Mechanistic studies on substrate inhibition and substrate activation of ribonuclease A: experimental and in silico investigation.

Ribonuclease A (RNase A) is an endonuclease that plays a significant role in antimicrobial activity by the cleavage and hydrolysis of ssRNA. In this study, the interactions between RNase A and cCMP have been investigated, through molecular docking and a 200 ns molecular dynamics simulation. The enzyme kinetic properties were analyzed using saturation curve, Eadie-Hofstee, and Hill plots. The docking results indicate that the cCMP-RNase A complexes are stabilized through hydrophobic interaction, hydrogen bonding, and π-π stacking interaction. The most binding site is observed in the catalytic cleft, especially at residue His12 and His119. Enzyme-ligand docking study indicates that cCMP binds to residues located in the internal cavity of the catalytic site and shows three phases of binding modes. The analysis of MD simulations shows that RMSD, Rg, and RMSF reach equilibrium. The ΔGbinding of the cCMP-RNase A was negative (-619.673 kJ/mol), The comparison between the results pre and post MD simulation showed that ΔGbinding after MD simulation causes to more stable the structure than before simulation. Experimental methods such as saturation, Eadie-Hofstee, and Hill plots confirm theoretical data and show three phases of binding modes as well. Two significant events are demonstrated in the interaction between RNase A and cCMP: substrate activation and substrate inhibition are observed in concentrations below and above 0.8 mM, respectively, for cCMP. Choosing the appropriate concentration of cCMP is very important in investigation of ribonuclease A's catalytic behavour, especially for exploration of the effects of some drugs on biological behaviours related to ribonuclease A.Communicated by Ramaswamy H. Sarma.

Dermokine mutations contribute to epithelial-mesenchymal transition and advanced melanoma through ERK/MAPK pathways.

To discover vulnerabilities associated with dermokine (DMKN) as a new trigger of the epithelial-mesenchymal transition (EMT) -driven melanoma, we undertook a genome-wide genetic screening using transgenic. Here, we showed that DMKN expression could be constitutively increased in human malignant melanoma (MM) and that this correlates with poor overall survival in melanoma patients, especially in BRAF-mutated MM samples. Furthermore, in vitro, knockdown of DMKN inhibited the cell proliferation, migration, invasion, and apoptosis of MM cancer cells by the activation of ERK/MAPK signaling pathways and regulator of STAT3 in downstream molecular. By interrogating the in vitro melanoma dataset and characterization of advanced melanoma samples, we found that DMKN downregulated the EMT-like transcriptional program by disrupting EMT cortical actin, increasing the expression of epithelial markers, and decreasing the expression of mesenchymal markers. In addition, whole exome sequencing was presented with p.E69D and p.V91A DMKN mutations as a novel somatic loss of function mutations in those patients. Moreover, our purposeful proof-of-principle modeled the interaction of ERK with p.E69D and p.V91A DMKN mutations in the ERK-MAPK kinas signaling that may be naturally associated with triggering the EMT during melanomagenesis. Altogether, these findings provide preclinical evidence for the role of DMKN in shaping the EMT-like melanoma phenotype and introduced DMKN as a new exceptional responder for personalized MM therapy.

Treatment of malignant pleural effusion in non-small cell lung cancer with VEGF-directed therapy.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a critical regulator of malignant pleural effusion (MPE) in non-small-cell lung cancer (NSCLC). Bevacizumab (BEV) and apatinib (APA) are novel VEGF blockers that inhibit lung cancer cell proliferation and the development of pleural effusion. METHODS: In this study, we established Lewis lung cancer (LLC) xenograft mouse models to compare the therapeutic effect of APA and BEV in combination with cisplatin (CDDP) against MPE. The anti-tumour and anti-angiogenic effects of this combination therapy were evaluated by 18F-FDG PET/CT imaging, TUNEL assay and Immunohistochemistry. RESULTS: The triple drug combination significantly prolonged the overall survival of the tumour-bearing mice by reducing MPE and glucose metabolism and was more effective in lowering VEGF/soluble VEGFR-2 levels in the serum and pleural exudates compared to either of the monotherapies. Furthermore, CDDP + APA + BEV promoted in vivo apoptosis and decreased microvessel density. CONCLUSIONS: Mechanistically, LLC-induced MPE was inhibited by targeting the VEGF-MEK/ERK pathways. Further studies are needed to establish the synergistic therapeutic effect of these drugs in NSCLC patients with MPE.KEY MESSAGESCombined treatment of MPE with apatinib, bevacizumab and cisplatin can prolong the survival time of mice, reduce the content of MPE, decrease the SUVmax of thoracic tumour tissue, down-regulate the content of VEGF and sVEGFR-2 in serum and pleural fluid, and promote the apoptosis of tumour cells. Angiogenesis and MPE formation can be inhibited by down-regulation of HIF-1α, VEGF, VEGFR-2, MEK1 and MMP-2 molecular signalling pathway proteins.

