Identification of Trichomonas Vaginalis Genotypes Using by Actin Gene and Molecular Based Methods in Southwest of Iran

Background:Trichomonas vaginalis (T. vaginalis) is a sexually transmitted protozoan parasite and the causative agent of trichomoniasis. The genetic characterization of T. vaginalis isolates shows notable genetic variation in this parasite. In the present study, we aimed to identify the T. vaginalis genotypes based on analyzing of actin gene in women specimens referred to health centers of Ilam city, southwest Iran.Methods:A total of 1765 female samples were collected from gynecology clinics in the city of Ilam. DNA was extracted from positive samples and nested polymerase chain reaction (Nested PCR) was used to amplify the actin gene. Then, partial sequencing and genotyping of the actin gene was performed. A phylogenetic tree was drawn using the detected genotypes of T. vaginalis and reference sequences.Results:Twenty-one of the 1765 urine and vaginal samples were positive for T. vaginalis. All infected individuals were married and their age in years was between 25 to 34. Further, the majority of infected women had cervical lesions, patchy erythema, and white color discharge. According to sequencing analysis, the isolates were identified as genotype G (n= 8) and genotype E (n= 2).Conclusion:From the collected samples, we were able to distinguish at least two genotypes (G and E) of T. vaginalis. However, lesser is known about these genotypes in the city of Ilam. Further studies with a higher number of isolates should be performed in order to understand the implications of these results in this region.Key Words: Actin gene, Genotypes, Ilam, Iran, Trichomonas vaginalis     

Complementing biological control with plant suppression: Implications for improved management of parthenium weed (Parthenium hysterophorus L.)

Parthenium hysterophorus L. is a weed of global significance that has become a major weed in Australia and many other parts of the world. A combined approach for the management of parthenium weed using biological control and plant suppression, was tested under field conditions over a two-year period in southern central Queensland. The six suppressive plant species, selected for their demonstrably suppressive ability in earlier glasshouse studies, worked synergistically with the biological control agents (Epiblema strenuana Walker, Zygogramma bicolorata Pallister, Listronotus setosipennis Hustache and Puccinia abrupta var. partheniicola) present in the field to reduce the growth (above ground biomass) of parthenium weed, by between 60–86% and 47–91%, in Years 1 and 2, respectively. The biomass of the suppressive plants was between 6% and 23% greater when biological control agents were present than when the biological control agents had been excluded. This shows that parthenium weed can be more effectively managed by combining the current biological control management strategy with selected sown suppressive plant species, both in Australia and elsewhere.     

Studies on the Refolding Process of Recombinant Horseradish Peroxidase

Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl₂ in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.     

Development of enzyme-linked immunosorbent assay for Δ12-prostaglandin J2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase

Peroxisome proliferator-activated receptor (PPAR)γ is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to J(2) series of PGs including 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2), which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ(12)-PGJ(2) has not been attempted during the life stage of adipocytes. In this study, we developed an enzyme-linked immunosorbent assay using mouse antiserum specific for Δ(12)-PGJ(2). According to the standard curve, the amount of Δ(12)-PGJ(2) can be measured from 0.5 pg to 14.4 ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ(2), while it only showed the cross-reaction of 28% with unstable PGJ(2). This immunological assay was applied to the determination of the endogenous formation of Δ(12)-PGJ(2) in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ(12)-PGJ(2) increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ(2). Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Δ(12)-PGJ(2) in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ(2) series at various doses on adipogenesis during the maturation phase. Although Δ(12)-PGJ(2) was slightly less potent than 15d-PGJ(2), each of these PGJ(2) series rescued effectively both the accumulation of fats and the gene expression of typical adipocyte-markers that were attenuated in the presence of aspirin. Taken together, our findings indicate that endogenous Δ(12)-PGJ(2) contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ(2) during the maturation phase of cultured adipocytes.     

