Gene regulation of pteridine reductase 1 in leishmania promasti-gotes and amastigotes using a full-length antisense construct

Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 micro g of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Double-stranded RNA viral infection in Tehran trichomonas vaginalis isolates

Trichomonas vaginalis is a pathogenic protozoon and may be contaminated with dsRNA virus called Trichomonas vaginalis virus [TVV]. The viral infection is an important factor for its pathogenesis and sensitivity to metronidazole. The presence of TVV is associated with qualitative and quantitative expression of cysteine proteinases and surface immunogenic; P270. The purpose of this study was to determine TVV frequency in T. vaginalis clinical isolates in Tehran, Iran. The 46 T. vaginalis isolates were collected from Tehran Province and cultured in TYI-S-33 culture medium. Viral RNA was extracted and RT-PCR was done. Of 46 T. vaginalis isolates, 8 isolates [17.39%] were infected with TVV-1. There was not any association between patient age and TVV- infected T. vaginalis. There were 17.39% viral infection in T. vaginalis isolates which was lower than that reported by other researchers. This is the first report on T. vaginalis isolates infection by TVV-1 in Iran

[ relationship between cardiovascular fitness and physical activity with obesity and changes in their pattern among 12-16 year-old boys]

This study aimed at determining the association between cardiovascular fitness and physical activity with obesity and changes in their patterns among 12-16 year-old boys. In this cross-sectional study, 275 boy students, 12-16 year-old; from Bardaskan city were investigated. Subjects were selected via random sampling. Underweight, overweight and obesity status were evaluated based on the 5[th], 85[th] and 95[th] percentiles of body mass index [BMI] for age and sex based on the United States' Center for Disease Control [CDC 2000] standards. Physical activity levels were estimated by the Physical Activity Questionnaire for Older Children [PAQ-C]. Cardiovascular fitness [VO2max] was assessed by a 20 m shuttle run test. Overall prevalence's of underweight, overweight and obesity among subjects were 3.6%, 10.5% and 4.7% respectively. There was significant positive correlation between physical activity level and cardiovascular fitness. Cardiovascular fitness and physical activity levels decreased significantly with aging. The overweight and obesity groups had lower levels of cardiovascular fitness than the normal and underweight groups. There was no statistically significant difference between the overweight-obese and the non overweight groups in physical activity levels. Moreover, There was a significant negative correlation between physical activity and cardiovascular fitness levels and subjects' BMIs. Considering the in adequate levels of cardiovascular fitness and physical activity in overweight and obese boys, programs increasing physical activity [endurance exercise], and diet and weight control are recommended for these groups

Occurrence of thermotolerant hartmannella vermiformis and naegleria spp. in hot springs of Ardebil province, northwest Iran

Geothermal waters could be suitable niches for thermophilic free living amoebae including Naegleria and Hartmannella. Ardebil Province, northwest Iran is popular for having many hot springs for recreational and health purposes activity. The present research is the first molecular based investigation regarding the presence of Naegleria and Hartmannella in the hot springs of Ardebil Province in Iran. Overall, 30 water samples were taken from waters of thermal hot springs in Ardebil Province, Iran during 2010-2011. All collected samples were transferred to Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Cultivation of concentrated water samples was performed using culture-enrichment method. Cloning of the target amoebae was obtained and morphological and molecular analysis was done using page key combined with two sets of primers, respectively. Sequence analysis and homology search was used for strains identification. Of 30 water samples, 8 [26.7%] were positive for thermotolerant Vahlkampfiids and Hartmannella based on morphological characteristics of vegetative form and double walled cysts. Cloning of the target amoebae were done successfully. Sequencing of the positive isolates revealed that the strains belonged to Naegleria [N. carteri and N. spp] and H. vermiformis. The result highlights a need for improved filtration and disinfection and periodic monitoring of recreational thermal waters in order to prevent disease related to free- living amoebae. This is the first comprehensive molecular study of thermophilic Naegleria and Hartmannella in hot springs of Iran

Inhibition of leishmania major PTR1 gene expression by antisense in escherichia coli

Protozoa related to Trypanosome family including Leishmania, synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase [DHFR]. Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more. PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression. Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells. Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid

[Hormonal responses to two programs of exhaustive resistance training of different intensities in male body builders]