Protective Effect of Low Dose of Methamphetamine on The Amount of Extracellular Glutamine in Primary Fetal Human Astrocytes Induced by Amyloid Beta.

Objective: Change in astrocytes is one of the first pathological symptoms of Alzheimer's disease (AD). Understanding the signaling pathways in astrocytes can be a great help in treating of AD. This study aimed to investigate signaling pathway relations between low dose of methamphetamine (METH), the apoptosis, cell cycle, and glutamine (Gln) pathways in the activated astrocyte. Materials and Methods: In this experimental study, the activated astrocyte cells were exposed to a low dose of METH (12.5 µM) which was determined by Thiazolyl blue tetrazolium bromide (MTT) method. The groups were: group 1 cells with Aß, group 2 cells with METH, group 3 cells with METH after 24 hours of adding Aß (Aß+METH, treated group), group 4 cells with Aß after 24 hours of adding METH (METH+Aß, prevention group), and group 5 as the control. The Gln was assayed by high-performance liquid chromatography (HPLC), and also the apoptosis, and cell cycle and BAX, BCL-X expression was evaluated. Results: The amount of Gln was increased, and the value of late and early apoptosis was reduced in the treatment groups, and necrosis is decreased in the prevention group (group 4 compared to group 1). Moreover, it was revealed through cell cycle analysis that G2 in group 4 was reduced compared to group 1 and the expression of BAX, BAX/ BCL-X, and BCL-X in group 3 and group 4, was decreased and increased, respectively compared to group 1. Conclusion: These findings suggest that perhaps a non-toxic dosage of METH (low dose) can reduce the amount of apoptosis and BAX expression and increase the expression of BCL-X. Furthermore, the cells are arrested in the G2 phase and can raise the amount of extracellular glutamine, which has a protective role in neuron cells. These findings may provide a new perspective to design a new drug with less toxic results.

Alteration of gene expression in reactive astrocytes induced by Aß1-42 using low dose of methamphetamine.

BACKGROUND: Alzheimer's disease (AD) is a degenerative brain disorder. Due to the relationship between the functional loss of astrocytes and AD, the present study aims to evaluate the effects of the low dose of methamphetamine (METH) on primary fetal human astrocytes under a stress paradigm as a possible model for AD. METHODS AND RESULTS: The groups in this study included Aß (Group 1), METH (Group 2), Aß + METH (METH after adding Aß for 24 h) (Group 3 as treated group), METH + Aß (Aß after adding METH for 24 h) (Group 4 as prevention group), and control group. Then, the gene expression of Bax, Bcl-X, PKCα, GSK3ß, and Cdk5 was evaluated. In addition, phosphorylated tau, p-GSK3ß, GSK3ß, and GSK3α proteins were assessed by western blotting. Further, cell cycle arrest and apoptosis were checked by flow cytometry and Hoechst staining. Based on the results, the expression of GSK3ß, Cdk5, and PKCα genes decreased in the prevention group, while GSK3ß and Cdk5 were amplified in the treatment group. Furthermore, the level of GSK3α and GSK3ß proteins in the treatment group increased, while it decreased in the prevention group. Additionally, a decrease occurred in the percentage of necrosis and early apoptosis in the treatment and prevention groups. The results of the cell cycle indicated that G1 increased, while G2 decreased in the prevention group. CONCLUSION: The pure form of METH can prevent from activating GSK-3ß and CdK-5, as well as enhanced activity of PKCα to inhibit phosphorylated tau protein. Therefore, a low dose of METH may have a protective effect or reducing role in the pathway of tau production in reactive astrocytes.

Co-expression Network Analysis Reveals Key Genes Related to Ankylosing spondylitis Arthritis Disease: Computational and Experimental Validation.

