inhibited mice, accumulation in the liver was increased, but accumulation in the gallbladder and intestines, the excretory organ, was delayed. Since only metabolites of 125I-BMQ are detected in bile, visualization and measuring of the accumulation of metabolites over time in the intestine, where bile is excreted, could predict the amount of metabolites produced in the body and evaluate CYP2D activity, which would be useful in determining the dosage of various drugs metabolized by CYP2D. Conclusion: 123/125I-BMQ is useful as a SPECT imaging probe for comprehensive and direct assessment of hepatic CYP2D activity in a minimally invasive and simple approach. [ABSTRACT FROM AUTHOR]

Polyunsaturated fatty acids (PUFAs) have been extensively studied for their health benefits because they can be oxidized by lipoxygenases to form bioactive oxylipins. In this study, we investigated the impact of double bond placement on the kinetic properties and product profiles of human platelet 12-lipoxygenase (h12-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), and human endothelial 15-lipoxygenase-2 (h15-LOX-2) by using 22-carbon (C22) fatty acid substrates with differing double bond content. With respect to kcat/KM values, the loss of Δ4 and Δ19 led to an 18-fold loss of kinetic activity for h12-LOX, no change in kinetic capability for h15-LOX-1, but a 24-fold loss for h15-LOX-2 for both C22-FAs. With respect to the product profiles, h12-LOX produced mainly 14-oxylipins. For h15-LOX-1, the 14-oxylipin production increased with the loss of either Δ4 and Δ19, however, the 17-oxylipin became the major species upon loss of both Δ4 and Δ19. h15-LOX-2 produced mostly the 17-oxylipin products throughout the fatty acid series. This study also investigated the effects of various 17-oxylipins on platelet activation. The results revealed that both 17(S)-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-DHA (17-HDHA) and 17-hydroxy-4Z,7Z,10Z,13Z,15E-DPAn6 (17-HDPAn6) demonstrated anti-aggregation properties with thrombin or collagen stimulation. 17-hydroxy-7Z,10Z,13Z,15E,19Z-DPAn3 (17-HDPAn3) exhibited agonistic properties, and 17-hydroxy-7Z,10Z,13Z,15E-DTA (17-HDTA) showed biphasic effects, inhibiting collagen-induced aggregation at lower concentrationsbut promoting aggregation at higher concentrations. Both 17-hydroxy-13Z,15E,19Z-DTrA (17-HDTrA), and 17-hydroxy-13Z,15E-DDiA (17-HDDiA) induced platelet aggregation. In summary, the number and placement of the double bonds affect platelet activation, with the general trend being that more double bonds generally inhibit aggregation, while less double bonds promote aggregation. These findings provide insights into the potential role of specific fatty acids and their metabolizing LOX isozymes with respect to cardiovascular health.

Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCβ-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

Objective To explore the effect of moderate fatty liver on cardiac function in orthotopic autologous liver transplantation model rats. Methods Sixty SPF-grade healthy male SD rats were randomly divided into three groups (20 rats in each group). Group A was fed ordinary diet for 6 weeks and underwent laparotomy followed by separation of the blood vessels and ligaments around the liver and abdominal closure; group B was fed ordinary diet for 6 weeks and underwent orthotopic autologous liver transplantation; group C was fed high-fat diet for 6 weeks and underwent orthotopic autologous liver transplantation. The body mass, liver wet mass, liver index, and serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) after 6 weeks of feeding, as well as intraoperative blood pressure, pulse rate, respiratory rate, proportion of arrhythmia occurring in the new liver stage, and serum levels of lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase (α-HBDH), creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and high-sensitivity cardiac troponin (hs-cTn) at 0, 5, 10, and 60 min in the new liver stage were compared. Results The body mass, liver wet mass, liver index, and serum AST and ALT levels, as well as intraoperative systolic blood pressure, diastolic blood pressure, pulse rate, respiratory rate, and proportion of arrhythmia occurring in the new liver stage in group C were significantly higher than those in groups A and B (F=36.40-4 060.54,χ2 =10.91,P0.05). The serum levels of LDH, α-HBDH, CK, CK-MB, and hs-cTn at different time points in the new liver stage were significantly higher in group C than in groups A and B (F=14.11-1 057.08,P

