In vitro study of the expression of autophagy genes ATG101, mTOR and AMPK in breast cancer with treatment of lactoferrin and in silico study of their communication networks and protein interactions.

Autophagy is a new window of science that has been noticed due to the importance of specific therapies in cancer. In this study, the effect of lactoferrin (Lf) on the expression level of ATG101, mTOR and AMPK genes in breast cancer cell line MCF7, as well as the interaction between lactoferrin protein and their protein were investigated. The expression level of the genes was measured using a real-time PCR method. PDB, UniProt, KEGG, and STRING databases and ClusPro webserver and PyMol software were used in silico study. The results showed that the expression level of the ATG101 gene in treatment with concentrations of 100, 400, 600, and 800 µg/ml Lf decreased by 0.05, 0.13, 0.54 and 0.77, respectively. The expression level of the mTOR gene in treatment with concentrations of 100, 400, 600, and 800 µg/ml Lf decreased by 0.07, 0.05, 0.13, and 0.49 times respectively. The level of the AMPK gene expression in treatment with concentrations of 100, 400, 600, and 800 µg/ml Lf decreased by 0.05, 0.01, 0.06, and 0.03, respectively. Virtualization of the interaction of Lf protein with ATG101, mTOR and AMPK proteins by Pymol software showed that the N lobe region of Lf interacted with the HORMA domain of ATG101 protein, the fat domain of mTOR protein, and the CTD domain of AMPK protein. Although Lf was not able to increase the expression of autophagy-inducing genes, it may be able to induce autophagy through protein interaction by activating or inhibiting proteins related to autophagy regulation.

Identification, Isolation, Cloning, and Expression of a New Alkaline Serine Protease Gene from Native Iranian Bacillus sp. RAM 53 for Use in the Industry.

Proteases are one of the most important and widely applicable proteolytic enzymes that are used in various industries. The aim of this study was to identify, isolate, characterize, and clone the new extracellular alkaline protease from the native bacterium Bacillus sp. RAM53 that was isolated from rice fields in Iran. In this study, first, the primary assay of protease production was performed. The bacteria were cultured in a nutrient broth culture medium at 37° C for 48 h, and then, the enzyme extraction was performed. Enzyme activity was measured by standard methods in the range of 20 to 60 °C and the range of pH 6.0 to 12. Degenerate primers were designed to alkaline protease gene sequences. The isolated gene was cloned into the pET28a+ vector, the positive clones were transferred to Escherichia coli BL21, and the expression of the recombinant enzyme was optimized. The results showed that the optimum temperature and pH of the alkaline protease were 40° C and 9.0, respectively, and were stable at 60° C for 3 h. The molecular weight of the recombinant enzyme was 40 kDa in SDS-PAGE. The recombinant alkaline protease was inhibited by the PMSF inhibitor, indicating that the enzyme was serine protease. The results showed that the sequence alignment of the enzyme gene with the other alkaline protease gene sequences related to Bacillus was 94% identity. The result of Blastx showed about 86% identity to the S8 peptidase family in Bacillus cereus and Bacillus thuringiensis and other Bacillus species. The enzyme may be useful for various industries.

Evaluation of the effect of nano-encapsulated lactoferrin on the expression of Bak and Bax genes in gastric cancer cell line AGS and study of the molecular docking of lactoferrin with these proteins.

lactoferrin (Lf) is a glycoprotein with various biological activities, including antibacterial, antiviral, anti-cancer, etc. In the present study, the effect of different concentrations of nano-encapsulated lactoferrin (NE-Lf) on the expression of Bax and Bak genes was evaluated in stomach cancer cell line AGS using real-time PCR technique and cytotoxicity of NE-Lf on the growth cells as well as the molecular mechanism of these two genes and their proteins in the apoptosis pathway and the relationship between lactoferrin and these proteins were investigated by bioinformatics studies. In the viability test, the results showed that the growth inhibition effect of nano-lactoferrin was greater than lactoferrin in both concentrations, and chitosan had no inhibitory effect on the cells. In concentrations of 250 and 500 µg of NE-Lf Bax gene expression increased by 2.3 and 5 times, respectively, and Bak gene expression increased by 1.94 and 1.74 times, respectively. Statistical analysis showed that there is a significant difference in the relative amount of gene expression between the treatments in both genes (P < 0.05). The binding mode of lactoferrin with Bax and Bak proteins was obtained using docking. According to docking results, the N-lobe region of lactoferrin interacts with the Bax protein, as well as the Bak protein. The results show that lactoferrin, in addition to acting on the gene, interacts with Bax and Bak proteins. Since two proteins are components of apoptosis, lactoferrin can induce apoptosis in this way.

