Seminal prolactin is associated with HSP90 transcript content in ejaculated spermatozoa / 대한생식의학회지

Objective@#Evidence indicates that an imbalance between the production of reactive oxygen species and defense ability of antioxidants has clinical significance in the pathophysiology of male infertility. To investigate the role of seminal prolactin (PRL) in the fertilizing capacity of men, the present study evaluated the associations of seminal PRL levels with semen parameters and heat shock protein 90 (HSP90) transcript abundance in ejaculated spermatozoa. @*Methods@#We assessed seminal PRL levels and the abundance of HSP90 transcripts in ejaculated spermatozoa from normozoospermic donors (n=18) and infertile men (n=18). The transcript content of HSP90 in ejaculated spermatozoa was analyzed using real-time polymerase chain reaction. @*Results@#Seminal PRL concentrations in infertile patients were significantly lower (p=0.004) than in fertile controls. Seminal PRL showed relatively good diagnostic power for discriminating infertile men (area under the curve=0.776; 95% confidence interval, 0.568 to 0.934; p=0.005). Significant positive correlations were seen between seminal PRL levels and sperm count (r=0.400, p=0.016) and progressive motility (r=0.422, p=0.010). Infertile patients showed a significantly higher abundance of sperm HSP90 than fertile controls (p=0.040). Sperm HSP90 transcript abundance was negatively correlated with sperm progressive motility (r=0.394, p=0.018). Men with higher seminal PRL levels exhibited a lower abundance of sperm HSP90 transcripts. @*Conclusion@#Our finding demonstrated associations among semen quality, seminal PRL levels, and the abundance of HSP90 transcripts in ejaculated spermatozoa. Seminal PRL may contribute to male fertility by maintaining the seminal antioxidant capacity and may have the potential to act as a diagnostic and prognostic biomarker.

first report of a 290-bp deletion in beta-globin gene in the South of Iran

Background: beta-thalassemia is one of the most widespread disease in the world, including Iran. In this study, we reported, for the first time, A 290-bp beta-globin gene deletion in the south of Iran

Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. beta-globin gene amplification, multiplex ligation-dependent probe ampli?cation [MLPA] and DNA sequencing were performed

Results: The PCR followed by sequencing and MLPA test of the beta-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels

Conclusions: This mutation causes beta0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different beta-globin gene mutations

novel defensin-like peptide associated with two other new cationic antimicrobial peptides in transcriptome of the Iranian scorpion venom

Introduction: Scorpion venom is a source of bioactive peptides, and some antimicrobial peptides [AMPs] have been found in the venom gland of scorpions. Therefore, the discovery of new anti-infective agents is an essential need to overcome the problem of antibiotic resistance of clinical isolates. Here, we describe three new cationic AMPs, including meuVAP-6, meuAP-18-1, and meuPep34 from the venom gland of the Iranian scorpion, Mesobuthus eupeus

Methods: The cDNA sequences encoding all the three peptides were obtained from the cDNA library of scorpion venom gland and were deposited in the GenBank database

Results: MeuVAP-6 and meuAP-18-1 are non-disulphide-bridged antimicrobial peptides, while meuPep34 is a cysteine-rich defensin-like peptide

Discussion: All three identified AMPs are rich in arginine and tryptophan. The overall results from the length, net charge, and hydrophobicity index suggested that meuPep34 could be the most active AMPs with the potential ability of biofilm inhibition. The data from molecular characterization of identified AMPs can provide a platform for further investigations in the drug design

Molecular Characterization and Biodiversity of a Putative Chlorotoxin from the Iranian Yellow Scorpion Odontobuthus doriae

Background: Chloride channels have already been over-expressed in the different types of cancer. Chlorotoxins, as the blocking agent of these channels, have been indicated to be an effective drug against tumors. In this study, we characterized a putative chlorotoxin from a cDNA library of the venom glands obtained from the Iranian scorpion Odontobuthus doriae