The genome of Trametes versicolor encodes multiple laccase isozymes, the expression of which is responsive to various conditions. Here, we set out to investigate the potential of orange peel extract as an inducer of laccase production in this white-rot fungus, in comparison to the previously identified inducing chemical compound, veratryl alcohol. For four geographically distinct T. versicolor strains, a positive correlation has been observed between their oxidative activity and incubation time in liquid cultures. The addition of 20% orange peel extract or 5 mM veratryl alcohol caused a rapid increase in the oxidative potential of T. versicolor M99 after 24 h, with a more pronounced effect observed for the orange peel extract. To elucidate the underlying molecular mechanisms of the induced laccase activity, a transcriptional gene expression analysis was performed for the seven individual laccase genes in T. versicolor, revealing the upregulation of several laccase genes in response to the addition of each inducer. Notably, the gene encoding TvLac5 demonstrated a substantial upregulation in response to the addition of 20% orange peel extract, likely contributing to the observed increase in its oxidative potential. In conclusion, our results demonstrate that orange peels are a promising agro-industrial side stream for implementation as inducing agents in large-scale laccase production with T. versicolor. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD) cofactor metabolism plays a significant role in cancer development. Tumor cells have an increased demand for NAD and ATP to support rapid growth and proliferation. Limiting the amount of available NAD by targeting critical NAD biosynthesis enzymes has emerged as a promising anticancer therapeutic approach. In mammals, the enzyme nicotinamide/nicotinic acid adenylyltransferase (NMNAT) catalyzes a crucial downstream reaction for all known NAD synthesis routes. Novel nicotinamide/nicotinic acid adenine dinucleotide (NAD/NaAD) analogues 1–4, containing a methyl group at the ribose 2′-C and 3′-C-position of the adenosine moiety, were synthesized as inhibitors of the three isoforms of human NMN-adenylyltransferase, named hNMNAT-1, hNMNAT-2, and hNMNAT-3. An NMR-based conformational analysis suggests that individual NAD-analogues (1–4) have distinct conformational preferences. Biological evaluation of dinucleotides 1–4 as inhibitors of hNMNAT isoforms revealed structural relationships between different conformations (North-anti and South-syn) and enzyme-inhibitory activity. Among the new series of NAD analogues synthesized and tested, the 2′-C-methyl-NAD analogue 1 (Ki = 15 and 21 µM towards NMN and ATP, respectively) emerged as the most potent and selective inhibitor of hNMNAT-2 reported so far. Finally, we rationalized the in vitro bioactivity and selectivity of methylated NAD analogues with in silico studies, helping to lay the groundwork for rational scaffold optimization. [ABSTRACT FROM AUTHOR]

Reproduction is a key feature of all organisms, yet the way in which it is achieved varies greatly across the tree of life. One striking example of this variation is the stick insect genus Bacillus , in which five different reproductive modes have been described: sex, facultative and obligate parthenogenesis, and two highly unusual reproductive modes: hybridogenesis and androgenesis. Under hybridogenesis, the entire genome from the paternal species is eliminated and replaced each generation by mating with the corresponding species. Under androgenesis, an egg is fertilized, but the developing diploid offspring bear two paternal genomes and no maternal genome, as a consequence of unknown mechanisms. Here, we reevaluate the previous descriptions of Bacillus lineages and the proposed F1 hybrid ancestries of the hybridogenetic and obligately parthenogenetic lineages (based on allozymes and karyotypes) from Sicily, where all these reproductive modes are found. We generate a chromosome-level genome assembly for a facultative parthenogenetic species (B. rossius) and combine extensive field sampling with RADseq and mtDNA data. We identify and genetically corroborate all previously described species and confirm the ancestry of hybrid lineages. All hybrid lineages have fully retained their F1 hybrid constitution throughout the genome, indicating that the elimination of the paternal genome in hybridogens is always complete and that obligate parthenogenesis in Bacillus hybrid species is not associated with an erosion of heterozygosity as known in other hybrid asexuals. Our results provide a stepping stone toward understanding the transitions between reproductive modes and the proximate mechanisms of genome elimination. [ABSTRACT FROM AUTHOR]

A pathway frequently altered in cancer is glutaminolysis, whereby glutaminase (GA) catalyzes the main step as follows: the deamidation of glutamine to form glutamate and ammonium. There are two types of GA isozymes, named GLS and GLS2, which differ considerably in their expression patterns and can even perform opposing roles in cancer. GLS correlates with tumor growth and proliferation, while GLS2 can function as a context-dependent tumor suppressor. However, both isoenzymes have been described as essential molecules handling oxidant stress because of their involvement in glutathione production. We reviewed the literature to highlight the critical roles of GLS and GLS2 in restraining ROS and regulating both cellular signaling and metabolic stress due to their function as indirect antioxidant enzymes, as well as by modulating both reductive carboxylation and ferroptosis. Blocking GA activity appears to be a potential strategy in the dual activation of ferroptosis and inhibition of cancer cell growth in a ROS-mediated mechanism. [ABSTRACT FROM AUTHOR]

Aim: Despite numerous reports of various neurological symptoms observed in Coronavirus disease-2019 (COVID-19) disease, the role of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in the neuropathogenesis and tropism of the central nervous system (CNS) has yet to be fully elucidated. In this study, we evaluated the development of CNS damage related to the severity of COVID-19 pneumonia with creatine kinase BB (CK-BB) serum levels. Material and Methods: The study included 55 patients hospitalized in the intensive care unit diagnosed with severe COVID-19 pneumonia, 79 in the inpatient service diagnosed with milder COVID-19 pneumonia, and 39 healthy volunteers. CK-BB levels were measured using a quantitative sandwich enzyme immunoassay technique with an Enzyme-Linked Immunosorbent Assay kit. Detection of SARS-CoV-2 was performed by real time-polymerase chain reaction from the respiratory tract (nasopharyngeal swab) according to current guidelines. Results: While markers that were shown to predict disease severity were higher in patients with severe COVID-19 pneumonia compared with patients with milder pneumonia and the control group, serum levels of the neurological damage marker CK-BB were found to be similar between the groups [respectively 6.84 (5.05-16.2), 7.48 (4.7-22.5), 6.7 (3.8-16.2), p>0.005]. In addition, there was no difference in serum CK-BB levels between patients who developed neurological symptoms and those who did not [respectively 6.78 (5.07-17.21), 7.43 (4.7-22.5), p>0.005]. Conclusion: In COVID-19 pneumonia, serum CK-BB levels do not increase with disease severity and the development of neurological symptoms. [ABSTRACT FROM AUTHOR]

