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Purpose: L-asparaginase has been widely recognized as a critical component in the treatment of various types of lymphoproliferative disorders, since its introduction in 1960s. However, its use in some cases leads to allergic reactions rendering the continuation of treatment unfeasible. Thus, the development of L-asparaginase from alternative sources or the production of engineered enzymes have always been considered. This study aimed to produce and evaluate a novel enzyme designed based on the sequence of L-asparaginase from Escherichia coli bacteria with Y176F/S241C mutations. Methods: The Y176F/S241C mutant L-asparaginase was successfully expressed as the GST-fusion protein in E. coli , and then was subjected to affinity and size exclusion chromatography. The activity of the purified enzyme was determined based on the released ammonia as the result of substrate hydrolysis using Nessler's reagent. Chemical denaturation experiment in the presence of increasing concentration of guanidinium chloride was applied to determine the folding stability of the purified enzyme. Results: The mutant enzyme was purified with an efficiency of 77-fold but at a low recovery of 0.7%. The determined kinetic parameters Km, Vmax, kcat, specific activity and catalytic efficiency were 13.96 (mM), 2.218 (mM/min), 273.9 (min-1), 237.8 (IU/mg) and 19.62 (mM-1 min-1), respectively. Moreover, unfolding free energy determined by guanidinium chloride induced denaturation for mutated and commercial L-asparaginase enzymes were 8421 J/mol and 5274 J/mol, respectively. Conclusion: The mutant enzyme showed improved stability over the wild-type. Although the expression level and recovery were low, the mutant L-asparaginase demonstrated promising activity and stability, with potential clinical and industrial applications. [ABSTRACT FROM AUTHOR]

Given multiple treatment strategies for prostate cancer, its mortality rate is still high; therefore, novel treatment strategies seem necessary. G2013 or α‐L‐guluronic acid is a new patented drug with immunomodulatory and anti‐inflammatory properties. This study aimed to evaluate the property of G2013 on inflammatory molecules involved in tumorigenesis of prostate cancer. MTT assay was used to assess the effect of the drug on the proliferation of PC‐3 cells. Expression of interleukin 8 (IL‐8), Nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB), myeloid differentiation factor 88 (MYD‐88), cyclooxygenase 2 (COX‐2), matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 genes were studied in the PC‐3 cells treated with 25 (low dose) or 50 (high dose) µg/mL of G2013 for 24 h using quantitative real‐time polymerase chain reaction (qRT‐PCR) technique. Protein expression of NF‐κB and protein activities of MMP‐2 and MMP‐9 were assayed using flow cytometry and gelatin zymography, respectively. The expression of COX‐2 (p = 0.007 at low dose), MMP‐2 (p = 0.023 at low dose, p = 0.002 at high dose), NF‐κB (p = 0.004 at low dose) and IL‐8 (p < 0.0001 in both doses) genes, NF‐κB protein (p < 0.0001 in both doses), and MMP‐2 activity (p < 0.0001 in both doses) were significantly reduced in the presence of G2013 as compared to the control group. Cancer cell proliferation was also inhibited under 10–500 µg/mL G2013 treatment. Our results revealed that G2013 has the potential to inhibit PC‐3 cell proliferation and reduce the expression of tumour‐promoting mediators, COX‐2, MMP‐2, NF‐κB, and IL‐8 involved in the progression and metastasis of prostate cancer. [ABSTRACT FROM AUTHOR]

Summary: In vitro experiments demonstrated that stimulation of Toll‐like receptor 9 (TLR‐9) by synthetic TLR‐9 ligands induces the invasion of TLR‐9‐expressing prostate cancer cells through matrix metalloproteinase 13 (MMP‐13). However, the clinical value of TLR‐9 and MMP‐13 co‐expression in the pathophysiology of the prostate is unknown. In the study, we evaluated the expression levels and clinical significance of the TLR‐9 and MMP‐13 in a series of prostate tissues. One hundred and eighty prostate tissues including prostate cancer (PCa) (n = 137), high‐grade prostatic intraepithelial neoplasia (HPIN) (n = 18) and benign prostatic hyperplasia (BPH) (n = 25) were immunostained for the TLR‐9 and MMP‐13 markers. Subsequently, the correlation between the TLR‐9 and MMP‐13 staining scores and clinicopathological parameters was obtained. Higher expressions of TLR‐9 and MMP‐13 were found in PCa and high‐grade prostatic intraepithelial neoplasia compared to benign prostatic hyperplasia tissues. Among PCa samples, a positive relationship was revealed between the MMP‐13 expression and Gleason score (P < 0.001). There was a significant correlation between TLR‐9 expression and regional lymph node involvement (P = 0.04). The expression patterns of TLR‐9 and MMP‐13 markers demonstrated a reciprocal significant correlation between the two markers in the same series of prostate samples (P < 0.001). Furthermore, the Gleason score of TLR‐9high/MMP‐13high and TLR‐9low/MMP‐13low phenotypes showed a significant difference (P = 0.002). Higher expressions of TLR‐9 and MMP‐13 can confer aggressive behaviour to PCa. Therefore, these markers may be used as a valuable target for tailored therapy of PCa. [ABSTRACT FROM AUTHOR]