The effect of lactoferrin on ULK1 and ATG13 genes expression in breast cancer cell line MCF7 and bioinformatics studies of protein interaction between lactoferrin and the autophagy initiation complex.

Recently, the study of autophagy and its mechanism on the cancer cell growth process has received much attention. lactoferrin (Lf) is a glycoprotein with various biological activities, including antibacterial, antiviral, anti-cancer, etc. In the present study, the effect of different concentrations of lactoferrin on the expression of ULK1 and ATG13 genes was evaluated in breast cancer cell line MCF7 using real-time PCR technique as well as the molecular mechanism of these two genes and their proteins in the autophagy pathway and the relationship between lactoferrin and these proteins were investigated by bioinformatics studies. The result showed that the expression of the ULK1 gene at a concentration of 500 µg/ml of lactoferrin was significantly (P < 0.007) increased compared to the control and two other concentrations. Also, the expression of the ATG13 gene at all three concentrations was not significantly different from each other and compared to the control (P = 0.635). In the immunoblot of ULK1 protein at a concentration of 500 µg, more protein expression was observed. The binding mode of lactoferrin with ULK1, ATG13, and ATG101 proteins was obtained using docking. According to docking results, the N-lobe region of lactoferrin interacts with the PS domain of the ULK1 protein, and the N-lobe region of lactoferrin interacts with the horma domain of the ATG 13 and ATG101 proteins. The results show that lactoferrin, in addition to acting on the gene, interacts with ULK1, ATG13, and ATG101 proteins. Since all three proteins are components of the autophagy initiation complex, lactoferrin can induce autophagy in this way.

Activation of periodate using ultrasonic waves and UV radiation for landfill leachate treatment.

In the present work, saline leachate of the Bushehr coastal city (Iran) was purified using the ultraviolet/ultrasonication wave/periodate process. The initial TDS and TOC values of the leachate studied were 7390 mg/L and 975 mg/L, respectively. During the effect of various parameters on leachate purification, the experiments were optimized at pH 3, oxidizer concentration of 4 mM, and treatment time of 120 min. The initial BOD5/COD ratio of 0.66 was reduced to 0.42 at the end of the purification time (120 min). After leachate treatment under optimal conditions, the amount of BOD5, COD, and UV254 were 451.5 mg/L, 1072 mg/L, and 12.69 cm-1, respectively. Concentrations of heavy metals in crude leachate by ICP-OES were checked. Also, the concentration of organic compounds before and after purification was determined using GC-Mass. The leachate purification kinetics followed the first-order model using the designed method. Based on the COD factor, the system energy consumption for leachate treatment was calculated to be 11.4 kWh/m3. The results showed that the system explored (UV/US/IO4-) can effectively purify high salinity waste leachate.

CYP1A1 contiguous hypermethylation within a putative CpG block is associated with breast cancer progression: Feasibility to define boundary motives.

Having broad specificity for xenobiotics metabolism throughout the body, cytochrome P450 (CYP) isoform 1A1 is of key relevance for carcinogenesis. However, the oncogenic potential of its altered transcription and the underlying mechanism has not been well-established in breast cancer. Direct bisulfite sequencing PCR (BSP) of the CYP1A1 promoter, enriched by 113 CpGs within and flanking the xenobiotic response elements (XREs) 2 to 10, in paired cancerous and normal tissues from 40 breast cancer patients revealed three distinctly methylated patterns; unmethylated (XREs 2 to 6) and completely methylated (XREs 7 and 8) CpGs, in common for the normal and cancerous tissues, and a putative 171bp CpG block (XREs 9 and 10) contiguously hypermethylated in the tumor tissues. Increased transcription of CYP1A1, observed for the cancerous tissues, was correlated with the hypermethylation of given CpG block, besides simultaneously being associated with upregulation of the anti-apoptotic BCL-2. Clinical value of the methylation changes, investigated based on the comparisons between the tissue cohorts of different clinicopathological features, exhibited gradual hypermethylation of the corresponding CpG block following disease progression as well as lymphatic involvement. Hypermethylation of given CpG block may has potential to be used as a biomarker for diagnosis and progression of breast cancer.