Long regarded to represent an undescribed species related to Sesarma reticulatum , warm-temperate populations of a common marsh crab from the Gulf of Mexico are herewith formally named Sesarma schubarti sp. nov. Records of S. reticulatum s. s. are restricted to the U.S. Atlantic seaboard from the Woods Hole region of Massachusetts to the Indian River region on the east coast of Florida. A diverse body of supportive evidence to underpin this separation has accrued over three decades, including previous observations based on physiology, ecology, reproduction, blood hemocyanin proteins, isozymes, and mtDNA sequences. Comparative morphological studies provide only minor structural characters to distinguish the new species from its Atlantic seaboard counterpart, including differences in the shape of the mature male first gonopod tip and positioning of a transverse crest on the male first pleonite. However, in fresh specimens colouration also differs, often strikingly so. Body surfaces that are variously purplish in mature specimens of S. reticulatum s. s., yielding the common name "purple marsh crab", are instead more commonly reddish to orange in S. schubarti sp. nov. Our analysis of new 16S and COI gene sequences, along with existing genetic data, clarifies relationships of the new species to both S. reticulatum s. s. and S. curacaoense. The new species is formally named in honor of the late Christoph D. Schubart, a cherished colleague who contributed extensively to knowledge of sesarmid and other brachyuran crabs over his unfortunately abbreviated career. [ABSTRACT FROM AUTHOR]

The tumor microenvironment is affected by reactive oxygen species and has been suggested to have an important role in ovarian cancer (OC) tumorigenesis. The role of glutathione transferases (GSTs) in the maintenance of redox balance is considered as an important contributing factor in cancer, including OC. Furthermore, GSTs are mostly encoded by highly polymorphic genes, which further highlights their potential role in OC, known to originate from accumulated genetic changes. Since the potential relevance of genetic variations in omega-class GSTs (GSTO1 and GSTO2), with somewhat different activities such as thioltransferase and dehydroascorbate reductase activity, has not been clarified as yet in terms of susceptibility to OC, we aimed to investigate whether the presence of different GSTO1 and GSTO2 genetic variants, individually or combined, might represent determinants of risk for OC development. Genotyping was performed in 110 OC patients and 129 matched controls using a PCR-based assay for genotyping single nucleotide polymorphisms. The results of our study show that homozygous carriers of the GSTO2 variant G allele are at an increased risk of OC development in comparison to the carriers of the referent genotype (OR1 = 2.16, 95% CI: 0.88–5.26, p = 0.08; OR2 = 2.49, 95% CI: 0.93–6.61, p = 0.06). Furthermore, individuals with GST omega haplotype H2, meaning the concomitant presence of the GSTO1*A and GSTO2*G alleles, are more susceptible to OC development, while carriers of the H4 (*A*A) haplotype exhibited lower risk of OC when crude and adjusted haplotype analysis was performed (OR1 = 0.29; 95% CI: 0.12–0.70; p = 0.007 and OR2 = 0.27; 95% CI: 0.11–0.67; p = 0.0054). Overall, our results suggest that GSTO locus variants may confer OC risk. [ABSTRACT FROM AUTHOR]

We established myocardial injury models in vivo and in vitro to investigate the cardioprotective effect of gomisin D obtained from Schisandra chinensis. Gomisin D significantly inhibited isoproterenol-induced apoptosis and hypertrophy in H9C2 cells. Gomisin D decreased serum BNP, ANP, CK-MB, cTn-T levels and histopathological alterations, and inhibited myocardial hypertrophy in mice. In mechanisms research, gomisin D reversed ISO-induced accumulation of intracellular ROS and Ca2+. Gomisin D further improved mitochondrial energy metabolism disorders by regulating the TCA cycle. These results demonstrated that gomisin D had a significant effect on isoproterenol-induced myocardial injury by inhibiting oxidative stress, calcium overload and improving mitochondrial energy metabolism. [ABSTRACT FROM AUTHOR]

The advancement of graph neural networks (GNNs) has made it possible to accurately predict metabolic sites. Despite the combination of GNNs with XGBOOST showing impressive performance, this technology has not yet been applied in the realm of metabolic site prediction. Previous metabolic site prediction tools focused on bonds and atoms, regardless of the overall molecular skeleton. This study introduces a novel tool, named D-CyPre, that amalgamates atom, bond, and molecular skeleton information via two directed message-passing neural networks (D-MPNN) to predict the metabolic sites of the nine cytochrome P450 enzymes using XGBOOST. In D-CyPre Precision Mode, the model produces fewer, but more accurate results (Jaccard score: 0.497, F1: 0.660, and precision: 0.737 in the test set). In D-CyPre Recall Mode, the model produces less accurate, but more comprehensive results (Jaccard score: 0.506, F1: 0.669, and recall: 0.720 in the test set). In the test set of 68 reactants, D-CyPre outperformed BioTransformer on all isoenzymes and CyProduct on most isoenzymes (5/9). For the subtypes where D-CyPre outperformed CyProducts, the Jaccard score and F1 scores increased by 24% and 16% in Precision Mode (4/9) and 19% and 12% in Recall Mode (5/9), respectively, relative to the second-best CyProduct. Overall, D-CyPre provides more accurate prediction results for human CYP450 enzyme metabolic sites. [ABSTRACT FROM AUTHOR]

