Publications by authors named "Amal Z Barakat"

6 Publications

  • Page 1 of 1

Purification and characterization of peroxidases from garden cress sprouts and their roles in lignification and removal of phenol and p-chlorophenol.

J Food Biochem 2021 Jan 2;45(1):e13526. Epub 2020 Nov 2.

Molecular Biology Department, National Research Centre, Dokki, Cairo, Egypt.

The study aims to evaluate the relation between peroxidases of day-6 garden cress sprouts and phenolic compounds. Three cationic, three anionic, and two unbounded peroxidases were separated from day-6 garden cress sprouts. Cationic (GCP1) and anionic (GCP2) peroxidases were purified with molecular masses of 25 and 40 kDa, respectively. The K values of GCP1 toward H O and guaiacol were lower than GCP2. The anionic GCP2 exhibited high affinity toward some lignin monomers, sinapyl alcohol, coniferyl alcohol, cinnamic and ferulic acids. Therefore, GCP2 is considered as a lignin peroxidase and contributed in lignin synthesis. The activity of GCP1 and GCP2 was stable at a wide pH range 5.5-8.0 and 6.0-7.5, respectively. Both peroxidases showed the same thermal stability range 20-50°C. GCP2 was more resistant against the effect of metal ions than GCP1. GCP2 showed high ability to remove of phenol and p-chlorophenol from effluent compared to GCP1. PRACTICAL APPLICATIONS: Generally, garden cress is used as a test plant to conduct biomonitoring of pollution in urban soil on a wide scale because of its simplicity, sensitivity, and cost-effectiveness. Peroxidase is an important antioxidant enzyme, which elevated when plant subjected to pollution. Recently, we reported that the increase of peroxidase activity was strongly correlated with high phenolic content and antioxidant activity during the germination of garden cress. In the present study, anionic peroxidase GCP2 may play an important role in lignification process and removal of phenol and p-chlorophenol from polluted soil/wastewater as well as resisted the harmful effect of heavy metals. Cationic peroxidase GCP1, as a natural scavenger, had high affinity toward H O coupled to oxidation of some plant phenolic compounds suggesting its role in consuming of excess H O .
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http://dx.doi.org/10.1111/jfbc.13526DOI Listing
January 2021

Diabetic complications and oxidative stress: The role of phenolic-rich extracts of saw palmetto and date palm seeds.

J Food Biochem 2020 11 4;44(11):e13416. Epub 2020 Sep 4.

Department of Molecular Biology, National Research Center, Cairo, Egypt.

Recently, we reported that the date palm seed (DP) and saw palmetto seed (SP) extracts possessed a great amount of phenolic contents with potent antioxidant, antimicrobial, and anti-inflammatory activities. Therefore, this study aimed to assess the role of DP and SP phenolic-rich extracts in modulating diabetic complications and oxidative stress in the STZ- diabetic rat. DP and SP extracts significantly inhibited both microbial and pancreatic α-amylases. The STZ-induced diabetic rat groups treated with DP and SP extracts exhibited reversed hyperglycemia (40% and 54%, p < .001-.01) and body weight (70%, p < .001) alteration close to normal. Moreover, DP and SP extracts modulated serious damages in the structures of the pancreas, kidney, and liver of diabetic rats. DP and SP extracts improved the decline of the activities of antioxidant enzymes: Catalase, glutathione-S-transferase, glutathione reductase, and glutathione peroxidase in liver, kidney, and pancreas of the diabetic rats. PRACTICAL APPLICATIONS: Generally, date seed is a rich source of dietary fibers, polyphenols, and antioxidants and used in foods and pharmaceuticals. Our study reported that date palm seed (DP) and saw palmetto seed (SP) phenolic-rich extracts attenuated diabetes and its complications, probably tissue regeneration and normalizing the oxidative stress in the STZ-induced diabetic rat.
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http://dx.doi.org/10.1111/jfbc.13416DOI Listing
November 2020

Revealing of a novel xylose-binding site of Geobacillus stearothermophilus xylanase by directed evolution.

J Biochem 2019 Feb;165(2):177-184

Molecular Biology Department, Genetic Engineering and Biotechnology Research Division, National Research Centre, 33 El Bohouth St., Dokki, Giza, Egypt.

Xylan saccharification is a key step in many important biotechnological applications. Xylose is the main product of xylan degradation and is a major xylanase inhibitor in a bioreactor; however, xylose-binding site of xylanase is not discovered yet. Evolving of xylose-tolerant xylanase variants will reduce the cost of xylanases in industry. Glycoside hydrolase family-10 thermostable Geobacillus stearothermophilus xylanase XT6 is non-competitively inhibited by xylose with inhibition constant ki equals to 12.2 mM. In the absence of X-ray crystallography of xylanase-xylose complex, unbiased random mutagenesis of the whole xylanase gene was done by error-prone polymerase chain reaction constructing a huge library. Screening a part of the library revealed xylose-tolerant mutants having three mutations, M116I, L131P and L133V, clustered in the N-terminus of α-helix 3. The best xylose-tolerant mutant showed higher ki and catalytic capability than that of the parent by 3.5- and 3-fold, respectively. In addition, kcat increased 4.5-fold and KM decreased 2-fold. The molecular docking of xylose into xylanase XT6 structure showed that xylose binds into a small pocket between N-terminus of α-helices 3 and 4 and close to the three mutations. Mobility of α-helices 3 and 4, which controls catalysis rate, is restricted by xylose binding and increased by these mutations.
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http://dx.doi.org/10.1093/jb/mvy092DOI Listing
February 2019

Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double-mutant mice.