Effective treatment of high-salinity landfill leachate using ultraviolet/ultrasonication/ peroxymonosulfate system.

Landfill leachate, as a complex medium with a high concentration of organic and mineral materials, is a serious problem for the environment. In the current study, the saline landfill leachate of Bushehr coastal city (Iran) was treated using ultraviolet/ultrasonic waves/peroxymonosulfate system. The initial TOC and TDS of the studied leachate was 915 mg/L and 7390 mg/L, respectively. The system had the maximum efficiency at conditions of pH 3, peroxymonosulfate (PMS) of 4 mM, and reaction time of 150 min. Based on the findings, the initial ratio of BOD5/COD (0.66) was reduced to 0.38 using the developed system. After treatment of the landfill leachate at the optimal condition, the values of COD, BOD5, and UV254 were reached to 983 mg/L, 348 mg/L, and 10.16 cm-1, respectively. The concentration of all studied elements (except Pb, As, and Ca) increased after purification. According to the GC-mass spectrometry, the molecular weight and concentration of organic matter in raw leachate were higher than that of the treated one. The studied system had an energy consumption value of 86 kW·h/m3 for the treatment of landfill leachate. The results confirm the effectiveness of the ultraviolet/ultrasonic waves/PMS system for the treatment of high saline landfill leachate.

Association of allelic polymorphisms of IGFALS gene with growth traits in Makouei and Ghezel sheep breeds.

Insulin-like growth factor-binding protein acid-labile subunit (IGFALS) encodes a protein which binds to IGF1 and IGFBP-3 to regulate the growth, differentiation, and other physiological processes. The aim of this study was the identification of allelic polymorphisms of the IGFALS gene using the PCR-RFLP technique and evaluation of their association with growth traits. For this end, 120 blood samples were randomly collected from each breed. Following amplification of an 1113-bp fragment of exon 1 and a part of intron 1 of the IGFLAS gene, genotyping was conducted by three restriction enzymes including HinfI, MscI, and PvuII. The results showed that only one allele was observed in IGFALS-PvuII site, while in IGFLAS-MscI site, three AA, AB, and BB genotypes with the frequencies of 17.5%, 32%, and 50.5% and 11%, 37.5%, and 51.5% were observed in Makouei and Ghezel sheep breeds, respectively. Additionally, in the IGFLAS-HinfI site, two AB and BB genotypes with the frequency of 34.2% and 65.8% were observed in Makouei sheep and AA, AB, and BB genotypes with the frequency of 9%, 21%, and 70% were observed in Ghezel sheep. So that, Makouei sheep with AB genotype had more chest girth (CG) compared with other genotypes. Furthermore, a significant association was observed between the genotypes of IGFLAS-HinfI with birth weight (BW) in Ghezel and BW, weaning weight (BW3), and CG in Makouei sheep. Haplotype analysis revealed an association between paternal haplotypes and BW in both Ghezel and Makouei breeds. So that, AAB and ABB haplotypes showed more BW than others in Makouei and Ghezel sheep, respectively.

Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish.

GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer half-life, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and Western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24 h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

Cloning, expression and characterization of a novel alkaline serine protease gene from native Iranian Bacillus sp.; a producer of protease for use in livestock.