(3aR,4S,7R,7aS)‐2‐Alkyl/aryl‐3a,4,7,7a‐tetrahydro‐1H‐4,7‐epoxyisoindole‐1,3(2H)‐diones, which are norcantharimide derivatives, were synthesized and their effects on carbonic anhydrase I (hCA I) and II (hCA II), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibitory activity were investigated. For enzyme activity studies, hCA I and II isoenzymes purified from human erythrocytes and the commercially available enzymes AChE and BChE, which are both markers and significantly affect the known symptoms of Alzheimer's disease, were used. The two derivatives exerted efficient inhibition with IC50=4.530 nM (Ki=4.483) and 4.426 nM (Ki=4.696) against hCA I and with IC50=3.825 nM (Ki=3.854) and 3.457 nM (Ki=3.292) against hCA II, respectively. The another two derivatives exerted considerable inhibition with IC50=0.526 nM (Ki=0.224) and 0.575 nM (Ki=0.292) against AChE and with IC50=0.135 nM (Ki=0.057) and IC50=0.180 nM (Ki=0.070) against BChE, respectively. The compounds showed activity at the nanomolar level. These remarkable inhibition results were compared with those of standard inhibitors (acetazolamide for hCA I and II and tacrine for AChE and BChE) of each enzyme, reported, and graphed. In addition, molecular docking studies were carried out by in silico methods and the structure–activity relationship was discussed. The poses of compound 4 c are presented along with the ligand–receptor interaction against all metabolic enzymes. [ABSTRACT FROM AUTHOR]

Superoxide dismutase (SOD) protects plants from abiotic stress-induced reactive oxygen species (ROS) damage. Here, the effects of cadmium (Cd) exposure on ROS accumulation and SOD isozymes, as well as the identification of significant SOD isozyme genes, were investigated under different Cd stress treatments to Zhe-Maidong (Ophiopogon japonicus). The exposure to Cd stress resulted in a notable elevation in the SOD activity in roots. Cu/ZnSODa and Cu/ZnSODb were the most critical SOD isozymes in response to Cd stress, as indicated by the detection results for SOD isozymes. A total of 22 OjSOD genes were identified and classified into three subgroups, including 10 OjCu/ZnSODs, 6 OjMnSODs, and 6 OjFeSODs, based on the analysis of conserved motif and phylogenetic tree. Cu/ZnSOD-15, Cu/ZnSOD-18, Cu/ZnSOD-20, and Cu/ZnSOD-22 were the main genes that control the increase in SOD activity under Cd stress, as revealed via quantitative PCR and transcriptome analysis. Additionally, under various heavy metal stress (Cu2+, Fe2+, Zn2+, Mn2+), Cu/ZnSOD-15, Cu/ZnSOD-18, and Cu/ ZnSOD-22 gene expression were significantly upregulated, indicating that these three genes play a critical part in resisting heavy metal stress. The molecular docking experiments performed on the interaction between oxygen ion (O2•-) and OjSOD protein have revealed that the critical amino acid residues involved in the binding of Cu/ZnSOD-22 to the substrate were Pro135, Ile136, Ile140, and Arg144. Our findings provide a solid foundation for additional functional investigations on the OjSOD genes, as well as suggestions for improving genetic breeding and agricultural management strategies to increase Cd resistance in O. japonicus. [ABSTRACT FROM AUTHOR]

The first and committed step in proline synthesis from glutamate is catalyzed by δ1‐pyrroline‐5‐carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress‐induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed KM values in the micro‐ to millimolar range, and its activity was inhibited by NADP+, ADP and proline, and by glutamine and arginine at high levels. Mg2+ ions were required for activity, which was further stimulated by K+ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cations and pyridine dinucleotides. Summary statement: In Arabidopsis thaliana, the two forms of the enzyme leading to proline synthesis, P5C synthetase, show different functional features and posttranslational regulation, suggesting activity modulation in vivo by an array of factors including related amino acids, cations and pyridine dinucleotides. [ABSTRACT FROM AUTHOR]

We aimed to test the relationship between serum lactate dehydrogenase (LDH) and its isoenzymes and treatment outcomes during hospitalization for acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Thirty-eight AECOPD patients were recruited from a tertiary hospital from December 2017 to June 2018. Serum LDH and LDH isoenzymes were measured on venous blood collected at admission. Treatment outcomes included duration of hospital stay, initiation of mechanical or non-invasive ventilation (NIV), initiation of antipseudomonal antibiotics, change in empirical antibiotic treatment, need for intravenous corticosteroids or methylxanthines, and percentage of change in C-reactive protein from admission to the third day. Multivariate linear and binary logistic regression analyses were used to test the study’s objectives. We found that, after adjusting for age, gender, comorbidities, COPD severity, level of hypoxemia, and inflammation markers, each 10 U/L increase in serum LDH was associated with prolongation of the hospital stay by 0.25 (0.03, 0.46) days, 42% higher odds [odds ratio (OR) 1.42 (1.00, 2.03)] for need of NIV, and 25% higher odds [OR 1.25 (1.04, 1.49)] for initiation of antipseudomonal treatment. LDH1 and LDH2 were the LDH isoenzymes that mainly drove these relationships. LDH release in the context of AECOPD could originate from lung, muscle, or heart tissue damage due to airway inflammation, respiratory muscle recruitment, and myocardial stress. Myocardial injury and aerobic adaptation in respiratory muscles may explain the predominance of LDH1 and LDH2 isoenzymes in these associations. [ABSTRACT FROM AUTHOR]