Methods: A cDNA library was constructed from venom gland transcriptome of six scorpions. The cDNA encoding Odontobuthus doriae chlorotoxin was isolated from the library, and its putative peptide was characterized by some bioinformatics software such as protein blast, SignalP4.0, DISULFIND and Clustal Omega

Results: The mature Odontobuthus doriae chlorotoxin peptide has a 35-amino-acid residue and four disulfide bounds. This putative chlorotoxin is a small, compact, and stable molecule. Moreover, based on the open reading frame sequence similarity, this peptide is similar to Buthus martensii Karsch chlorotoxin-like toxin and Bml2-b neurotoxins from the Chinese scorpion Mesobuthus martensii

Conclusion: The small size of this putative chlorotoxin and its stability make it as a suitable candidate for medical and pharmacological research, especially in the cancer research

Association study of rs1333040 and rs1004638 polymorphisms in the 9p21 locus with coronary artery disease in southwest of Iran

Background: Coronary artery disease [CAD] is a multifactorial and heterogenic disease. Recently, genome-wide association studies have reported that rs1333040 [C/T] and rs1004638 [A/T] single nucleotide polymorphisms [SNPs] in the 9p21 locus have very strong association with CAD. This study aimed to examine these associations in Southwest of Iran

Methods: Blood samples were collected from 200 CAD patients and 110 healthy individuals with no CAD. The association of two SNPs with CAD was evaluated by PCR and restriction fragment length polymorphism

Results: Chi-square test showed no association between rs1333040 SNP and CAD [X[2]: 4.66, df: 2, P=0.09]. Also, there was no association between rs1004638 SNP and CAD [X[2]: 0.27, df: 2, P=0.88]

Conclusion: No association was observed between rs1333040 and rs1004638 SNPs in the 9P21 region and CAD in Southwest of Iran

no Asssciatian sf androgen receptor GGN repeat length polymorphism and male infertility in Khuzestan, Iran

Background: Androgens play critical role in secoidary sexual and male gonadsdifferentiations such as spermatogenesis, via androgen receptor. The human androgen receptor [AR] encoding gens contains two regions with three nucleotide polyinorphic repeats [CAG and GGN] in the first exon. Unlike the CAG repeats, the I GGN has been less studied because of technical difficulties, so the functional role of: these polyniorphic repeats is still unclear. Objective: The goal of this study was to investigate any relationship betweell GGNrepeat length in the iirst exon ofAR gene and idiopathic male infertility in southwest of Iran. Materials and Metlrods: This is the first study on GGN repeat of AR gene in; infertile illale ill IChuzestan, iran. We used polymerase chain reactioi [PCR] and: polyacrylamide gel electrophoresis to categorize GGN repeat lengthsin 72 infertile and 72 fertile men. Afterwards we sequeliced the PCR products to determine theexact length of GGN repeat in each category. Our samples included 36 azoospernicaiid 36 oligozoospeniiic men as cases and 72 fertile men as control group.Results: We foulid that the numbers of repeats in the cases range from 18 to 2while iu the controls this range is fiom 20 to 28. The results showed a significantrelation between the length OF GGN repeat and fertility [p=0.015]. The most I fiequent alleles were alleles with 24 and 25 repeats respectively in case and control groups. On the other hand no significant differences were found between Arab and non-Arab cases by considering GGN repeat lengths [p=0.234].Conclusion: Due to our results, there is a significant association between the: presence ofallele with 24 repeats and susceptibilitv to Inale infertilitv. Therefore thispolylnorphism should be considered in future studies to clarify etiolbgy of disorders: related to androgen receptor activity

Association study between coronary artery disease and rs1333049 and RS10757274 polymorphisms at 9P21 locus in south-west Iran