BACKGROUND: Ankylosing spondylitis (AS) is a type of arthritis which can cause inflammation in the vertebrae and joints between the spine and pelvis. However, our understanding of the exact genetic mechanisms of AS is still far from being clear. OBJECTIVE: To study and find the mechanisms and possible biomarkers related to AS by surveying inter-gene correlations of networks. MATERIALS AND METHODS: A weighted gene co-expression network was constructed among genes identified by microarray analysis, gene co-expression network analysis, and network clustering. Then receiver operating characteristic (ROC) curves were conducted to identify a significant module with the genes implicated in the AS pathogenesis. Real-time PCR was performed to validate the results of microarray analysis. RESULTS: In the significant module obtained from the network analysis there were eight AS related genes (LSM3, MRPS11, NSMCE2, PSMA4, UBL5, RPL17, MRPL22 and RPS17) which have been reported in previous studies as hub genes. Further, in this module, eight significant enriched pathways were found with adjusted p-values < 0.001 consisting of oxidative phosphorylation, ribosome, nonalcoholic fatty liver disease, Alzheimer's, Huntington's, and Parkinson's diseases, spliceosome, and cardiac muscle contraction pathways which have been linked to AS. Furthermore, we identified nine AS related genes (UQCRB, UQCRH, UQCRHL, UQCRQ, COX7B, COX5B, COX6C, COX6A1 and COX7C) in these pathways which can play essential roles in controlling mitochondrial activity and pathogenesis of autoimmune diseases. Real-time PCR results showed that three genes including UQCRH, MRPS11, and NSMCE2 in AS patients were significantly differentially expressed compared with normal controls. CONCLUSIONS: The results of the present study may contribute to understanding of AS molecular pathogenesis, thereby aiding the early prognosis, diagnosis, and effective therapies of the disease.

In vitro investigation of the effect of mesalazine on amyloid fibril formation of hen egg-white lysozyme and defibrillation lysozyme fibrils.

In certain conditions (such as fever and stress) mutated lysozyme enzyme deposits in different tissues and organs in the form of amyloid fibrils (plaques). These aggregates lead to tissue destruction and the pathogenesis of a disease. In this study, we investigated the in vitro effects of mesalazine drug on both preventions of lysozyme aggregation and the removal of lysozyme fibrils. With this regard, hen egg-white lysozyme (HEWL) was incubated in the absence and presence of mesalazine in high temperature and acidic pH conditions. The influence of mesalazine was surveyed by Congo red (CR) absorbance, Circular dichroism spectroscopy (CD), Thioflavin T (ThT) fluorescence assay, 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence test, and Field-emission scanning electron microscopy (FE-SEM). Our results demonstrate that mesalazine, in all concentrations, especially in 1:1 and higher drug to protein ratios, has a strong inhibitory effect on protein fibrillation. Additionally, mesalazine does not show an acceptable impact on the reversibility of HEWL fibril in any of the related concentrations. Based on the obtained results, we conclude that mesalazine can be used as a drug for the prevention of amyloid-fibrils formation in hereditary lysozyme amyloidosis and other systemic non-neuropathic hereditary amyloidosis.

The Implication of Androgens in the Presence of Protein Kinase C to Repair Alzheimer's Disease-Induced Cognitive Dysfunction

Aging, as a major risk factor of memory deficiency, affects neural signaling pathways in hippocampus. In particular, age-dependent androgens deficiency causes cognitive impairments. Several enzymes like protein kinase C (PKC) are involved in memory deficiency. Indeed, PKC regulatory process mediates α-secretase activation to cleave APP in ß-amyloid cascade and tau proteins phosphorylation mechanism. Androgens and cortisol regulate PKC signaling pathways, affecting the modulation of receptor for activated C kinase 1. Mitogen-activated protein kinase/ERK signaling pathway depends on CREB activity in hippocampal neurons and is involved in regulatory processes via PKC and androgens. Therefore, testosterone and PKC contribute in the neuronal apoptosis. The present review summarizes the current status of androgens, PKC, and their influence on cognitive learning. Inconsistencies in experimental investigations related to this fundamental correlation are also discussed, with emphasis on the mentioned contributors as the probable potent candidates for learning and memory improvement.

Protein Kinase Cɛ in the Platelet and Hippocampal Tissue as a Diagnostic Biological Marker in Alzheimer Disease.