Lipid metabolism is a complex process crucial for energy production resulting in high levels of acyl-coenzyme A (acyl-CoA) molecules in the cell. Acyl-CoAs have also been implicated in inflammation, which could be possibly linked to lipoxygenase (LOX) biochemistry by the observation that an acyl-CoA was bound to human platelet 12-lipoxygenase via cryo-EM. Given that LOX isozymes play a pivotal role in inflammation, a more thorough investigation of the inhibitory effects of acyl-CoAs on lipoxygenase isozymes was judged to be warranted. Subsequently, it was determined that C18 acyl-CoA derivatives were the most potent against h12-LOX, human reticulocyte 15-LOX-1 (h15-LOX-1), and human endothelial 15-LOX-2 (h15-LOX-2), while C16 acyl-CoAs were more potent against human 5-LOX. Specifically, oleoyl-CoA (18:1) was most potent against h12-LOX (IC50 = 32 μM) and h15-LOX-2 (IC50 = 0.62 μM), stearoyl-CoA against h15-LOX-1 (IC50 = 4.2 μM), and palmitoleoyl-CoA against h5-LOX (IC50 = 2.0 μM). The inhibition of h15-LOX-2 by oleoyl-CoA was further determined to be allosteric inhibition with a Ki of 82 +/- 70 nM, an α of 3.2 +/- 1, a β of 0.30 +/- 0.07, and a β/α = 0.09. Interestingly, linoleoyl-CoA (18:2) was a weak inhibitor against h5-LOX, h12-LOX, and h15-LOX-1 but a rapid substrate for h15-LOX-1, with comparable kinetic rates to free linoleic acid (kcat = 7.5 +/- 0.4 s-1, kcat/KM = 0.62 +/- 0.1 µM-1s-1). Additionally, it was determined that methylated fatty acids were not substrates but rather weak inhibitors. These findings imply a greater role for acyl-CoAs in the regulation of LOX activity in the cell, either through inhibition of novel oxylipin species or as a novel source of oxylipin-CoAs.

Abstract Hypostomus is distributed by Central and South America basins, with diverse species with taxonomic conflicts. This way, the integration of auxiliary techniques contributes to understanding the systematics and phylogeny of the group. Thus, this study aimed to investigate the Hypostomus cochliodon and H. boulengeri from the Onça stream (Paraguai River basin) by allozyme and cytogenetic techniques. Hypostomus boulengeri showed a diploid number of 68 chromosomes (14m+14sm+18st+22a), multiple NOR revealed by Ag-NOR and 18S rDNA FISH, a polymorphism of heterochromatin in acrocentrics and the presence of B microchromosome. Hypostomus cochliodon showed a diploid number of 64 chromosomes (16m+26sm+14st+8a); despite the single NOR, some individuals showed NOR in both telomeres detected by Ag-NOR and 18S rDNA FISH. Isozyme identified two diagnostic loci (Idh-A and Gdh-A) between the two species and multiple loci with unique alleles in H. boulengeri. The genetic variability indicated by the mean heterozygosity (He) was 0.2461 and 0.0309 in H. boulengeri and H. cochliodon,respectively.Thus, this study reports the first cytogenetic data for H. boulengeri and the first isozymatic data for H. boulengeri and H. cochliodon. The two species presented evident cytogenetic and isoenzymatic differences with the obtaining of exclusive genetic markers providing support for future evolutionary studies in the group.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of analgesics that inhibit the activity of cyclooxygenase isoenzymes, which drive tissue inflammation pathways. Caution should be exercised when taking these drugs during pregnancy as they increase the risk of developmental defects. Due to the high rates of NSAID use by individuals, possibilities for in utero exposure to NSAIDs are high, and it is vital that we define the potential risks these drugs pose during embryonic development. In this review, we characterize the identified roles of the cyclooxygenase signaling pathway components throughout pregnancy and discuss the effects of cyclooxygenase pathway perturbation on developmental outcomes.

The Cytochrome P450 (CYP) enzymes metabolize a variety of drugs, which may potentially lead to toxicity or reduced efficacy when drugs are co-administered. These drug-drug interactions are often manifested by CYP3A4, the most prevalent of all CYP isozymes. We carried out multiple MD simulations employing CAVER to quantify the channels, and Hidden Markov Models (HMM) to characterize the behavior of the gating residues. We discuss channel properties, bottleneck residues with respect to their likelihood to deem the respective channel ingress or egress, gating residues regarding their open or closed states, and channel location relative to the membrane. Channels do not display coordinated motion and randomly transition between different conformations. Gateway residues also behave in a random fashion. Our findings shed light on the equilibrium behavior of the gating residues and channels in the apo state. [ABSTRACT FROM AUTHOR]