Coronary artery disease [CAD] is a multi-factorial and heterogenic disease with atherosclerosis plaques formation in internal wall of coronary artery. Plaque formation results to limitation of the blood reaching to myocardium leading to appearance of some problems, such as ischemia, sudden thrombosis veins and myocardial infarction [MI]. Several environmental and genetic factors are involved in prevalence and incident of CAD as follows: hypertension, high low density lipoprotein-cholesterol [LDL-C], age, diabetes mellitus, family history of early-onset heart disease and smoking. According to genome wide association studies [GWAS], five polymorphisms in the 9p21 locus seem to be associated with the CAD. We aimed to evaluate the remarkable association of two polymorphisms at 9p21 locus, rs1333049 and rs10757274, with CAD. This experimental study was conducted in Golestan, Aria Hospitals and Genetics Lab of Shahid Chamran University in the city of Ahvaz, Iran, in 2010- 2011. The collected blood samples belonging to 170 CAD patients [case group] and 100 healthy individuals [control group] were analyzed by tetra-primer amplification refractory mutation system [ARMS]-polymerase chain reaction [PCR] technique. The results were analyzed using software package used for statistical analysis [SPSS; SPSS Inc., USA] version 16. A value of p<0.05 and an odd ratio [OR] with 95% confidence intervals [CI] were considered significant. The frequencies of CC, CG and GG genotypes for rs1333049 polymorphism in patients were 18.2, 65.3 and 16.5%, while in controls, the related values were 25, 67 and 8%, respectively. GG genotypes of rs1333049 polymorphism in CAD patients were more than control cases [OR: 0.354, 95%CI: 0.138-0.912, p=0.032]. The frequencies of AA, AG and GG genotypes for rs10757274 in CAD patients were 8.2, 58.3 and 33.5%, while in controls, the related values were 35, 63 and 2%, respectively. GG Genotype in rs10757274 polymorphism in CAD patients was found more than control cases [OR: 0.014, 95% CI: 0.003 -0.065, p=0.0001]. The rs1333049 polymorphism at 9p21 locus shows a weak association with CAD, whereas rs10757274 polymorphism reveals a significant association with CAD. These variants may help the identification of patients with increased risk for coronary artery disease

Expression optimizing and purification of recombinant human leukemia inhibitory factor produced in E. coli strain BL21

Leukemia inhibitory factor [LIF] is a glycoprotein, categorized as a subfamily of interleukin 6 cytokines which is known in many mammals. A pluripotent cytokine with a wide biological function range has numerous effects on target cells. The LIF regulates neuron survival, hematopoiesis and seen in LIF[-/-] knockout mice affects blastocyst implantation, also acts as pre-inflammatory cytokine, and regulates immune response. Further, it is able to maintain stem cells poly potency. The main object of present work was expression, optimizing, and purification of recombinant human leukemia inhibitory factor [rhLIF]. In this experimental study, Pet28 [+] carrying the LIF gene and kanamycin resistance marker was cloned in E. coli strain BL21. The induction was optimized by altering 3 factors including the temperature, the induction time, and the concentration of the Isopropyl beta-D-1-thiogalactopyranoside [IPTG] as inducer. The purification of the recombinant human LIF [rhLIF] was done by single step affinity chromatography. After the purification, method accuracy was proved by Sodium dodecyl sulfate [SDS] -PAGE electrophoresis and Western blotting. Optimizing of the expression was reached by changing various parameters, and purification has been done successful. rhLIF undergoes modification by glycosylation to get its full functionality. The produced rhLIF in prokaryotic host in this work is lacking of glycosylation. However, its proper function should be evaluated in further studies

unique profile of adenomatous polyposis coli gene mutations in iranian patients suffering sporadic colorectal cancer