The use of proteases in the last decade has been welcomed in livestock and poultry industries and has led to significant results such as improved feed conversion ratio, weight gain and increased growth performance. In the present study, isolation and identification of a novel alkaline protease from Iranian Bacillus species was performed in order to use in livestock feed. After primary isolation of bacteria from soil samples of rice fields and early detection of bacterial genus, the zymogram plate was performed for evaluation of production extracellular proteases. Of the 11 strains producing protease, one strain that produced more enzymes was selected to continue the work. Characterization of alkaline protease was done using specific enzyme assays. To confirm the genus of isolates as well as to identify the species close to, molecular analysis of 16S rRNA gene sequence was done. After that, bioinformatics analysis carried out in NCBI database for searching bacterial alkaline proteases gene sequences. The primer designed based on gene homology of close species for extraction of alkaline protease gene. The results showed that the enzyme extract had the highest activity at pH 9.0 and 50 °C. The 16S rRNA gene sequence was submitted for the strain called Bacillus sp. RAM on the NCBI site. According to the results of the phylogenetic tree, the bacterium was belonged to Bacillus genus and Bacillus sp. RAM was close to Thuringiensis C405. The isolated alkaline protease gene successfully cloned in pET28a and transferred to the expression host E.coli BL21. The expression of the protease gene was evaluated by SDS-PAGE electrophoresis. The induced recombinant cells expressed the protease and revealed molecular weight band of about 38 kDa. According to the enzyme properties, this alkaline protease can useful for application in animal industry.

Isolation, purification and characterization of a surfactants-, laundry detergents- and organic solvents-resistant alkaline protease from Bacillus sp. HR-08.

Bacillus sp. HR-08 screened from soil samples of Iran, is capable of producing proteolytic enzymes. 16S rDNA analysis showed that this strain is closely related to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, and Bacillus atrophaeus. The zymogram analysis of the crude extract revealed the presence of five extracellular proteases. One of the proteases was purified in three steps procedure involving ammonium sulfate precipitation, DEAE-Sepharose ionic exchange and Sephacryl S-200 gel filtration chromatography. The molecular mass of the enzyme on SDS-PAGE was estimated to be 29 kDa. The protease exhibited maximum activity at pH 10.0 and 60 degrees C and was inhibited by PMSF but it was not affected by cysteine inhibitors, suggesting that the enzyme is a serine alkaline protease. Irreversible thermoinactivation of enzyme was examined at 50, 60, and 70 degrees C in the presence of 10 mM CaCl(2). Results showed that the protease activity retains more than 80% and 50% of its initial activity after incubation for 30 min at 60 and 70 degrees C, respectively. This enzyme had good stability in the presence of H(2)O(2), nonionic surfactant, and local detergents and its activity was enhanced in the presence of 20% of dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and isopropanol. The enzyme retained more than 90% of its initial activity after pre-incubation 1 h at room temperature in the presence of 20% of these solvents. Also, activation can be seen for the enzyme at high concentration (50%, v/v) of DMF and DMSO.

The crystal structures of ornithine carbamoyltransferase from Mycobacterium tuberculosis and its ternary complex with carbamoyl phosphate and L-norvaline reveal the enzyme's catalytic mechanism.

Mycobacterium tuberculosis ornithine carbamoyltransferase (Mtb OTC) catalyzes the sixth step in arginine biosynthesis; it produces citrulline from carbamoyl phosphate (CP) and ornithine (ORN). Here, we report the crystal structures of Mtb OTC in orthorhombic (form I) and hexagonal (form II) space groups. The molecules in form II are complexed with CP and l-norvaline (NVA); the latter is a competitive inhibitor of OTC. The asymmetric unit in form I contains a pseudo hexamer with 32 point group symmetry. The CP and NVA in form II induce a remarkable conformational change in the 80s and the 240s loops with the displacement of these loops towards the active site. The displacement of these loops is strikingly different from that seen in other OTC structures. In addition, the ligands induce a domain closure of 4.4 degrees in form II. Sequence comparison of active-site residues of Mtb OTC with several other OTCs of known structure reveals that they are virtually identical. The interactions involving the active-site residues of Mtb OTC with CP and NVA and a modeling study of ORN in the form II structure strongly rule out an earlier proposed mechanistic role of Cys264 in catalysis and suggest a possible mechanism for OTC. Our results strongly support the view that ORN with an already deprotonated N(epsilon) atom is the species that binds to the enzyme and that one of the phosphate oxygen atoms of CP is likely to be involved in accepting a proton from the doubly protonated N(epsilon) atom of ORN. We have interpreted this deprotonation as part of the collapse of the transition state of the reaction.

