Structural Characterization of the Heterobactin Siderophores from Rhodococcuserythropolis PR4 and Elucidation of their Biosynthetic Machinery

Summary The genus Rhodococcus belong to the order actinomycetes, which are gram-positive bacteria with high GC content. They produce a broad range of bioactive secondary metabolites that found use in the pharmaceutical industry and in other biotechnological applications. Most of these bioactive metabolites were derived from nonribosomal peptides (NRP) or polyketides (PK). However, only few natural products have been isolated and characterized so far. In particular, within the Rhodococcus genus, substantial chemical diversity has been observed among the iron-chelating siderophores through the structure elucidation of rhodochelin, rhodobactin and heterobactin A1. Therefore this work was focused on isolation and structural characterization of further new iron-chelating molecules to explore the possible chemical potential of this genus on secondary metabolite production. In this study we accomplished the isolation, the structural characterization and the elucidation of the biosynthetic origin of heterobactins, a catecholate-hydroxamate mixed-type siderophores from Rhodococcus erythropolis PR4. The structure elucidation of the extracted and purified siderophore heterobactin A was accomplished via MSn analysis and NMR spectroscopy and revealed the noteworthy presence of a peptide bond between the guanidine group of an arginine residue and a 2,3- dihydroxybenzoate moiety. The two other purified siderophores heterobactin S1 and S2 were found to be derivatives of heterobactin A that have sulfonation modifications on the aromatic rings. The bioinformatic analysis of the R. erythropolis PR4 genome and the subsequent genetic and biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for the biosynthesis of the heterobactins to encode the three modules comprising nonribosomal peptide synthetase (NRPS) HtbG. Interestingly, the HtbG NRPS contains an unprecedented C-PCP-A domain organization within the second module of the HtbG-synthetase that may help the correct elongation of the peptide intermediate. The present work also revises the structure of heterobactin A that was described by Carrano et al. in 2001. Also, the biochemical characterization of the monooxygenase HMO (encoded by the hmo gene within the gene cluster) established a route for the biosynthesis of the non- proteinogenic amino acid L-hOrn, prior to its incorporation by the NRPS HtbG into the siderophore scaffold. The insights gained from the structural and biochemical characterization of the siderophore heterobactins, together with the genetic and biochemical characterization of the respective biosynthetic gene clusters, allowed us to establish a biosynthetic model for heterobactins assembly. The iron-siderophore binding protein HtbH (encoded by htbH gene within the gene cluster) was also biochemically characterized and was shown to display a novel mix-type catecholate-hydroxamate binding behavior

Possible hypocholesterolemic effect of ginger and rosemary oils in rats

Background: Hypercholesterolemia is a major risk factor for development of atherosclerosis. The present study was conducted to evaluate the potential effect of ginger oil alone or combined with rosemary oil as hypocholesterolemic agent in rats fed high fat diet.Materials and methods: Healthy female albino rats (n=80) weighting about (150-180 g) were included in this study divided into two equal groups; Group (I): were fed on the basal diet. Group (I) were divided into 4 subgroups each 10: Group (Ia): negative control. Group (Ib): Rats received i.p 2.5 g/Kg b.w of ginger oil. Group (Ic): rats received i.p 2.5 g/Kg b.w of rosemary oil. Group (Id): Rats received i.p 5 g/Kg b.w mixture of ginger oil and rosemary oil (1:1). The second main groups; Group (II): high fat diet (HFD) were fed on the basal diet plus cholesterol (1%), bile salt (0.25%) and animal fat (15%) to induce hypercholesterolemia for six weeks. Group (II) was divided into 4subgroups: Group (IIa): HFD. Group (IIb): HFD were treated with i.p 2.5 g/Kg b.w ginger oil. Group (IIc): (n=10) HFD were treated with i.p 2.5 g/Kg b.w rosemary oil. Group (IId): (n=10) HFD were treated with i.p 5 g/Kg b.w mixture of oils.Results: It was found that HFD rats showed a significant elevation in glucose, total cholesterol, triglyceride, GOT, GPT, alkaline phosphatase and a reduction in serum HDL-c compared with negative control. Treatment with ginger oil, rosemary oil and their mixture modulated the elevation of these parameters. Histopathological examination of the liver tissue of HFD rats showed a lipid deposition and macrophage infiltration and stenosis of hepatic vein. Treatment with mixture oils preserves normal structure of liver.Conclusion: It was concluded that, hypocholesterolemic effect was related to the active oil content as Rosemary oil contain - α-pinene, Camphor, cineole, borneol and Ginger oil contain Linalool, Terpineol ,Borneol , Eucalyptol.Keywords: Ginger Oil, Rosemary Oil, Rats, Hypocholesterolemi