15-Deoxy-Δ(12,14)-prostaglandin J(2) interferes inducible synthesis of prostaglandins E(2) and F(2α) that suppress subsequent adipogenesis program in cultured preadipocytes

Cultured preadipocytes enhance the synthesis of prostaglandin (PG) E(2) and PGF(2α) involving the induction of cyclooxygenase (COX)-2 during the growth phase upon stimulation with a mixture of phorbol 12-myristate 13-acetate, a mitogenic factor, and calcium ionophore A23187. Here, we studied the interactive effect of 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) on the inducible synthesis of the endogenous PGs in cultured preadipocytes and its implication in adipogenesis program. 15d-PGJ(2) interfered significantly the endogenous synthesis of those PGs in response to cell stimuli by suppressing the induction of COX-2 following the attenuation of NF-κB activation. In contrast, Δ(12)-PGJ(2) and troglitazone had almost no inhibitory effects, indicating a mechanism independent of the activation of peroxisome proliferator-activated receptor γ for the action of 15-PGJ(2). Pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor, effectively inhibited on the inducible synthesis of those PGs in preadipocytes. Endogenous PGs generated by preadipocytes only during the growth phase in response to the cell stimuli autonomously attenuated the subsequent adipogenesis program leading to the differentiation and maturation of adipocytes. These effects were prevented by additional co-incubation of preadipocytes with either 15d-PGJ(2) or PDTC although 15d-PGJ(2) alone has no stimulatory effect. Moreover, 15d-PGJ(2) did not block the inhibitory effects of exogenous PGE(2) and PGF(2α) on the adipogenesis program in preadipocytes. Taken together, 15d-PGJ(2) can interfere the COX pathway leading to the induced synthesis of endogenous PGs that contribute to negative regulation of adipogenesis program in preadipocytes.     

Requirement of argininosuccinate lyase for systemic nitric oxide production

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.     

Disruption of a mitochondrial protease machinery in Plasmodium falciparum is an intrinsic signal for parasite cell death

The ATP-dependent ClpQY protease system in Plasmodium falciparum is a prokaryotic machinery in the parasite. In the present study, we have identified the complete ClpQY system in P. falciparum and elucidated its functional importance in survival and growth of asexual stage parasites. We characterized the interaction of P. falciparum ClpQ protease (PfClpQ) and PfClpY ATPase components, and showed that a short stretch of residues at the C terminus of PfClpY has an important role in this interaction; a synthetic peptide corresponding to this region antagonizes this interaction and interferes with the functioning of this machinery in the parasite. Disruption of ClpQY function by this peptide caused hindrance in the parasite growth and maturation of asexual stages of parasites. Detailed analyses of cellular effects in these parasites showed features of apoptosis-like cell death. The peptide-treated parasites showed mitochondrial dysfunction and loss of mitochondrial membrane potential. Dysfunctioning of mitochondria initiated a cascade of reactions in parasites, including activation of VAD–FMK-binding proteases and nucleases, which resulted in apoptosis-like cell death. These results show functional importance of mitochondrial proteases in the parasite and involvement of mitochondria in programmed cell death in the malaria parasites.     

Spatial and temporal variations of pollen concentrations in Islamabad (Pakistan): effect of meteorological parameters and impact on human health

This study focuses on the identification and quantification of airborne pollen grains from allergenic plant species and their relationship with meteorological factors, i.e. maximum and minimum daily temperature, relative humidity, rainfall and wind speed in the city of Islamabad, Pakistan. An aerobiological data set (2010–2012), collected using rotorod samplers in five different sectors of the city, was supplied by the Pakistan Meteorological Department. Pollen of eight allergenic species was identified amongst which Broussonetia papyrifera exceeded the highest pollen level and, therefore, likely played a key role in aggravating the symptoms of pollen allergy in the city. The mean weekly pollen counts were next correlated with the weekly number of allergic patients visiting hospitals during 2010–2011. Clinical data were acquired from the Pakistan Institute of Medical Sciences. The highest number of allergic patients visiting hospital was usually observed during weeks with high pollen level. These results suggest a close relationship between the pollen concentration in the air and the allergy symptoms. Spearman’s rank correlation analysis was performed to establish the relationships between meteorological parameters and daily average pollen counts. A pollen calendar for the Islamabad city was also prepared to provide a guide for the timing and duration of season for all encountered pollen types.     

Activin A is essential for Feeder‐free culture of human induced pluripotent stem cells

Feeder‐free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder‐free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA‐1‐60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA‐1‐60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder‐free culture of hiPS cells requires A or AC to maintain pluripotency. J. Cell. Biochem. 114: 584–588, 2013. © 2012 Wiley Periodicals, Inc.