Laccases are multicopper oxidases able to oxidize several phenolic compounds and find application in numerous industrial applications. Among laccase producers, white-rot fungi represent a valuable source of multiple isoforms and isoenzymes of these multicopper oxidases. Here we describe the identification, biochemical characterization, and application of laccase 2 from Trametes polyzona (TP-Lac2), a basidiomycete fungus emerged among others that have been screened by plate assay. This enzyme has an optimal temperature of 50 °C and in acidic conditions it is able to oxidize both phenolic and non-phenolic compounds. The ability of TP-Lac2 to decolorize textile dyes was tested in the presence of natural and synthetic mediators at 30 °C and 50 °C. Our results indicate that TP-Lac2 most efficiently decolorizes (decolorization rate > 75%) malachite green oxalate, orange G, amido black10B and bromocresol purple in the presence of acetosyringone and 2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonate)—ABTS. Overall, the laccase mediator system consisting of TP-Lac2 and the natural mediator acetosyringone has potential as an environmentally friendly alternative for wastewater treatment in the textile industry. Key points: A novel laccase from T. polyzona was induced by the addition of veratryl alcohol TP-Lac2 was heterologous expressed in S. cerevisiae and biochemically characterized TP-Lac2 efficiently decolorizes textile dyes using acetosyringone as mediator [ABSTRACT FROM AUTHOR]

Backgroud: Endometriosis remains a diagnostic challenge, both clinically and economically, affecting 6% to 15% of women of child-bearing potential. We have attempted to determine whether testing serum concentrations and activity of arginase isoenzymes could be useful for the non-invasive diagnosis of endometriosis. Methods: This study involved 180 women (105 endometriosis subjects—study group B; 22 subjects with other benign gynaecological conditions—control group 1—K1, both undergoing surgery; and 53 healthy subjects without features of endometriosis—control group 2—K2). Results: Preoperative and postoperative arginase-1 (Arg-1) concentrations were significantly higher in patients, as compared with the control groups K1 (p < 0.0001 and p = 0.0005, respectively) and K2 (both p < 0.0001). Similarly, arginase activity was significantly higher in patients than in the control group K1 before surgery and higher than in both control groups after surgery. No significant differences in either Arg-1 concentrations or arginase activity were noted between the operated control group K1 and the non-operated control group K2. A significant postoperative decrease in Arg-1 concentration was observed within both patient (p < 0.0001) and control group K1 (p = 0.0043). Diagnostic performance was assessed using the receiver operating characteristic (ROC) method. The threshold for differentiation between endometriosis patients and healthy non-operated controls was 42.3 ng/mL, with a sensitivity of 90% and specificity of 81%. For differentiation of patients and operated controls with benign gynaecological conditions, the threshold was 78.4 ng/mL, with a sensitivity of 61% and specificity of 95%. Conclusions: We, therefore, conclude that Arg-1 serum concentrations and arginase activity could be considered potential biomarkers for endometriosis but require further studies on larger cohorts of patients. [ABSTRACT FROM AUTHOR]

Infection biology and defence responses of watermelon attacked by Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae) were studied in two genotypes, accessions PI189225 (moderately resistant) and 232-0125/B (susceptible). On intact leaf surfaces, spores started to germinate 14 h after inoculation (hai) with one to three germ tubes, which subsequently developed and formed appressoria. Invasion of the host tissue started at 20 hai by direct penetration from appressoria or occasionally indirectly through stomata. In the susceptible accession, a significantly higher number of direct penetrations were observed than in the moderately resistant. After invasion, hyphal colonisation was restricted in the intercellular spaces in the moderately resistant accession, whereas they developed extensively, causing tissue decay, in the susceptible accession. Macroscopic symptoms were seen in leaves of the moderately resistant accession as small and dry lesions, whereas big, water-soaked lesions developed on the susceptible accession within 48 hai. Investigations of the defence responses of the two accessions showed accumulation of H2O2 at penetration sites beneath appressoria in the moderately resistant, but to a lesser extent in the susceptible accession. Such H2O2 accumulation correlated with a reduction in penetration frequency and a lower level of hyphal growth after infection in the moderately resistant accession. There was a rapid and early increase in total peroxidase as well as β-1,3-glucanase activity in the moderately resistant compared to the susceptible accession. These results indicate that fungal penetration and development in watermelon are inhibited by a consorted action of different responses including accumulation of H2O2, peroxidase and β-1,3-glucanase. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the demographic, clinical, and laboratory characteristics of children with benign transient hyperphosphatasemia (BTH) and to increase awareness of BTH. Children with BTH followed up in a tertiary pediatric endocrinology outpatient clinic were included in this retrospective study. Children who were aged under 5 years, who had no evidence of liver, kidney, or bone disease, who had serum total alkaline phosphatase (ALP) levels above 1000 IU/L, and whose ALP values resolved within 4 months, were included in the study. The study included 10 infants and children (4 females) with BTH who were referred to our clinic during 1 year for high ALP levels. The mean age was 2.4±0.5 years. The mean weight SDS was -0.24±1.27 and the mean height SDS was -0.51±0.67. The mean ALP level was 2.395±934 IU/L. The mean normalization time for serum ALP was 2.4±1.1 months. Benign transient hyperphosphatasemia is an entity that occurs in healthy infants and children without evidence of liver or bone disease and resolves without intervention. [ABSTRACT FROM AUTHOR]