INTRODUCTION: Alzheimer Disease (AD) is a neurodegenerative disorder characterized by the progressive loss of memory and other cognitive functions. Protein Kinase Cɛ (PKCɛ) is an isoform that most effectively suppresses Amyloid Beta (Aß) production and synaptic loss. METHODS: In this study, spatial learning and memory for treated rats were evaluated by the Morris water maze test. The activity (total PKC), mRNA expression, and protein level of PKCɛ in the platelet and hippocampal tissue were evaluated using immunosorbent assay, real-time qPCR, and western blotting analysis, respectively. RESULTS: The traveled distance was significantly prolonged, and escape latency significantly increased in Aß-treated groups. PKC activity assay showed that there was a remarkable difference between the Aß-treated and sham-operated groups on days 10 and 30 in the hippocampus and also day 30 in platelet after the injection of Aß. A significant effect in PKC activity was observed between days 0 and 10, days 0 and 30, as well as days 5 and 30. Aß significantly downregulated the PKCɛ mRNA expression in the hippocampus of rats on day 30; however, no significant difference was observed in platelet. Western blot analysis demonstrated that Aß significantly reduced PKCɛ protein expression in the hippocampus of treated groups on day 30. CONCLUSION: The expression level of PKCɛ was downregulated following the injection of Aß in the hippocampus, but no significant difference was observed between the AD and sham groups in platelet that may be due to the low concentration of PKCɛ or duration of Aß exposure in the rat brain.

The importance of α-CT and Salt bridges in the Formation of Insulin and its Receptor Complex by Computational Simulation.

Insulin hormone is an important part of the endocrine system. It contains two polypeptide chains and plays a pivotal role in regulating carbohydrate metabolism. Insulin receptors (IR) located on cell surface interacts with insulin to control the intake of glucose. Although several studies have tried to clarify the interaction between insulin and its receptor, the mechanism of this interaction remains elusive because of the receptor's structural complexity and structural changes during the interaction. In this work, we tried to fractionate the interactions. Therefore, sequential docking method utilization of HADDOCK was used to achieve the mentioned goal, so the following processes were done: the first, two pdb files of IR i.e., 3LOH and 3W11 were concatenated using modeller. The second, flexible regions of IR were predicted by HingeProt. Output files resulting from HingeProt were uploaded into HADDOCK. Our results predict new salt bridges in the complex and emphasize on the role of salt bridges to maintain an inverted V structure of IR. Having an inverted V structure leads to activate intracellular signaling pathway. In addition to presence salt bridges to form a convenient structure of IR, the importance of α-chain of carboxyl terminal (α-CT) to interact with insulin was surveyed and also foretokened new insulin/IR contacts, particularly at site 2 (rigid parts 2 and 3). Finally, several conformational changes in residues Asn711-Val715 of α-CT were occurred, we suggest that α-CT is a suitable situation relative to insulin due to these conformational alterations.

Effects of Herbal Compound (IMOD) on Behavior and Expression of Alzheimer's Disease Related Genes in Streptozotocin-Rat Model of Sporadic Alzheimer's Disease.

Purpose: Sporadic Alzheimer's disease (AD) accounts for over 95% of cases. Possible mechanisms of AD such as inflammation and oxidative stresses in the brain motivate researchers to follow many therapies which would be effective, especially in the early stages of the disease. IMOD, the herbal extract of R. Canina, T. Vulgare and U. Dioica plant species enriched with selenium, has anti-inflammatory, immunoregulatory and protective effects against oxidative stress. Methods: In this study three AD-related genes, DAXX, NFκß and VEGF, were chosen as candidate to investigate the neuroprotective effect of the extract by comparing their expression levels in the hippocampus of rat model of sporadic AD, using qPCR in the herbal-treated and control groups. The therapeutic effects on learning and memory levels were evaluated by Morris Water Maze (MWM) test. Results: Gene expression results were indicative of significant up-regulation of Vegf in rat's hippocampus after treatment with the herbal extract comparing to model group (P-value= 0.001). The MWM results showed significant changes in path length and time for finding the hidden platform in all groups during test and the same change in the treated comparing to the control group in memory level. Conclusion: It could be concluded that the herbal extract may have significant effect on gene expression but not on behavioral level.

The Effects of Melilotus officinalis Extract on Expression of Daxx, Nfkb and Vegf Genes in the Streptozotocin-Induced Rat Model of Sporadic Alzheimer's Disease.