Background: The CCR5 is a chemokine receptor that serves as a co-receptor for HIV-1 attachment and entry to T lymphocytes. A 32bp deletion (Δ32) in this gene is believed to be associated with resistance to infection and delay disease progression. The aim of this study was to determine the Δ32 allele frequency in healthy individuals and HIV-infected individuals with AIDS. Methods: In this experiment, 530 normal individuals from healthy Iranian population and 40 HIV-infected samples from Western Clinic of Tehran were examined for Δ32 in CCR5 gene using polymerase chain reaction (PCR) techniques followed by agarose gel electrophoresis. Results: Allele frequencies of the CCR5Δ32 in normal individuals were calculated to be 1.1% for heterozygous genotype and 0.19% for homozygous genotype. None of the co-receptor gene in HIV cases was found to be mutated in this study. Conclusion: Based on the findings of this study and the literature in Iran, we could conclude that Iranian people similar to neighbor countries such as Arabs are susceptible to HIV virus infection. [ABSTRACT FROM AUTHOR]

Background: The main property of a successful conjugation of antibodies to nanoparticles is keeping the potency of antibody for binding the antigen, and an oriented conjugation can do that. Under such ground, this study was carried out to explore the efficiency of two conjugation methods in binding iron nanoparticles to an antibody produced against PSCA (prostate stem cell antigen) using in vitro labeling of PC3 cells. Methods: In this experimental study, we conjugated dextran-superparamagnetic iron oxide nanoparticles (dexSPIONs) to anti-PSCA antibody by two different methods, including targeting carbohydrate moieties in FC domain and the free amine group of amino acid side chains. Ultimately, Iron staining was done by anti-PSCA antibody-dexSPIONs in PC3 cells to detect antibody binding to the cells. Results: A strong blue dye was induced by iron staining in conjugated dexSPIONs on the membrane of PC3 cells by the former method than the second one. Moreover, cells treated with 20 nm diameters of dexSPIONs showed higher resolution of blue color than those treated with 100 nm nanoparticles. Conclusion: This oriented conjugation method promoted the efficiency of targeting tumor antigens, and the presence of iron particles might enhance MRI image intensity in vivo by targeting PSCA-overexpressing cells in future studies.

Proteins expressed in prostate cancer, including prostate stem cell antigen (PSCA), have been investigated as biomarkers for diagnosis and therapy of prostate cancer. Immunohistochemical analysis of PSCA expression was performed on tissue microarrays of 185 paraffin-embedded tissues of Iranian patients, including 114 prostate cancers (PCa), 21 High Grade Prostatic Intraepithelial Neoplasias (HGPIN) and 50 samples of benign prostate tissue. The level of PSCA expression was compared between benign tissues, HGPIN and PCa. Then the correlations of PSCA expression with clinicopathologic parameters were assessed in PCa. The PSCA expression was detected in the membrane and cytoplasm of epithelial secretory cells in normal prostate tissues, HGPIN and PCa with a variety of intensities. The intensity of PSCA staining was significantly increased in the PCa group as compared with HGPIN and benign prostate tissues (P-value<0.05). Moreover, the level of PSCA expression was increased with higher Gleason score of PCa (P-value=0.036). The data presented here revealed that expression of PSCA as a cell surface marker increased from benign prostate tissues (BPH) and HGPIN to PCa, and its expression in PCa was positively associated with poor cell differentiation, suggesting that PSCA could be considered as a valuable target for diagnosis and therapy of PCa., (Copyright © 2013 Elsevier GmbH. All rights reserved.)