Phosphatidic acid (PA) is the precursor of most phospholipids like phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In bacteria, its biosynthesis begins with the acylation of glycerol‐3‐phosphate to lysophosphatidic acid (LPA), which is further acylated to PA by the PlsC enzyme. Some bacteria, like the plant pathogen Xanthomonas campestris, use a similar pathway to acylate lysophosphatidylcholine to phosphatidylcholine (PC). Previous studies assigned two acyltransferases to PC formation. Here, we set out to study their activity and found a second much more prominent function of these enzymes in LPA to PA conversion. This PlsC‐like activity was supported by the functional complementation of a temperature‐sensitive plsC‐deficient Escherichia coli strain. Biocomputational analysis revealed two further PlsC homologs in X. campestris. The cellular levels of the four PlsC‐like proteins varied with respect to growth phase and growth temperature. To address the question whether these enzymes have redundant or specific functions, we purified two recombinant, detergent‐solubilized enzymes in their active form, which enabled the first direct biochemical comparison of PlsC isoenzymes from the same organism. Overlapping but not identical acyl acceptor and acyl donor preferences suggest redundant and specialized functions of the X. campestris PlsC enzymes. The altered fatty acid composition in plsC mutant strains further supports the functional differentiation of these enzymes. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase (CA, EC 4.2.1.1) is an enzyme that catalyzes the reversible reaction of carbon dioxide to bicarbonate and a proton under physiological conditions. Pharmaceutical research has gained importance since the design of novel compounds that inhibit CA I–II isoenzymes has a promising approach for pharmacological intervention in many diseases. Triazole derivatives have attracted attention due to their chemotherapeutic, antifungal, antiviral, antibiotic, analgesic, and antifungal activities. Therefore, in this study, the effect of 1,2,4‐triazole and thiazole ring‐containing compounds on human carbonic anhydrase I (hCA I) and II (hCA II) isoenzymes were investigated in vitro. For this purpose, hCA I and hCA II isoenzymes were purified by Sepharose‐4B affinity column chromatography. Estimation of inhibition mechanism and drug‐likeness characteristics of compounds were also determined using molecular docking simulation. The inhibitory effects of ten compounds were investigated. Activity vs. concentration graphs were prepared for each compound and IC50 values or AC50 were calculated from these graphs. It was revealed that some of the compounds exhibited selective inhibition on carbonic anhydrase isoenzymes. The studied compounds are considered to be drug candidates. [ABSTRACT FROM AUTHOR]

Histone deacylases are erasers of Nε-acyl-lysine post-translational modifications and have been targeted for decades for the treatment of cancer, neurodegeneration and other disorders. Due to their relatively promiscuous activity on peptide substrates in vitro, it has been challenging to determine the individual targets and substrate identification mechanisms of each isozyme, and they have been considered redundant regulators. In recent years, biochemical and biophysical studies have incorporated the use of reconstituted nucleosomes, which has revealed a diverse and complex arsenal of recognition mechanisms by which histone deacylases may differentiate themselves in vivo. In this review, we first present the peptide-based tools that have helped characterize histone deacylases in vitro to date, and we discuss the new insights that nucleosome tools are providing into their recognition of histone substrates within chromatin. Then, we summarize the powerful semi-synthetic approaches that are moving forward the study of chromatin-associated factors, both in vitro by detailed single-molecule mechanistic studies, and in cells by live chromatin modification. We finally offer our perspective on how these new techniques would advance the study of histone deacylases. We envision that such studies will help elucidate the role of individual isozymes in disease and provide a platform for the development of the next generation of therapeutics. [ABSTRACT FROM AUTHOR]

The secondary sulfonamide derivatives containing benzothiazole scaffold (1–10) were synthesized to determine their inhibition properties on two physiologically essential human carbonic anhydrases isoforms (hCAs, EC, 4.2.1.1), hCA I, and hCA II. The inhibitory effects of the compounds on hCA I and hCA II isoenzymes were investigated by comparing their IC50 and Ki values. The Ki values of compounds (1–10) against hCA I and hCA II are in the range of 0.052 ± 0.022–0.971 ± 0.280 and 0.025 ± 0.010–0.682 ± 0.335, respectively. Some of these inhibited the enzyme more effectively than the standard drug, acetazolamide. In particular, compounds 5 and 4 were found to be most effective on hCA I and hCA II. [ABSTRACT FROM AUTHOR]

Railroadworms luciferases emit the widest range of bioluminescence colors among beetles, ranging from green to red, being model enzymes to investigate the structure and bioluminescence colors relationships. Only three active railroadworms luciferases from the larval stage have been cloned and investigated: the Phrixothrix hirtus head lanterns red-emitting luciferase (PhRE); the Phrixothrix vivianii lateral lanterns green emitting luciferases (PvGR) and the Phengodes sp. dorsal lanterns yellow-green emitting luciferase (Ph). No active luciferase emitting in the yellow–orange region, however, has been cloned yet. Here we report the cloning and characterization of the orange emitting luciferase from the adult males of a rare Brazilian Cerrado railroadworm, Euryopa clarindae, and the transcriptional identification of two isozymes from the Amazon forest Mastinomorphus sp. railroadworm. The luciferase of E. clarindae has 548 residues, emits orange bioluminescence (600 nm), and displays intermediate kinetic values [KM(luciferin) = 50 µM, KM(ATP) ~ 170 µM] between those reported for green-emitting lateral lanterns and red emitting head lanterns luciferases. It displays 74–78% identity with the lateral lanterns luciferases of other railroadworms and 70% with the head lantern PhRE luciferase, and 96% with the larval Mastinomorphus sp. Mast-1, suggesting that this larva could be from the Euryopa genus. The phylogenetic analysis and kinetic/functional properties, place this orange-emitting enzyme as an intermediate form between the green-emitting lateral lanterns and red-emitting head lanterns luciferases. Major structural differences that could be associated with bioluminescence color determination are a relatively larger cavity size, and substitutions in the loops 223–235 and 311–316, especially N/C/T311, and their interactions which may help to close the bottom of LBS. [ABSTRACT FROM AUTHOR]