Crystal structure of N-acetyl-gamma-glutamyl-phosphate reductase from Mycobacterium tuberculosis in complex with NADP(+).

The enzyme N-acetyl-gamma-glutamyl-phosphate reductase (AGPR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductive dephosphorylation of N-acetyl-gamma-glutamyl-phosphate to N-acetylglutamate-gamma-semialdehyde. This reaction is part of the arginine biosynthetic pathway that is essential for some microorganisms and plants, in particular, for Mycobacterium tuberculosis (Mtb). The structures of apo MtbAGPR in the space groups P2(1)2(1)2(1) and C2 and the structure of MtbAGPR bound to the cofactor NADP(+) have been solved and analyzed. Each MtbAGPR subunit consists of alpha/beta and alpha+beta domains; NADP(+) is bound in the cleft between them. The hydrogen bonds and hydrophobic contacts between the enzyme and cofactor have been examined. Comparison of the apo and the bound enzyme structures has revealed a conformational change in MtbAGPR upon NADP(+) binding. Namely, a loop (Leu88 to His92) moves more than 5 A to confine sterically the cofactor's adenine moiety in a hydrophobic pocket. To identify the catalytically important residues in MtbAGPR, a docking of the substrate to the enzyme has been performed using the present structure of the MtbAGPR/NADP(+) complex. It reveals that residues His217 and His219 could form hydrogen bonds with the docked substrate. In addition, an ion pair could form between the substrate phosphate group and the guanidinium group of Arg114. These interactions optimally place and orient the substrate for subsequent nucleophilic attack by Cys158 on the substrate gamma-carboxyl group. His219 is the most probable general base to accept a proton from Cys158 and an adjacent ion pair interaction with the side-chain carboxyl group of Glu222 could help to stabilize the resulting positive charge on His219. For this catalytic triad to function efficiently it requires a small conformational change of the order of 1 A in the loop containing His217 and His219; this could easily result from the substrate binding.

Expression, purification, crystallization and preliminary X-ray analysis of two arginine-biosynthetic enzymes from Mycobacterium tuberculosis.

The gene products of two open reading frames from Mycobacterium tuberculosis (Mtb) have been crystallized using the sitting-drop vapour-diffusion method. Rv1652 encodes a putative N-acetyl-gamma-glutamyl-phosphate reductase (MtbAGPR), while the Rv1656 gene product is annotated as ornithine carbamoyltransferase (MtbOTC). Both MtbAGPR and MtbOTC were expressed in Escherichia coli, purified to homogeneity and crystallized. Native data for each crystal were collected to resolutions of 2.15 and 2.80 A, respectively. Preliminary X-ray data are presented for both enzymes.

Thiol-dependent serine alkaline proteases from Bacillus sp. HR-08 and KR-8102: isolation, production, and characterization.

Two Bacillus sp. strains, HR-08 and KR-8102, isolated from soil of the west and north parts of Iran were screened on gelatin agar medium for their ability to produce alkaline protease. The enzymes were active in a wide pH range (6.0-11.0) and stable in the alkaline range (7.0-12.0). The optimum temperatures for the protease from HR-08 and KR-8102 were 65 and 50 degrees C, respectively. The irreversible thermoinactivation of HR-08 and KR-8102 proteases showed that the stability of HR-08 enzyme was higher than that of KR-8102 and the half-lives of these enzymes were 95 and 32 min at 50 degrees C, respectively. In the presence of 10 mM Ca(2+), HR-08 retained 100, 90, and 20% of its initial activity after heating for 30 min at 50, 60, and 70 degrees C, respectively. Enzymes were inhibited by phenylmethylsulfonyl fluoride and iodoacetate. After inhibition by iodoacetate, both enzymes were reactivated by dithiothreitol. These data show that the enzymes seem to be thiol-dependent serine alkaline proteases. The enzymes especially from HR-08 were stable in the presence of H(2)O(2), surfactants, and local detergents; their activities were enhanced in the presence of 5 mM Fe(2+); and the presence of 5 mM metal ions such as Mg(2+), Cu(2+), and Mn(2+) produced almost no effect.