The purpose of this cross-over study was to investigate of hormonal responses to two programs of exhaustive resistance training with different intensities in male body builders. Participants were 13 men [age 23.8 +/- 5.53 years, height 177.53 +/- 5.69 cm, body weight 76.13 +/- 8.91 kg, waist to hip ratio 0.85 +/- 0.33] who had regular resistance training at least 3 times a week for more than 3 months. Study design was crossover. Subjects participated in three states of control, moderate resistance exercise [with 65% intensity, one repetition maximum] and high resistance exercise [90% intensity, one repetition maximum] modes, in 5 sessions. Blood samples were taken before exercise in fasting state, immediately after and one hour after exercise protocol. The data were analyzed using the analyses of variance method [ANOVA] with repeated measures and multiple analyses variance [MANOVA]. After adjusting the results relative to plasma volume changes, no significant differences were observed between the three groups in hormonal responses of testosterone, cortisol, growth, insulin, epinephrine and norepinephrine at the time points of immediately after and one hour after exercise protocol. It can be concluded that resistance exercise until exhaustion with moderate and high intensity, does not induce significant changes in acute and chronic responses of circulating anabolic and catabolic hormones in male body builders.

Development of sensitive detection of cryptosporidum and giardia from surface water in Iran

The protozoan parasites Cryptosporidium spp. and Giardia are known to occur widely in both raw and drinking waters. They are two of the causative agents of waterborne outbreaks of gastroenteritis throughout the world. In the present study, a PCR assay and FA were developed for detection of Cryptosporidium oocysts and Giardia cyst in environmental samples. We have detected Cryptosporidium spp. oocysts and Giardia cysts in seeded and unseeded environmental water samples by PCR method. Water samples were spiked with oocysts [50, 100, 300, 500] and filtrated with a 1.2- micro m pore size cellulos nitrate and follow by DNA extraction and purification by QIA amp DNA mini kit. Nested -PCR assay amplified an 850 bp fragment of 18s rDNA gene specific for Cryptosporidium and 453 bp fragment of glutamate dehydrogenase [GDH] target genre for Giardia. Also many river water from north of Iran, be checked by these methods. Cryptosporidium and Giardia DNAs were detected in seeded water sample and Giardia was detected in all 5 water samples from river in north of Iran by nested-PCR and FA. Also in one river water sample, Cryptosporidium was detected. This protocol if effective for detection of these waterborne parasites in treated and untreated water samples. This study can also serve as a platform for further investigations and research water source in Iran

Sequence diversity in tRNA gene locus A-L among Iranian isolates of entamoeba dispar

A number of methods for detecting diversity in Entamoeba have been described over the years. In the present study the genetic polymorphism of noncoding locus A-L was analyzed using PCR and sequencing in order to clarify the genotypic differences among E. dispar isolates. A total of 28 E. dispar from patients with gastrointestinal symptoms were determined and the genomic DNA was extracted directly from stool. For genotype analysis; Locus A-L was amplified by PCR and PCR products were sequenced .The sequences obtained were edited manually and aligned using Gene Runner software. With sequencing of PCR products a reliable genetic diversity in size, number and position of the repeat units were observed among the Iranian E. dispar isolates in locus A-L gene. Sequences showed variation in length from 448bp to 507bp and seven distinct types were identified. The genetic diversity of loci like A-L shows them to be suitable for epidemiological studies such as the characterization of the routes of transmission of these parasites in Iran

Molecular epidemiology of cryptosporidiosis in Iranian children, Tehran, Iran

Cryptosporidium is a worldwide protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was determination of subtypes of Cryptosporidium among children with diarrhea in Tehran by sequence analysis of the highly polymorphic 60-kDa glycoprotein [GP60] gene. Fecal samples were collected from 794 diarrheic children. Initial identification of Cryptosporidium was carried out on stool samples by Ziehl-Neelsen acid-fast staining method. DNA was extracted from positive microscopically samples and Cryptosporidium genotypes and subtypes were determined, accordingly. Out of 794 collected samples, 19 [2.40%] were positive for Cryptosporidium oocysts. Sequences analysis of GP60 gene showed that 17 [89.47%] of the positive isolates were Cryptosporidium parvum and 2 [10.52%] were C. hominis. All subtypes of C. parvum isolates belonged to allele families IIa [6/17] and IId [11/17]. The most common allele in all 17 isolates belonged to IId A20G1a [41.18%]. A22G1 [IF] subtype was detected in two C. hominis isolates of the children. The predominancy of C. parvum species [specially, IId A20G1a subtype] in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran

Prevalence of intestinal parasites in bakery workers in Khorramabad, Lorestan Iran