BACKGROUND: Possible mechanisms of Alzheimer Disease (AD) such as inflammation and oxidative stresses in the brain led us to investigate potential AD therapeutics of Melilotus officinalis, an herbal extract, with possible role as an anti-inflammatory and anti-oxidant agent. Among different genes which had important role in Sporadic AD (SAD), three genes including DAXX, NFkB and VEGF have shown significant statistical diversity in the brains of Alzheimer patients. METHODS: These genes were chosen to be investigated for neuroprotective effects of the extract by comparing the expression level in the hippocampus of Sporadic AD (SAD) rat model using quantitative polymerase chain reaction (qPCR) in the treated and untreated groups. In addition, therapeutic effects at the behavioral, learning and memory level by Morris Water Maze (MWM) test were investigated. RESULTS: The results represented significant decreased expression in Daxx, Nfkb and Vegf genes in the SAD rat's model treated with the herbal extract compared to the Streptozotocin-induced (STZ-induced) rats. Furthermore, no significant changes were seen in swimming distance and time for finding the hidden platform in the herbal-treated compared to the STZ-induced group. In memory level, no significant changes were observed among treated and untreated groups. CONCLUSION: It seems that the herbal extract may have significant effect on Alzheimer-related gene expression changes but not on clinical levels.

The Association Between Effective Dose of Magnesium and Mild Compulsive Exercise on Spatial Learning, Memory, and Motor Activity of Adult Male Rats.

One of the most important survival mechanisms is learning and memory processes. To emphasize the role of physical exercises and magnesium (Mg) in improvement of cognitive performance, we planned to investigate the effect of Mg and mild compulsive exercise on spatial learning and memory of adult male rats. Accordingly, we divided male Wistar rats into four groups: (I) control, (II) Mg treatment, (III) exercise, and (IV) Mg-exercise in the different dosages of Mg (0.5, 1, 1.5, and 2 mmol/kbw) were injected in the form of gavage during 1 week. Also, 1-week mild running on treadmill was used for exercise treatment. The Morris water maze (MWM) test and open field tool were used to evaluate spatial learning, memory, and motor activity, respectively. Our results clearly showed that 1 mmol/kbw Mg was applied as an effective dosage. Strikingly, 1-week mild exercise on treadmill had no significant effect on spatial motor activity, learning, and memory. Feeding 1 mmol/kbw Mg for a week showed a significant difference in learning and exploration stages. Compared to control animals, these results reveal exercise and Mg simultaneously had effect on learning and reminding. As a consequence, although mild exercise had no effect on motor activity and memory, Mg intake improved spatial learning, memory, and locomotor activity. The Mg feeding could be a promising supplemental treatment in the neurodegenerative disease. It is worthwhile to mention consumption of Mg leads to enhancement of memory, so animals find the hidden platform with the highest velocity.

Effects of Ectoine on Behavior and Candidate Genes Expression in ICV-STZ Rat Model of Sporadic Alzheimer's Disease.

Purpose: Alzheimer's disease (AD) is pathologically defined by the presence of amyloid plaques and tangles in the brain, therefore, any drug or compound with potential effect on lowering amyloid plaques, could be noticed for AD management especially in the primary phases of the disease. Ectoine constitutes a group of small molecule chaperones (SMCs). SMCs inhibit proteins and other changeable macromolecular structures misfolding from environmental stresses. Ectoine has been reported successfully prohibit insulin amyloid formation in vitro. Methods: We selected eight genes, DAXX, NFκß, VEGF, PSEN1, MTAP2, SYP, MAPK3 and TNFα genes which had previously showed significant differential expression in Alzheimer human brain and STZ- rat model. We considered the neuroprotective efficacy by comparing the expression of candidate genes levels in the hippocampus of rat model of Sopradic Alzheimer's disease (SAD), using qPCR in compound-treated and control groups as well as therapeutic effects at learning and memory levels by using Morris Water Maze (MWM) test. Results: Our results showed significant down-regulation of Syp, Mapk3 and Tnfα and up-regulation of Vegf in rat's hippocampus after treatment with ectoine comparing to the STZ-induced group. In MWM, there was no significant change in swimming distance and time for finding the hidden platform in treated comparing to STZ-induced group. In addition, it wasn't seen significant change in compound-treated comparing to STZ-induced and control groups in memory level. Conclusion: It seems this compound may have significant effect on expression level of some AD- related genes but not on clinical levels.

Neuroprotective Effects of Herbal Extract (Rosa canina, Tanacetum vulgare and Urtica dioica) on Rat Model of Sporadic Alzheimer's Disease.

