Arsenic Induced Inhibition of δ-aminolevulinate DehydrataseActivity in Rat Blood and its Response To Meso2,3-dimercaptosuccinic Acid andMonoisoamyl DMSA

Objective The objective of this study was to investigate arsenic induced changes in blood 8-aminolevulinic acid dehydratase (ALAD) after in vitro and in vivo exposure to this element and its response to co-administration of meso 2,3-dimercaptosuccinic acid (DMSA) and monoisoamyl DMSA (MiADMSA) either individually or in combination. Methods Rat whole blood was exposed to varying concentrations (0.1, 0.2 and 0.5 mmol/L) of arsenic (III) or arsenic (V), to assess their effects on blood ALAD activity. Varying concentrations of MiADMSA and DMSA (0.1,0.5 and 1.0 mmol/L) were also tried in combination to determine its ability to mask the effect of arsenic induced (0.5 mmol/L) inhibition of blood ALAD in vitro. In vitro and in vivo experiments were also conducted to determine the effects of DMSA and MiADMSA either individually or in combination with arsenic, on blood ALAD activity and blood arsenic concentration. Results In vitro experiments showed significant inhibition of the enzyme activity when 0.1-0.5 mmol/L of arsenic (III and V) was used. Treatment with MiADMSA increased ALAD activity when blood was incubated at the concentration of 0.1 mmol/L arsenic (III) and 0.1 mmol/L MiADMSA. No effect of 0.1 mmol/L MiADMSA on ALAD activity was noticed when the arsenic concentration was increased to 0.2 and 0.5 mmol/L. Similarly, MiADMSA at a lower concentration (0.1 mmol/L) was partially effective in the turnover of ALAD activity against 0.5 mmol/L arsenic (III), but at two higher concentrations (0.5 and 1.0 mmol/L) a complete restoration of ALAD activity was observed. DMSA at all the three concentrations (0.1,0.5 and 1.0 mmol/L) was effective in restoring ALAD activity to the normal value. Conclusions The results thus suggest that arsenic has a distinct effect on ALAD activity. Another important toxicological finding of the present study, based on in vivo experiments further suggests that combined administration of DMSA and MiADMSA could be more beneficial for reducing blood ALAD inhibition and blood arsenic concentration than the individual treatment.

Mechanistic Investigations into the Catalytic Mode of a Dehydratase Complex Involved in the Biosynthesis of Lantibiotic Cacaoidin

Dehydration of serine/threonine residues necessitates the activity of a dehydratase enzyme(domain)during the biosynthesis of RiPP.Recently,it was reported that dehydration in the thioviridamide pathway relies on a distinct dehydratase complex that showcases the activities of a phosphotransferase TvaC for serine/threonine phosphorylation and a lyase TvaD for subsequent phosphate elimination.Herein,we report that dehydration reactions in the pathway of lantibiotic cacaoidin involves a similar dehydratase complex,CaoK/CaoY.Remarkably,this dehydratase complex exhibits flexible enzymatic activity and tolerates significant variations in its substrate peptide sequence.By binding with the leader peptide(LP)sequence of precursor peptide CaoA,the dehydration reactions proceed in a directional manner from the C-terminus of the core peptide(CP)to the N-terminus,and C-terminally truncated variants of CP are acceptable.We show that fusing CaoK to CaoY in a 1:1 molar ratio enables the resulting enzyme CaoYK to exert enhanced dehydration activity.CaoK binds with the LP to improve its own solubility and to ensure the phosphate transfer activity,while CaoY functions in a manner independently of LP.This work advances our understanding of the dehydration process during cacaoidin formation,and provides useful enzymes and methods for the studies of the rapidly emerging RiPPs.

Characterization of Three Homoeologous cDNAs Encoding Chloroplast-targeted Aminolevulinic Acid Dehydratase in Common Wheat

In the tetrapyrrole biosynthetic pathway of higher plants, 5-aminolevulinic acid （ALA） is metabolized by ALA dehydratase （ALAD）. Here, we isolated ALAD1 cDNA from common wheat （Triticum aestivum L.） and its diploid progenitors, and produced transgenic tobacco plants expressing the wheat ALAD1 gene. The ALAD1 genes were highly conserved among wheat relatives, and three homoeologous loci of wheat ALAD1 （TaALAD1） were equally transcribed in common wheat. A transient expression assay of a TaALAD1-GFP （green fluorescent protein） fusion protein suggested that TaALAD1 is localized in chloroplasts. Overexpression of TaALAD1 in transgenic tobacco resulted in a significant increase in ALAD activity in leaves. Moreover, the transgenic tobacco showed vigorous growth and increased survival rate on medium containing ALA at herbicidal concentrations. These results indicate that wheat ALAD1 has catalytic activity in metabolizing ALA in plastids, and that ectopic expression of TaALAD1 in transgenic plants increases their tolerance to ALA application at high concentrations.