Food contamination may occur through production, processing, distribution and preparation. In Iran especially in Khorramabad, 33[degree sign] 29' 16" North, 48[degree sign] 21' 21" East, due to kind of nutrition, culture and economic status of people, bread is a part of the main meal and the consumption of bread is high. In this study, the bakery workers were studied for determining of intestinal parasites prevalence. The study was carried out during September to November 2010 in Khorramabad. All the 278 bakeries and the bakery workers including 816 people were studied in a census method and their feces were examined for the presence of parasites by direct wet-mount, Lugol's iodine solution, and formaldehyde- ether sedimentation, trichrome staining, and single round PCR [For discrimination of Entamoeba spp]. Ninety-six [11.9%] stool specimens were positive for different intestinal parasites. Intestinal parasites included Giardia lamblia 3.7%, Entamoeba coli 5.5%, Blastocystis sp. 2.1%, Entamoeba dispar 0.4%, Hymenolepis nana 0.1%, and Blastocystis sp. 0.1%. In order to reduce the contamination in these persons, some cases such as stool exam every three months with concentration methods, supervision and application of accurate health rules by health experts, training in transmission of parasites are recommended

First report of Vannellidae amoebae [vannella spp.] isolated from biofilm source

Members of the Vannellidae family are free-living amoebae [FLA] distributed mainly in water and soil sources. The present study reports the first isolation of this genus in the biofilm source from hospital environment in Tehran, Iran. Biofilm samples were collected from hospital environment. Cultivation was performed in non-nutrient agar covered with a heat-killed Escherichia coli. Cloning of the suspected amoebae was done. PCR amplification and Homology analysis using the Basic Local Alignment Search Tool [BLASTn] was performed to search for the most similar reference sequences. Microscopic examination showed numerous fan-shaped amoebae and peculiar cysts different to the usual shape of typical FLA. Sequence analysis of the PCR- product revealed that the suspected amoebae are highly homologous with Vannella spp. gene [99% identity and 100% query coverage] available in the gene bank database. Although Vannella spp. is not proved to be pathogenic itself, but they are capable of harboring pathogenic intracellular organisms such as Microsporidian parasites. Thus, identification of such amoebae can be of clinical importance, as they could lead to transmission of other pathogens to human

[Cryptosporidium genetic diversity based on analysis of COWP, SSU-rRNA and TRAP-C2 polymorphic genes in children with diarrhea in Tehran and qazvin provinces: a survey from Iran]

Since Cryptosporidium is a worldwide distributed protozoan parasite and is considered as one of the most common causes of infection and diarrhea in humans with autoimmune deficiency, as well as in young live stock, molecular epidemiologic studies of cryptosporidiasis will be helpful for underlying transmission and molecular pathogenesis of Cryptosporidium in humans. The aim of the present study was to determine the species and genotypes of Cryptosporidium among children with diarrhea in Tehran and Qazvin provinces by PCRRFLP using the three polymorphic regions of SSU-rRNA, COWP and TRAP-C2 genes. 1263 stool samples were collected from the children less than 12 years with diarrhea who referred to Pediatrics Medical Centers in Gazvin and Tehran Provinces, Iran, during 2005-2007. After determination of the presence of Cryptosporidium oocytes by ZiehlNeelsen acid, fast staining genomic DNA was extracted. Nested PCR-RFLP was performed by -rRNA, COWP and TRAP-C2 genes. Results of microscopically positive samples showed that the overall prevalence of infection in children was 31 [2.5%]. Results of nested PCR amplification showed that of 31 isolates of children, all of three targeted gene were successfully amplified. Our results indicated that the zoonotic transmission is the main mode of infection in Iran and indicates that direct or indirect contact with animals, especially calf, is possibly the main route of human infection

Seroprevalence of acanthamoeba antibodies in rheumatoid arthritis patients by IFAT, Tehran, Iran 2007

This preliminary study was conducted to discriminate the prevalence of Acanthamoeba antibodies in rheumatoid arthritis [RA] patients and healthy controls to analyze the correlation between these two groups. From October 2006 to August 2007 a total of 121 serum samples from RA patients attending the Rheumatolgy Department at Shariati Hospital in Tehran were obtained and stored at -20°C until using by indirect fluorescent-antibody test [IFAT]. RA was diagnosed according to the American Collage of Rheumatology classification criteria. The organism used in this study was isolated from various water resources in Tehran, Iran cultured axenically and then went on a PCR assay based on 18S rRNA to identify the genus Acanthomoeba. Indirect immunofluorescence antibody [IFA] staining of serum samples was carried out to detect anti Acanthomoeba antibodies. In culture, out of 22 samples, 13 [59%] were grown in xenic but only two in axenic medium. PCR amplified a 904bp fragment, specific for Acanthamoeba. Of examined serum samples, Acanthamoeba antibodies were present in 70 [57.8%] and 52 [41.2%], respectively. The highest titer of antibodies [1:320] was detected in one patient with RA. Our study supports the hypothesis that some parasitic microorganisms can involve and contribute toward the development of rheumatoid syndromes