Colorectal cancer [CRC] is one of the most common and aggressive cancers worldwide. The majority of CRC cases are sporadic that caused by somatic mutations. The Adenomatous Polyposis Coli [APC; OMIM 611731] is a tumor suppressor gene of Wnt pathway and is frequently mutated in CRC cases. This study was designed to investigate the spectrum of APC gene mutations in Iranian patients with sporadic colorectal cancer. In this descriptive study, Tumor and normal tissue samples were obtained from thirty randomly selected and unrelated sporadic CRC patients. We examined the hotspot region of the APC gene in all patients. Our mutation detection method was direct DNA sequencing. We found a total of 8 different APC mutations, including two nonsense mutations [c.4099C>T and c.4348C>T], two missense mutations [c.3236C>G and c.3527C>T] and four frame shift mutations [c.2804dupA, c.4317delT, c.4464_4471delATTACATT and c.4468_4469dupCA]. The c.3236C>G and c.4468_4469dupCA are novel mutations. The overall frequency of APC mutation was 26.7% [8 of 30 patients]. This mutation rate is lower in comparison with previous studies from other countries. The findings of present study demonstrate a different APC mutation spectrum in CRC patients of Iranian origin compared with other populations

Exon sequencing of PKD1 gene in an Iranian patient with autosomal-dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease [ADPKD] is one of the most common genetic kidney disorders with the incidence of 1 in 1,000 births. ADPKD is genetically heterogeneous with two genes identified: PKD1 [16p13.3, 46 exons] and PKD2 [4q21, 15 exons]. Eighty five percent of the patients with ADPKD have at least one mutation in the PKD1 gene. Genetic studies have demonstrated an important allelic variability among patients, but very few data are known about the genetic variation among Iranian populations. In this study, exon direct sequencing of PKD1 was performed in a seven-year old boy with ADPKD and in his parents. The patient's father was ADPKD who was affected without any kidney dysfunction, and the patient's mother was congenitally missing one kidney. Molecular genetic testing found a mutation in all three members of this family. It was a missense mutation GTG>ATG at position 3057 in exon 25 of PKD1. On the other hand, two novel missense mutations were reported just in the 7-year-old boy: ACA>GCA found in exon 15 at codon 2241 and CAC>AAC found in exon 38 at codon 3710. For checking the pathogenicity of these mutations, exons 15, 25, and 38 of 50 unrelated normal cases were sequenced. Our findings suggested that GTG>ATG is a polymorphism with high frequency [60%] as well as ACA>GCA and CAC>AAC are polymorphisms with frequencies of 14% and 22%, respectively in the population of Southwest Iran

Detection of a novel mutation in the GAA gene in an Iranian child with glycogen storage disease type II

Glycogen storage disease II [GSDII or Pompe disease, OMIM # 232300] is an autosomal recessive hereditary lysosomal disorder. Mutations in the GAA gene usually lead to reduced acid alpha-glucosidase [acid maltase, GAA, OMIM [asterisk] 606800, EC 3.1.26.2] activity, which results in impaired degradation and subsequent accumulation of glycogen within lysosomes. We present an Iranian boy, who was diagnosed with GSDII based upon clinical and biochemical findings. A single adenine insertion [insA] was detected at codon 693 that leads to a predicted premature stop codon at codon 736 in the GAA gene. The parents were heterozygous for the same change. According to the human genome mutation database [www.hgmd.org] and lecture reviews, the detected change is a novel mutation. We suppose that the discovered insertion in the GAA gene might lead to a reduced activity of the gene product. This assumption is in agreement with biochemical and clinical signs in the patient

New single nucleotide deletion in the SMPD1 gene causes Niemann Pick disease type A in a child from southwest Iran: a case report