The effect of Kamchatka crab hepatopancreas containing three collagenolytic isoenzymes Collagenase KK and proteinases of Streptomyces lavendulae on metabolic activity and cell death were carried out on in vitro models. It was shown that changes in the protein structure under the influence of Collagenase KK occurred earlier than under the effect of bacterial proteinases. At the same time, activity of Collagenase KK was significantly higher than that of bacterial proteinases (p<0.01). Both preparations had a pronounced time- and dose-dependent effects on metabolic activity of cells. Collagenase KK had low cytotoxic effect, and cells mainly died by apoptosis. Thus, hepatopancreas collagenase has a high activity and proapoptotic effect on cells and can be used in low concentrations for enzymatic disaggregation of tissues. [ABSTRACT FROM AUTHOR]

d‐Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l‐serine by serine racemase and degraded by d‐amino acid oxidase. In the present study, we identified a new d‐serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l‐serine and tetrahydrofolate (THF) to glycine and 5,10‐methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d‐serine dehydratase activity, which degrades d‐serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d‐serine dehydration and did not exhibit dehydratase activity toward l‐serine. Furthermore, SHMT did not use d‐serine as a substrate in the canonical hydroxymethyltransferase reaction. The d‐serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d‐serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat/Km) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d‐serine dehydratase activity of SHMT was physiologically important because d‐serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild‐type strain. Collectively, these results suggest that SHMT is involved not only in l‐ but also in d‐serine metabolism through the degradation of d‐serine. [ABSTRACT FROM AUTHOR]

• This work provides molecular level explanation for existence of isoenzymes in collagen prolyl 4-hydroxylation. • X position amino acid in collagen -X-Pro-Gly-triplets is the major determinant for hydroxylation. • Collagen prolyl 4-hydroxylation has sequence specificity. • Deletion of P4ha2 leads to collagen underhydroxylation at specific EPG and DPG sites and leads to reduced collagen melting temperature, reduced collagen fibril diameter and affects basement membranes. • Collagen prolyl 4-hydroxylase mRNA expression is not tightly coordinated with collagen expression. Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures. [ABSTRACT FROM AUTHOR]

Para-hydroxybenzoate hydroxylase (PHBH) is a group A flavoprotein monooxygenase that hydroxylates p-hydroxybenzoate to protocatechuate (PCA). Despite intensive studies of Pseudomonas aeruginosa p-hydroxybenzoate hydroxylase (PaPobA), the catalytic reactions of extremely diverse putative PHBH isozymes remain unresolved. We analyzed the phylogenetic relationships of known and predicted PHBHs and identified eight divergent clades. Clade F contains a protein that lacks the critical amino acid residues required for PaPobA to generate PHBH activity. Among proteins in this clade, Xylophilus ampelinus PobA (XaPobA) preferred PCA as a substrate and is the first known natural PCA 5-hydroxylase (PCAH). Crystal structures and kinetic properties revealed similar mechanisms of substrate carboxy group recognition between XaPobA and PaPobA. The unique Ile75, Met72, Val199, Trp201, and Phe385 residues of XaPobA form the bottom of a hydrophobic cavity with a shape that complements the 3-and 4-hydroxy groups of PCA and its binding site configuration. An interaction between the d-sulfur atom of Met210 and the aromatic ring of PCA is likely to stabilize XaPobA-PCA complexes. The 4-hydroxy group of PCA forms a hydrogen bond with the main chain carbonyl of Thr294. These modes of binding constitute a novel substrate recognition mechanism that PaPobA lacks. This mechanism characterizes XaPobA and sheds light on the diversity of catalytic mechanisms of PobA-type PHBHs and group A flavoprotein monooxygenases. [ABSTRACT FROM AUTHOR]

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Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

Laccases are important and valuable enzymes with a great potential for biotechnological applications. In this study, two novel laccases, LacHU1 and LacHU2, from Alternaria sp. HU have been purified and characterized. The molecular mass of each isoenzyme was ~66 kDa. LacHU1 laccases was yellow and had no typical blue oxidase spectra and LacHU2 had a blue color and characteristic absorption spectra. The catalytic efficiency of LacHU1 for most substrates was higher than that of LacHU2 laccase. Both isoenzymes effectively oxidize flavonoids. Alternaria sp. laccases were successfully immobilized on magnetic nanoparticles. The thermostability of immobilized laccases increased and optimal pH shifted to more alkaline compared to the free laccases. Potential applications of laccases from Alternaria sp. HU are in the oxidation of flavonoids in cotton or in water treatment processes.