Am J Respir Cell Mol Biol 2014 Apr;50(4):817-24

1 Institute of Human Genetics, and.

Heat shock proteins HSPA4L and HSPA4 are closely related members of the HSP110 family and act as cochaperones. We generated Hspa4l(-/-)Hspa4(-/-) mice to investigate a functional complementarity between HSPA4L and HSPA4 during embryonic development. Hspa4l(-/-)Hspa4(-/-) embryos exhibited marked pulmonary hypoplasia and neonatal death. Compared with lungs of wild-type, Hspa4l(-/-), and Hspa4(-/-) embryos, Hspa4l(-/-)Hspa4(-/-) lungs were characterized by diminished saccular spaces and increased mesenchymal septa. Mesenchymal hypercellularity was determined to be due to an increased cell proliferation index and decreased cell death. A significant increase in expression levels of prosurvival protein B cell leukemia/lymphoma 2 may be the cause for inhibition of apoptotic process in lungs of Hspa4(-/-)Hspa4l(-/-) embryos. Accumulation of glycogen and diminished expression of surfactant protein B, prosurfactant protein C, and aquaporin 5 in saccular epithelium suggested impaired maturation of type II and type I pneumocytes in the Hspa4l(-/-)Hspa4(-/-) lungs. Further experiments showed a significant accumulation of ubiquitinated proteins in the lungs of Hspa4l(-/-)Hspa4(-/-) embryos, indicating an impaired chaperone activity. Our study demonstrates that HSPA4L and HSPA4 collaborate in embryonic lung maturation, which is necessary for adaptation to air breathing at birth.
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http://dx.doi.org/10.1165/rcmb.2013-0132OCDOI Listing
April 2014

Targeted disruption of Hspa4 gene leads to cardiac hypertrophy and fibrosis.

J Mol Cell Cardiol 2012 Oct 1;53(4):459-68. Epub 2012 Aug 1.

Institute of Human Genetics, University of Göttingen, Germany.

Failure of molecular chaperones to direct the correct folding of newly synthesized proteins leads to the accumulation of misfolded proteins in cells. HSPA4 is a member of the heat shock protein 110 family (HSP110) that acts as a nucleotide exchange factor of HSP70 chaperones. We found that the expression of HSPA4 is upregulated in murine hearts subjected to pressure overload and in failing human hearts. To investigate the cardiac function of HSPA4, Hspa4 knockout (KO) mice were generated and exhibited cardiac hypertrophy and fibrosis. Hspa4 KO hearts were characterized by a significant increase in heart weight/body weight ratio, elevated expression of hypertrophic and fibrotic gene markers, and concentric hypertrophy with preserved contractile function. In response to pressure overload, cardiac hypertrophy and remodeling were further aggravated in the Hspa4 KO compared to wild type (WT) mice. Cardiac hypertrophy in Hspa4 KO hearts was associated with enhanced activation of gp130-STAT3, CaMKII, and calcineurin-NFAT signaling. Protein blot and immunofluorescent analyses showed a significant accumulation of polyubiquitinated proteins in cardiac cells of Hspa4 KO mice. These results suggest that the myocardial remodeling of Hspa4 KO mice is due to accumulation of misfolded proteins resulting from impaired chaperone activity. Further analyses revealed a significant increase in cross sectional area of cardiomyocytes, and in expression levels of hypertrophic markers in cultured neonatal Hspa4 KO cardiomyocytes suggesting that the hypertrophy of mutant mice was a result of primary defects in cardiomyocytes. Gene expression profile in hearts of 3.5-week-old mice revealed a differentially expressed gene sets related to ion channels, muscle-specific contractile proteins and stress response. Taken together, our in vivo data demonstrate that Hspa4 gene ablation results in cardiac hypertrophy and fibrosis, possibly, through its role in protein quality control mechanism.
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http://dx.doi.org/10.1016/j.yjmcc.2012.07.014DOI Listing
October 2012

Heat-shock protein HSPA4 is required for progression of spermatogenesis.

Reproduction 2011 Jul 12;142(1):133-44. Epub 2011 Apr 12.

Institute of Human Genetics, University of Göttingen, Göttingen, Germany.

Heat-shock protein 110 (HSP110) family members act as nucleotide exchange factors (NEF) of mammalian and yeast HSP70 chaperones during the ATP hydrolysis cycle. In this study, we describe the expression pattern of murine HSPA4, a member of the HSP110 family, during testis development and the consequence of HSPA4 deficiency on male fertility. HSPA4 is ubiquitously expressed in all the examined tissues. During prenatal and postnatal development of gonad, HSPA4 is expressed in both somatic and germ cells; however, expression was much higher in germ cells of prenatal gonads. Analyses of Hspa4-deficient mice revealed that all homozygous mice on the hybrid C57BL/6J×129/Sv genetic background were apparently healthy. Although HSPA4 is expressed as early as E13.5 in male gonad, a lack of histological differences between Hspa4(-/-) and control littermates suggests that Hspa4 deficiency does not impair the gonocytes or their development to spermatogonia. Remarkably, an increased number of the Hspa4-deficient males displayed impaired fertility, whereas females were fertile. The total number of spermatozoa and their motility were drastically reduced in infertile Hspa4-deficient mice compared with wild-type littermates. The majority of pachytene spermatocytes in the juvenile Hspa4(-/-) mice failed to complete the first meiotic prophase and became apoptotic. Furthermore, down-regulation of transcription levels of genes known to be expressed in spermatocytes at late stages of prophase I and post-meiotic spermatids leads to suggest that the development of most spermatogenic cells is arrested at late stages of meiotic prophase I. These results provide evidence that HSPA4 is required for normal spermatogenesis.
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http://dx.doi.org/10.1530/REP-11-0023DOI Listing
July 2011