Recombinant SAG1 antigen to detect Toxoplasma gondii specific immunoglobulin G in human sera by ELISA test

Although some serological tests for the detection of Toxoplasma gondii-specific immunoglobulin are commercially available, better diagnostic tools are needed. The aim of present study was to evaluate the usefulness of the recombinant Toxoplasma gondii SAG1 antigen for the recognition of toxoplasmosis by ELISA. This study was conducted in Cellular and Molecular Biology Research Centers, Shahid Beheshti University, M.C., Tehran, Iran in 2008-2009. Surface antigen 1 [SAG1], a tachyzoite stage-specific protein, was subcloned into an expression vector and was subsequently transformed into BL21 [DE3] pLyss competent bacterial cells. After inducing expression of the recombinant antigen, the protein product was purified using Ni-affinity chromatography. The immunoreactivity of recombinant SAG1 [rSAG1] was analyzed by SDS-PAGE and western blotting. The reactivity of the rec-SAG1 protein was evaluated using an ELISA. Sensitivity and specificity of the generated recombinant-ELISA [rec-ELISA] compared to a commercially available ELISA [com-ELISA] were 88.4% and 88%, respectively. Recombinant SAG1 produced in E. coli is a promising antigen that can be used in diagnostic assays for the detection of specific antibodies against T. gondii

Prevalence of thyroid disorders in systemic lupus erythematosus and rheumatoid arthritis

Autoimmune diseases are caused by immune systems reacting against self antigens. One important feature of autoimmune diseases is the tendency for overlap, such that an individual with a specific syndrome is more likely to simultaneously develop a second syndrome. Autoimmune thyroid disorders are frequently associated with autoimmune diseases of other organs such as Rheumatoid Arthritis [RA] and Systemic Lupus Erythematosus [SLE]. The aim of this study was to evaluate the occurrence of thyroid disorders in patients with RA or Systemic Lupus Erythematosus [SLE] In this case-control study, 200 patients with diagnoses of RA or SLE, referred to the rheumatology clinic of Shahid Mohammadi hospital [Bandar abbas- Iran] between May 2004 and December 2006 and following thyroid investigations were categorized in 3 groups [T3, T4, TSH, Anti TPO, and Anti TG] 57 patients with RA, 59 patients with SLE and 66 patients with mechanical low back pain or osteoarthritis as controls. Sub clinical hypothyroidism was more frequent in the RA [10.7%] and SLE [10.2%] groups than in controls [0%] [P < 0.05]. Clinical hypothyroidism was found in 8% of RA patients and 15.2% of SLE patients. None of the controls had clinical hypothyroidism [P < 0.02]. Hyperthyroidism [Graves disease] was found in only 1 patient with RA [1.13%] [P>0.05]. Anti TG and Anti TPO were found more frequently in RA and SLE cases than in controls. Autoimmune thyroid disorders are more frequent in patients with RA and SLE than in controls

[Differentiation of Entamoeba histolytica and Entamoeba dispar by single polymerase chain reaction]

In most part of the world detection of cysts and trophozoites of Entamoeba is based on morphological structure of this species in stool sample by microscopy. However, microscopic examination is unable to distinguish between similar morphological protozoa such as Entarnoeba histolytica and Entamoeba dispar. A simple and cost-effective method is needed in medical laboratories for detection and differentiation of these two species. Stool samples of patients who were referred from health care centers were examined by direct microscopy and trichrome stain. Polymerase chain reaction [PCR] utilizing pEd30F and pEd21AS primers from Peroxiredoxin gene, was used for differentiation of E. histolytica and E. dispar. Genomic DNA from samples was amplified by these primers. The fragment under 100 bp was related to E. histolytica and in contrast the fragment above the 100 bp was related to E. dispar. In this study from 22 microscopic positive samples, E. histolytica was observed only in one patient and E. dispar was detected in the other 21 samples. The result of this study indicate that the PCR reaction could amplify E. dispar and E. histolytica with just one primer pair and this is a cost-effective method for distinguishing between these two species