Niemann Pick disease [NPD] type A [NPA: MIM #257200] is a lipid storage disorder with an autosomal recessive inheritance and occurrs by defect of the SMPD1 gene encoding sphingomyelinase. Disruption of this enzyme leads to the accumulation of sphingomyelin in brain and liver, which in turn causes dysfunction or damage of tissue. We report firstly a 2.5 year old boy with NPA in southwest Iran. Initially, the diagnosis was resulted on the basis of clinical symptoms. The genomic DNA of the suspected individual was subjected to exon sequencing of the SMPD1 gene. According to the human reference sequence NM_000543.4, a novel single guanine deletion resulting in a frameshift mutation [p.Gly247Alafs*9] was observed in the SMPD1 gene that might be causative for the outcome of the disease. The present report is the first molecular genetics diagnosis of the NPA in southwest Iran. The detected deletion in the SMPD1 gene is remarkable because of its novelty. Despite similar morbidity SGA infants exhibited higher lethal complication rates following delayed meconium passage compared to AGA infants

study of the association between SNP8NRG241930 in the 5' end of neuroglin 1 gene with schizophrenia in a group of Iranian patients

Neuregulin1 [NRG1] gene is among the most promising candidate genes for schizophrenia. This gene is located on 8p22-p12, a region with a reported linkage to schizophrenia. Several studies have reported an association between schizophrenia and the 5? end polymorphisms in this gene. However, some studies have failed to confirm the role of NRG1 gene in the pathogenesis of schizophrenia. In the current study, we attempt to examine the association of SNP8NRG241930 from the NRG1 gene with schizophrenia in an Iranian population. It is noteworthy that there has been no report on the NRG1 association with schizophrenia in a population from the Middle East region. Genomic DNA samples were obtained via isolation from the peripheral blood cells of 95 unrelated subjects with schizophrenia and 95 matched healthy controls from southwest Iran. SNP8NRG241930 was genotyped by PCRRFLP using ScaI as a restriction endonuclease enzyme. Association of the SNP with schizophrenia was examined using the chi-square test. The frequency difference of alleles and genotypes between the two groups were compared. P?0.05 was considered significant. Statistical analysis on the studied polymorphism showed that both case and control groups were in Hardy-Weinberg equilibrium. The frequency of high risk allele [G allele] was 72.6% in patients, while this number was 56.8% in controls. The genotype frequencies in the patient group were as follows: GG [54%], GT [38%] and TT [8%] vs. genotype frequencies in the control group of: GG [26%], GT [63%] and TT [11%]. Considering allele and genotype frequencies, a significant association was observed between schizophrenia and SNP8NRG241930.The current study adds weight to the idea that some functional polymorphisms could exist in the 5? end of the NRG1 gene which increase susceptibility to schizophrenia. This is the first time that supportive evidence shows an involvement of the NRG1 locus in schizophrenia in an Iranian sample population

SMN1 and NAIP genes deletions in different types of spinal muscular atrophy in Khuzestan province, Iran

Spinal muscular atrophy [SMA] is the second most common lethal autosomal recessive disease. It is a neuromuscular disorder caused by degenerative of lower motor neurons and occasionally bulbar neurons leading to progressive limb paralysis and muscular atrophy. The SMN1 gene is recognized as a SMA causing gene while NAIP has been characterized as a modifying factor for the clinical severity and age at disease onset in SMA patients [SMA subtypes]. The relationship between NAIP deletion and type of SMA remains to be clarified; we investigated this gene alteration in all types of SMA patients. Molecular analysis was performed on fifty patients with a clinical diagnosis of SMA in Khuzestan province. In addition to common PCR-RFLP analysis for exon 7 and 8 of SMN1 gene, as an internal control we analysed NAIP deletion with PCR of exon 5 of this gene in a multiplex PCR with exon 13 of it. Homozygous-deletion frequency rate for the telomeric copy of SMN [SMN1] exon 7 in all types [type I, II, III] of SMA was approximately 90% and the frequency of deletion in exon 7 and 8 together in all types estimated about 70%. Moreover NAIP gene was deleted in about 60% of these patients and this shows deletion in 91% of type I SMA patients. The correlation between NAIP-deletion and SMN1 mutation showed a high frequency rate. In this study, high frequency of NAIP gene deletion in all type of disease shows the importance role of it in disease pathogenesis. High frequency of NAIP deletion in SMA type I, also shows the importance of the gene in type and severity of disease so it may be a modifier factor in severity of disease