POSSIBLE REGULATION OF LDL-RECEPTOR BY NARINGENIN IN HEPG2 HEPATOMA CELL LINE

Background: High plasma concentration of low-density lipoprotein cholesterol (LDL-c) plays a significant role in the incidence of atherosclerosis and coronary heart diseases (CHD). Materials and Methods: The purpose of this study was to investigate the mechanism by which citrus flavonoids, naringenin regulate the LDL receptor (LDLr) gene in human liver using the human hepatoma cell line, HepG2 as a model. Results: Time-course transient transfection of HepG2 cells with luciferase reporter-gene constructs incorporating the promoters of SREBP-1a,-1c, -2 and LDLr, revealed that in lipoprotein-deficient medium (LPDM), only SREBP-1a promoter activity was increased significantly after 4h exposure to 200μM naringenin respectively. However, after 24h incubation with 200μM naringenin the gene expression activities of all the SREBP-1a, -1c, -2 and LDLr promoterconstructs were increased significantly. The effects of both 200μM naringenin on elevating LDLr mRNA are possibly due to regulation of gene transcription by SREBP-la and SREBP-2. However, the suppression effect of 200μM naringenin on hepatic SREBP-1c mRNA expression is likely associated with the reduction in mRNA expression of both acetyl-CoA carboxylase and fatty acid synthase in human hepatoma HepG2 cells. It was found that, 200μM naringenin was likely to stimulate LDLr gene expression via increase phosphorylation of PI3K and ERK1/2 which enhance the transcription factors SREBP-1a and SREBP-2 mRNA levels and increased their protein maturation in human hepatoma HepG2 cell. Conclusion: Diets supplemented with naringenin could effectively reduce mortality and morbidity from coronary heart diseases and as cardio-protective effects in humans

The protective role of almond and thyme in carbonated beverage-induced osteoporosis in male albino rats

Osteoporosis is a common health problem distinguished by low bone mass and microarchitectural deterioration of bone tissue, thus increase bone fragility and fracture. Nutrition possesses a critical role in reducing osteoporosis risks among its impact on fragility factors, particularly on bone mass development and maintenance. The current study was carried out to assess and compare the protective effect of almond and thyme against osteoporosis induced by excessive intake of carbonated beverage (Coca-Cola) in rats. Forty-eight adult male Sprague–Dawley rats were divided into six groups: G1, control negative fed balanced diet; G2, control positive fed balanced diet and received Coca-Cola orally by gavage (20 ml/day) (CB); G3, almond powder (5g/100g diet) (A); G4, thyme powder (5g/100g diet) (T) and G5, (CB) +(A); 6, (CB)+(T)). Almond and thyme supplementation significantly reduced the development of osteoporosis as well as inflammation and oxidative stress. Our findings show that almond and thyme can effectively reduce soft drink-induced bone loss and could be used as dietary supplements to prevent bone resorption and osteoporosis

Structural Characterization of the Heterobactin Siderophores from Rhodococcuserythropolis PR4 and Elucidation of their Biosynthetic Machinery

Summary The genus Rhodococcus belong to the order actinomycetes, which are gram-positive bacteria with high GC content. They produce a broad range of bioactive secondary metabolites that found use in the pharmaceutical industry and in other biotechnological applications. Most of these bioactive metabolites were derived from nonribosomal peptides (NRP) or polyketides (PK). However, only few natural products have been isolated and characterized so far. In particular, within the Rhodococcus genus, substantial chemical diversity has been observed among the iron-chelating siderophores through the structure elucidation of rhodochelin, rhodobactin and heterobactin A1. Therefore this work was focused on isolation and structural characterization of further new iron-chelating molecules to explore the possible chemical potential of this genus on secondary metabolite production. In this study we accomplished the isolation, the structural characterization and the elucidation of the biosynthetic origin of heterobactins, a catecholate-hydroxamate mixed-type siderophores from Rhodococcus erythropolis PR4. The structure elucidation of the extracted and purified siderophore heterobactin A was accomplished via MSn analysis and NMR spectroscopy and revealed the noteworthy presence of a peptide bond between the guanidine group of an arginine residue and a 2,3- dihydroxybenzoate moiety. The two other purified siderophores heterobactin S1 and S2 were found to be derivatives of heterobactin A that have sulfonation modifications on the aromatic rings. The bioinformatic analysis of the R. erythropolis PR4 genome and the subsequent genetic and biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for the biosynthesis of the heterobactins to encode the three modules comprising nonribosomal peptide synthetase (NRPS) HtbG. Interestingly, the HtbG NRPS contains an unprecedented C-PCP-A domain organization within the second module of the HtbG-synthetase that may help the correct elongation of the peptide intermediate. The present work also revises the structure of heterobactin A that was described by Carrano et al. in 2001. Also, the biochemical characterization of the monooxygenase HMO (encoded by the hmo gene within the gene cluster) established a route for the biosynthesis of the non- proteinogenic amino acid L-hOrn, prior to its incorporation by the NRPS HtbG into the siderophore scaffold. The insights gained from the structural and biochemical characterization of the siderophore heterobactins, together with the genetic and biochemical characterization of the respective biosynthetic gene clusters, allowed us to establish a biosynthetic model for heterobactins assembly. The iron-siderophore binding protein HtbH (encoded by htbH gene within the gene cluster) was also biochemically characterized and was shown to display a novel mix-type catecholate-hydroxamate binding behavior