Detection of acanthamoeba from fresh water using polymerase chain reaction

Acanthamoeba is a genus of amoeba, one of the most common protozoa found worldwide in soil, and also frequently found in fresh water. In healthy individuals, Acanthamoeba spp. can cause ulcerating keratitis which is often associated with the use of improperly sterilized contact lenses. The aim of this study was to detect Acanthamoeba from fresh water collected from some town squares of Tehran by polymerase chain reaction [PCR]. In this study, 22 samples were collected from fresh water. They were cultured on NNA medium after filtration. Culture samples positive for Acanthamoeba were assessed using polymerase chain reaction [PCR]. Thirteen samples [59%] were recognized as Acanthamoeba on culture. Using species-specific primers which amplified a 903 bp fragment of 188 rRNA, 6 [27%] samples from 13 samples which were positive on culture were identified as Acanthamoeba. Acanthamoeba has been recognized as an etiologic agent of Keratitis in people who use contact lenses and also in immunocompromised individuals. So, detection of this organism in water resources and exact assessment of this parasite could have a significant role in prevention of disease

Subcloning and expression of recombinant Echinococcus granulosus antigen B, in Pqe-30 expression Vector

Echinococcosis or hydatid disease is a zoonotic infection caused by larval [metacestode] stages of cestodes belonging to the genus Echinococcus, family Taeniidae. We aimed to subclone antigen B gene in pQE-30 plasmid, its expression, and purification. We subcloned HI gene into pQE-30 expression vector. The recombinant vector was transformed into E. coli, M15 and mass cultured. The subcloned gene was expressed by IPTG. Subcloning of gene was confirmed by both PCR and enzyme digestion. Production of recombinant protein was confirmed by SDS-PAGE. Western blot analysis was carried out by both His-Tag monoclonal Ab and human serum to estimate the expressed potein in E. coli cells. Recombinant protein was purified and its specificity was proved by Western blotting. Production of this recombinant protein can increased sensitivity and specificity in serological test [ELISA]

Molecular characterization of dihydrofolate reductase-thymidylate synthase gene concerning antifulate resistance of Plasmodium vivax

The recently reported resistance to antimalarials contributes to making the control of malaria more difficult. There is a need to evaluate the current antimalaria regiments to prevent this emerging problem. The aim of this study was to determine dihydrofolate reductase-thymidylate synthase gene mutation [pvdhfr] regarding antifulate resistance in Plasmodium vivax. From 2007 to 2009, 117 P.vivax infected blood samples collected from two regions of Hormozgan Province, south of Iran were analyzed using PCR, semi-nested-PCR and RFLP methods. Eighty four isolates [71.8%] showed no mutation in pvdhfr gene of P. vivax known as wild type and 33 [28.2%] of the samples revealed nine single [7.7%], twenty two double [18.8%] and two [1.7%] triple mutations. Genetic diversity was observed by molecular methods in pvdhfr gene of p.vivax in Hormozgan Province suggests that the antifolate falciparum malaria drug [fansidar] is proportionally affecting P.vivax dhfr mutation. Therefore, more studies to evaluate antimalarial drugs that should preferably be effective against both P.vivax and P.falciparum are recommended

Genetic diversity among entamoeba histolytica and entamoeba dispar strains in gastrointestinal disorder patients in Tehran, Iran

Since the first description of Amebiasis, we still do not have a proper answer to the question of why disease and symptoms develop in only 5 to 10% of those infected with E. histolytica. It has been speculated that a spectrum of virulence levels among the E. histolytica strains contribute to the outcome of amebic infection. In this study, beside determination of prevalence rate of E.histolytica and E.dispar in gastrointestinal disorder patients, genetic diversity in non-coding locus 1-2 was investigated to identify genetic differentiation of Entamoeba in positive isolates. A total of 1700 stool samples were checked from patients referred to clinical laboratories affiliated with Shahid Beheshti Medical University; samples were examined by direct and formalin detergent methods. Twenty seven cases of E. histolytica/E. dispar were detected and total genomic DNA was extracted from stool samples. E. histolytica/E. dispar complex were determined by PCR with two sets of species-specific primers from locus 1-2 gene. The purified PCR products were sequenced and the results were compared with known E. histolytica and E. dispar sequenced data. PCR for locus 1-2 gene amplified a fragment of about 430 bp in 21 out of 27 samples and was identified as E. dispar. One isolate showed a band of about 340 bp and was identified as E. histolytica. PCR were negative in five samples which were discarded. With PCR and sequencing of the PCR products a reliable genetic diversity in size, number and position of the repeat units were seen among the Iranian E. dispar isolates in locus 1-2 gene. Eight new E. dispar genotypes were found in this study and submitted to the Gen Bank/EMBL/DDBJ. The only Iranian E. histolytica isolate [NH1 E.h IR] was completely similar with the KU2 [Accession No. AB075706] strain reported from Japan