[ first survey of distribution of inherited deafness patterns in individuals referred to genetic center of Ahvaz welfare organization, Southern Iran]

Deafness is a heterogeneous disorder induced by genetic and environmental factors. It is the most common hereditary sensory-neural disorder that affects 1/1000 to 1/2000 of the newborns. More than 70% of hearing loss cases are caused by genetic disorders, 85% of which result from nonsyndromic autosomal recessive sensory-neural hearing loss. Up to now, more than 100 genes contributing in hearing loss have been determined. Alteration of these genes may result in hearing loss. This study was performed to identify the inheritance patterns of deafness and its relation with ethnicity, gender and consanguineous marriages. In this survey, data from 356 families affected by hearing loss and referred to welfare organization of Ahvaz during the time were collected based on sex, ethnic groups and relativeness. The results state a high frequency of autosomal recessive deafness caused by consanguineous marriages within Arab and non-Arab ethnic groups [p<0.05]. But no significant difference in gender. In conclusion, the high frequency of autosomal recessive deafness among the population with a high frequency of consanguineous marriages is considerable. The dominant pattern of deafness observed in this population was autosomal recessive

Cloning and expression of Thermus aquaticus DNA polymerase in Escherichia coli

Thermostable DNA polymerase gene from Thermus aquaticus was cloned into constructed Taq from Thermus a Qaticus [pTTQ] plasmid using EcoRI and Sa/I sites with subsequent transformation in Escherichia coli strain [TOP10]. The use of Isopropyl-beta-D-thiogalactopyranosid [IPTG] as inducer of interested gene expression under control of the lac promoter was investigated. The optimization of enzyme induction by IPTG was determined at shake flask level to be 0.52mM at exponential growth phase. Enzyme preparation was performed by lysis the cultured cells. Afterwards, the cell suspension was incubated at 75°C to denature all heat sensitive proteins in the cell suspension that have been removed by subsequent centrifugation. Finally, the clarified supernatant containing heat resistant Taq DNA polymerase was collected and stored at -80°C. The activity of enzyme was compared with commercial Taq DNA polymerase, which remained when stored in buffer containing 50% glycerol, at -20°C. The purified enzyme had a molecular weight of 94 KDa, as estimated by SDS-PAGE and yielded appropriate enzyme activity comparing to the commercial Taq DNA polymerase

Cloning and expression of VP2 gene of infectious bursal disease virus in eukaryotic cells

Infectious bursal disease [IBD] is an economically important viral disease of chickens with worldwide distribution which suppresses the immune system of young chickens. VP2 is the major host-protective protein of infectious bursal disease virus [IBDV]. The encoding region of VP2 protein was PCR amplified from a plasmid containing a cDNA fragment of large genomic segment of IBDV, strain D78. This region of 1356 bp was inserted into a eukaryotic expression plasmid, pCDNA4, under the control of human cytomegalovirus [hCMV] immediate early enhancer and promoter. Plasmid DNA was transfected into COS-7 cell line and transient expression of VP2 from the constructed plasmid was characterized by dot blotting with a polyclonal antibody to IBDV

Perform gene analaysis in an Iranian family with familial hemophagocytic lytnphohistiocytosis

Perform gene [PRF1] mutations have been reported in 20-30% of patients with familial hemophagocytic lymphohistiocytosis [FHL], an immune disorder of infancy and early childhood. Cytotoxic T and natural killer [NK] cell activities are remarkably reduced or absent in FHL patients. We report the first cases of familial hemophagocytic lymphohistiocytosis in an Iranian family with two siblings. Exons 2 and 3 of the PRF1 gene were analyzed by polymerase chain reaction [PCR] amplification and direct sequencing. Perform gene mutation[s] were detected in none of the cases. The result of our study indicates that not much evidence is present concerning a correlation between perforin gene defects and familial hemophagocytic lymphohistiocytosis etiology in these cases