BACKGROUND: Sporadic Alzheimer's Disease (SAD) is caused by genetic risk factors, aging and oxidative stresses. The herbal extract of Rosa canina (R. canina), Tanacetum vulgare (T. vulgare) and Urtica dioica (U. dioica) has a beneficial role in aging, as an anti-inflammatory and anti-oxidative agent. In this study, the neuroprotective effects of this herbal extract in the rat model of SAD was investigated. METHODS: The rats were divided into control, sham, model, herbal extract -treated and ethanol-treated groups. Drug interventions were started on the 21(st) day after modeling and each treatment group was given the drugs by intraperitoneal (I.P.) route for 21 days. The expression levels of the five important genes for pathogenesis of SAD including Syp, Psen1, Mapk3, Map2 and Tnf-α were measured by qPCR between the hippocampi of SAD model which were treated by this herbal extract and control groups. The Morris Water Maze was adapted to test spatial learning and memory ability of the rats. RESULTS: Treatment of the rat model of SAD with herbal extract induced a significant change in expression of Syp (p=0.001) and Psen1 (p=0.029). In Morris Water Maze, significant changes in spatial learning seen in the rat model group were improved in herbal-treated group. CONCLUSION: This herbal extract could have anti-dementia properties and improve spatial learning and memory in SAD rat model.

Determination of the Best Concentration of Streptozotocin to Create a Diabetic Brain Using Histological Techniques.

Alzheimer's disease (AD) is one of the most important disorders among neurodegenerative diseases which is characterized by neurofibrillary tangles and senile plagues. Intercerebroventricular (ICV) streptozotocin administration is a form of sAD which was applied to examine different factors following AD. Previous reports used different doses of streptozotocin (STZ) to create Alzheimer's model, but no standard dose has been introduced. Therefore, we decided to investigate the best concentration of STZ to induce a diabetic brain with lowest mortality rate and high severity of destruction. We treated rats with three different doses of STZ (STZ 1.5, 2.25, and 3 mg/kg, ICV). Spatial memory for treated rats was evaluated by Morris water maze (MWM). Locomotor activities of rats were assessed by open field test. Histological observation such as immunohistochemistry, immunofluorescence, and Nissl staining were performed on the brain especially in CA1, CA3, and DG regions of hippocampal neurons at residues P-ser396 and P-ser404. Our data suggest that although the percentage hyperphosphorylation of tau protein by injection of STZ 3 mg/kg was about 10 % more than STZ 2.25 mg/kg compared to the control group, we considered the latter doses due to no effect on motor activities and enhance the number of glial cells.

Heterotropic Effect of ß-lactam Antibiotics on Antioxidant Property of Haptoglobin (2-2)-Hemoglobin Complex.

Haptoglobin (Hp) is a mammalian serum glycoprotein showing a genetic polymorphism with three types, 1-1, 2-2 and 1-2. Hp appears to conserve the recycling of heme-iron by forming an essentially irreversible but non-covalent complex with hemoglobin which is released into the plasma by erythrocyte lysis. As an important consequence, Haptoglobin-Hemoglobin complex (Hp-Hb) shows considerable antioxidant property. In this study, antioxidant activity of Hp (2-2)-Hb complex on hydrogen peroxide has been studied and analyzed in the absence and presence of two beta-lactam antibiotics in-vitro. For this purpose, non-Michaelis behavior of peroxidase activity of Hp (2-2)-Hb complex was analyzed using Eadie-Hofstee, Clearance and Hill plots, in the absence and presence of pharmaceutical dose of ampicillin and coamoxiclav. The results have shown that peroxidase activity of Hp (2-2)-Hb complex is modulated via homotropic effect of hydrogen peroxide as an allostric substrate. On the other hand antioxidant property of Hp (2-2)-Hb complex increased via heterotropic effect of both antibiotics on the peroxidase activity of the complex. Both drugs also have mild effect on quality of homotropic property of the peroxidase activity of Hp (2-2)-Hb complex. Therefore, it can be concluded from our study that both beta-lactam antibiotics can increase peroxidase activity of Hp (2-2)-Hb complex via heterotropic effect. Thus, the two antibiotics (especially ampicillin) may help those individuals with Hp (2-2) phenotype to improve the Hp-Hb complex efficiency of removing hydrogen peroxide from serum under oxidative stress. This can be important in the individuals with phenotype Hp 2-2 who have less antioxidant activity relative to other phenotypes and are susceptible to cardiovascular disorders, as has been reported by other researchers.