Molecular basis for the inhibition of β-hydroxyacyI-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors

Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobac- terium tuberculosis （Mtb）. Consequently, stalling dehy-dration cures tuberculosis （TB）. Clinically used anti-TB drugs like thiacetazone （TAC） and isoxyl （ISO） as well as flavonoids inhibit the enzyme activity of the β-hydroxy- acyI-ACP dehydratase HadAB complex. How this inhi- bition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2＇,4,4＇-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon muta- tion to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site, The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs,

Glutathione peroxidase mimicry of diphenyl diselenide: Plausible contribution of proteins’ thiols

Organoseleniums are a class of compounds attracting attention across the globe owing to their Glutathione peroxidase(GPx)mimicry,which confers on them a strong antioxidant activity.Diphenyl diselenide(DPDS)is an Organoselenium whose GPx mimetic property has been suggested to rely on the oxidation of non-protein or protein thiols critical to the activities of some sulfhydryl enzymes.This study,therefore investigated the GPx mimic/antioxidant property of DPDS as well as the role of thiols of two key sulfhydryl enzymes,cerebral Na^(+)/K^(+)-ATPase(sodium pump)and hepatic delta-aminolevulinic acid dehydratase(δ-ALAD)in the GPx mimicry of DPDS.Albino Wistar rats were euthanized,and the liver and brain were removed and used to assay for the effect of DPDS on lipid peroxidation induced by two prooxidants[Fe2^(+)(10μM)and H2O2,(1 mM)]as well as the activities of the sulfhydryl enzymes.The results revealed that DPDS profoundly(P<0.05)counteracted Fe2^(+)and H2O2-induced lipid peroxidation in the rats’hepatic and cerebral tissues.Furthermore,the results of assay systems for lipid peroxidation and sodium pump revealed that DPDS inhibited Na^(+)/K^(+)-ATPase and lipid peroxidation in the brain tissue homogenates in the same reaction system.A similar result was obtained in the assay system for lipid peroxidation and hepaticδ-ALAD as DPDS simultaneously inhibited the enzyme’s activity and lipid peroxidation.This suggests that the GPx mimetic property of DPDS may be linked to the enzymes’loss of activity,which further validates the suggestions that the enzymes’inhibition,as well as the antioxidant action of DPDS,rely on the oxidation of critical thiols of the enzymes.However,the GPx mimicry of DPDS should be investigated in the presence of thiol-blocking or oxidizing agents in biological systems in order to further ascertain the role of protein thiols.

Studies on Heavy Metals Levels in Hepatic Tissues of Cat Fishes(Clarias gariepinus)from River Ibi,Donga and Gindin-Dorowa in Taraba State Nigeria,in Relation to key Enzyme of heme Biosynthetic Pathway

Fishes are excellent markers of the extent of pollution from heavy metals in aquatic environments given that they are found at various levels of the food chain.This study aimed to investigate the bioaccumulation of heavy metals(Zn,Pb,Cd,As,and Hg)as well as the activity of delta-aminolevulinic acid dehydratase(δ-ALA-D)in the livers of cat fishes(Clarias gariepinus)collected from three rivers(Donga,Ibi and Gindin-Dorowa)in Taraba State,Nigeria.The concentrations of heavy metals in the liver tissues were determined using an atomic absorption spectrophotometer in accordance with the method of AOAC(2019),while theδ-ALA-D activity was assayed using the method of Sassa(1982).Results revealed that only Zn and As were present in the liver samples from the three rivers.Pb was found only in the liver from Gindin-Dorowa at the concentration of 0.0012mg/kg which is not significant(P<0.05)when compared with other locations,while Hg and Cd were absent in all the liver samples.The liver sample from Gindin-Dorowa had the highest concentration of Zn(4.2500 mg/kg),followed by Ibi(3.2067 mg/kg),and Donga having the least(2.5500 mg/kg),which were all substantially(P<0.05)different from one another.However,there was no significant(P<0.05)difference in the As composition of liver from Donga(0.0013 mg/kg),Ibi(0.0012 mg/kg)and Gindin-Dorowa(0.0010 mg/kg).The result ofδ-ALA-D activity showed that the highest enzymatic activity was found in the liver sample from Donga which has the least Zn and no Pb content,followed by Ibi sample.This validates the report that heavy metals impairδ-ALA-D activity.Nonetheless,the concentrations of all metals in fish livers from all regions do not exceed the acceptable limits set by international law,making them safe for human consumption and possibly not having a negative impact on public health.Since there is little or no industrial activity in the studied locations,these levels may be consequent to low anthropogenic inputs.The current situation should be safeguarded to prevent pollution of the river’s aquatic biota in the near future,and more appropriate steps should be made to guarantee higher fish quality and life in the rivers.