Metformin’s dual impact on Gut microbiota and cardiovascular health: A comprehensive analysis

Cardiovascular diseases (CVD) cause significant global morbidity, mortality and public health burden annually. CVD alters richness, diversity, and composition of Gut microbiota along with RAS and histopathological differences. Present study explores Metformin role in mitigating doxorubicin induced cardiovascular toxicity/remodeling. Animals were divided into 4 groups with n=6: Group I (N. Control) free access to diet and water; Group II (MET. Control) on oral Metformin (250 mg/kg) daily; Group III (DOX. Control) alternate day intraperitoneal Doxorubicin (3 mg/kg) totaling 18 mg/kg; Group IV (DOX. MET. Control) received both daily oral Metformin (250 mg/kg) and alternate day Doxorubicin (3 mg/kg). Gut microbial analysis was made from stool before animals were sacrificed for biochemical and histopathological analysis. Significant alterations were observed in ɑ and β-diversity with new genus from Firmicutes, specifically Clostridia_UCG-014, Eubacterium ruminantium, and Tunicibacter, were prevalent in both the DOX. Control and DOX.MET groups. Proteobacteria, represented by Succinivibrio, were absent in all groups. Additionally, Parabacteroides from the Bacteroidia phylum was absent in all groups except the N. control. In the DOX.MET Control group, levels of Angiotensin II ( 7.75± 0.49 nmol/min, p<0.01) and Renin (2.60±0.26 ng/ml/hr) were significantly reduced. Conversely, levels of CK-MB, Fibrinogen, Troponin, CRP ( p < 0.0001), and TNFɑ (p < 0.05) were elevated. Histopathological examination revealed substantial cardiac changes, including Fibrinogen and fat deposition and eosinophilic infiltration, as well as liver damage characterized by binucleated cells and damaged hepatocytes, along with altered renal tissues in the DOX.MET.Control group. The findings suggest that MET. significantly modifies gut microbiota, particularly impacting the Firmicutes and Proteobacteria phyla. The reduction in Angiotensin II levels, alongside increased inflammatory markers and myocardial damage, highlights the complex interactions and potential adverse effects associated with MET therapy on cardiovascular health

POSSIBLE HYPOCHOLESTEROLEMIC EFFECT OF GINGER AND ROSEMARY OILS IN RATS

Background: Hypercholesterolemia is a major risk factor for development of atherosclerosis. The present study was conducted to evaluate the potential effect of ginger oil alone or combined with rosemary oil as hypocholesterolemic agent in rats fed high fat diet. Materials and methods: Healthy female albino rats (n=80) weighting about (150-180 g) were included in this study divided into two equal groups; Group (I): were fed on the basal diet. Group (I) were divided into 4 subgroups each 10: Group (Ia): negative control. Group (Ib): Rats received i.p 2.5 g/Kg b.w of ginger oil. Group (Ic): rats received i.p 2.5 g/Kg b.w of rosemary oil. Group (Id): Rats received i.p 5 g/Kg b.w mixture of ginger oil and rosemary oil (1:1). The second main groups; Group (II): high fat diet (HFD) were fed on the basal diet plus cholesterol (1%), bile salt (0.25%) and animal fat (15%) to induce hypercholesterolemia for six weeks. Group (II) was divided into 4subgroups: Group (IIa): HFD. Group (IIb): HFD were treated with i.p 2.5 g/Kg b.w ginger oil. Group (IIc): (n=10) HFD were treated with i.p 2.5 g/Kg b.w rosemary oil. Group (IId): (n=10) HFD were treated with i.p 5 g/Kg b.w mixture of oils. Results: It was found that HFD rats showed a significant elevation in glucose, total cholesterol, triglyceride, GOT, GPT, alkaline phosphatase and a reduction in serum HDL-c compared with negative control. Treatment with ginger oil, rosemary oil and their mixture modulated the elevation of these parameters. Histopathological examination of the liver tissue of HFD rats showed a lipid deposition and macrophage infiltration and stenosis of hepatic vein. Treatment with mixture oils preserves normal structure of liver. Conclusion: It was concluded that, hypocholesterolemic effect was related to the active oil content as Rosemary oil contain - α-pinene, Camphor, cineole, borneol and Ginger oil contain Linalool, Terpineol ,Borneol , Eucalyptol