Influence of Montmorillonite Nanocomposite on Tissue Levels of Lead and Blood Biochemical Parameters in Pigs Exposed to Lead

ABSTRACT： Eighteen （Duroc x Landrace x Yorkshire） castrated boars, 55 days of age and weig- hing approximately 19 kg, were used to investigate the effects of montmorillonite nanocomposite on tissue levels of lead as well as changes in blood biochemical parameters in pigs fed lead contaminated diets. The barrows were randomly assigned to one of three treat- ments （n = 6） including a control which were fed a basal diet based on corn and soybean meal. The re- maining two groups were fed the basal diet supple- mented with 10 mg/kg lead either with or without 0.5% montmorillonite nanocomposite for 50 days. Pigs treated with lead had significantly increased lev- els of lead in their tissues. Lead accumulated in all tis- sues of the body with the highest accumulation found in the hair. When montmorillonite nanocomposite was given along with lead, tissue concentrations of lead did not differ from the control. Fecal excretion of leadincreased significantly in lead-exposed pigs treated with montmorillonite nanocomposite. A progressive and appreciable accumulation of lead was seen in blood with a concomitant increase in zinc protopor- phyrin levels during the course of treatment. Howev- er, zinc protoporphyrin levels did not differ from the control when montmorillonite nanocomposite was ad- ministered along with lead. The activity of ^-amino le- vulinic acid dehydratase in blood was significantly in- creased in lead treated pigs compared with the con- trol. However, in pigs treated with lead and montmo- riltonite nanocomposite in combination, the activity of δ-amino levulinic acid dehydratase was similar to con- trol pigs. Our results indicate that montmorillonite nanocomposite treatment of pigs exposed to lead re- suited in reduced lead concentrations in body tissues. It appears that montmorillonite nanocomposite func- tions by increasing fecal excretion of lead.

Impact of Essential Micronutrient, Zn, on Growth and Chlorophyll Biosynthesis in Young <i>Zea mays</i>Seedlings

The present study analyses growth and chlorophyll biosynthesis in young maize seedlings in response to Zn supply over a wide range of concentrations. Supply of 0 - 5 mM ZnCl2 to 3 days old light grown maize seedlings led to gradually increased accumulation of Zn in the shoot tissue, while in the root tissue substantial increase was observed at/and above 0.1 mM ZnCl2. Zn supply significantly reduced the overall growth of maize seedlings mostly at 1 - 5 mM ZnCl2 exerting strong correlation and the observed effect was more substantial for root tissue. Amongst the biochemical parameters, increase in protein and proline content was more prominent in root tissue than the shoot, while RNA content was reduced in shoot tissue. Zn treatment to light grown seedlings significantly increased the chlorophyll, carotenoid content, while in dark grown seedlings it had marginal/no effect. Delta amino levulinic acid (ALA) content in both the regimes was increased at higher Zn concentrations. Also ALA synthesis was increased in both the regimes, but non significantly. Zn enhanced ALA dehydratase (ALAD) activity of light as well as dark grown seedlings being significant in former. The results demonstrate that the Zn accumulation and growth effect at higher Zn concentrations in maize depend upon the tissue with root as the target site and shoot growth are mainly influenced by ALA and subsequently ALAD in maize seedlings.

PcTGD,a highly expressed gene in stem,is related to water stress in reed (Phragmites communis Trin.)

To compare differential gene expression among three ecotypes of reed (Phragmites communis Trin.), dune reed (DR), heavy-salt meadow reed (HSR) and light-salt meadow reed (LSR), mRNA transcripts were displayed by cDNA-AFLP (amplified fragment length polymorphisms). The result revealed that a relatively small number of genes are likely involved in adaptations of DR and HSR to stresses. A full-length cDNA encoding dTDP-D-glucose dehydratase gene (PcTGD) was subsequently cloned from DR. Northern blot analysis showed that it is highly expressed in stem as well as rhizoma of the three ecotypes. However, its expression in DR stem was much higher than that of the other two ecotypes. After the removal of water stress, the expression of PcTGD was significantly reduced, suggesting that it possibly plays a role in adaptation of DR to water stress through an osmotic regulation mechanism.