A role for endocannabinoids in acute stress-induced suppression of the hypothalamic-pituitary-gonadal axis in male rats / 대한생식의학회지

OBJECTIVE: Stress is known to be an inhibitor of the reproductive hypothalamic-pituitary-gonadal (HPG) axis. However, the neural and molecular connections between stress and reproduction are not yet understood. It is well established that in both humans and rodents, kisspeptin (encoded by the kiss1 gene) is a strong stimulator of the HPG axis. In the present study we hypothesized that endocannabinoids, an important neuromodulatory system in the brain, can act on the HPG axis at the level of kiss1 expression to inhibit reproductive function under stress. METHODS: Adult male Wistar rats were unilaterally implanted with an intracerebroventricular cannula. Afterwards, the animals were exposed to immobilization stress, with or without the presence of the cannabinoid CB1 receptor antagonist AM251 (1 microg/rat). Blood samples were collected through a retro-orbital plexus puncture before and after stress. Five hours after the stress, brain tissue was collected for reverse transcriptase-quantitative polymerase chain reaction measurements of kiss1 mRNA. RESULTS: Immobilization stress (1 hour) resulted in a decrease in the serum luteinizing hormone concentration. Additionally, kiss1 gene expression was decreased in key hypothalamic nuclei that regulate gonadotrophin secretion, the medial preoptic area (mPOA), and to some extent the arcuate nucleus (ARC). A single central administration of AM251 was effective in blocking these inhibitory responses. CONCLUSION: These findings suggest that endocannabinoids mediate, at least in part, immobilization stress-induced inhibition of the reproductive system. Our data suggest that the connection between immobilization stress and the HPG axis is kiss1 expression in the mPOA rather than the ARC.

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

Downregulation of calcineurin gene is associated with glucan-time[registered sign] resiatance in leishmania infantum

Pentavalent antimonials are the first line drugs for the treatment of leishmaniasis. Unresponsiveness of Leishmania spp. to antimonial drugs is a serious problem in some endemic areas. Investigations on molecular mechanisms involved in drug resistance are essential for monitoring and managing of the disease. Cal-cineurin is an essential protein phosphatase for number of signal transduction pathways in eukaryotic cells and it has a mediated role in apoptosis. This study aimed to determine of biomarker[s] in Glucantime[registered sign] resiatance strain of L. infan-tum. We used cDNA amplified fragment length polymorphism [cDNA-AFLP] and real time-RT PCR assays to compare gene expression profiles at the mRNA levels in resistant and susceptible L. infantum field isolates. The cDNA-AFLP results showed downlegulation of calcineurin in resis-tant isolate in comparison with susceptible one. Significant downregulation of cal-cineurin [0.42 fold] [P<0.05] was found in resistant isolate compared to susceptible one by Real time-RT PCR. This is the first report of calcineurin implication in Glucantime[registered sign] drug resistance of field [natural] isolate of L. infantum. Downregulation of calcineurin could protect parasites from antimonial-induced apoptosis

Differential expression of human homeodomain TGIFLX in brain tumor cell lines

Glioblastoma is the most common and the most lethal primary brain cancer. This malignancy is highly locally invasive, rarely metastatic and resistant to current therapies. Little is known about the distinct molecular biology of glioblastoma multiforme [GBM] in terms of initiation and progression. So far, several molecular mechanisms have been suggested to implicate in GBM development. Homeodomain [HD] transcription factors play central roles in the expression of genomic information in all known eukaryotes. The TGIFX homeobox gene was originally discovered in human adult testes. Our previous study showed implications of TGIFLX in prostate cancer and azoospermia, although the molecular mechanism by which TGIFLX acts is unknown. Moreover, studies reported that HD proteins are involved in normal and abnormal brain developments. We examined the expression pattern of TGIFLX in different human brain tumor cell lines including U87MG, A172, Daoy and 1321N1. Interestingly, real time RT-PCR and western blot analysis revealed a high level of TGIFLX expression in A172 cells but not in the other cell lines. We subsequently cloned the entire coding sequence of TGIFLX gene into the pEGFP-N1 vector, eukaryotic expressionvector encoding eGFP, and transfected into the U-87 MG cell line. The TGIFLX-GFP expression was confirmed by real time RT-PCR and UV-microscopic analysis. Upon transfection into U87 cells, fusion protein TGIFLX-GFP was found to locate mainly in the nucleus. This is the first report to determine the nuclear localization of TGIFLX and evaluation of its expression level between different brain tumor cell lines. Our data also suggest that TGIFLX gene dysregulation could be involved in the pathogenesis of some human brain tumors

Identification of Xq22.1-23 as a region linked with hereditary recurrent spontaneous abortion in a family

Recurrent spontaneous abortion [RSA] is one of the most common health complications with a strong genetic component. Several genetic disorders were identified as etiological factors of hereditary X linked RSA. However, more genetic factors remain to be identified. In this study we performed linkage analysis on a large X linked RSA pedigree to find a novel susceptibility locus for RSA. A linkage scan using 11 microsatellites was performed in 27 members of a large pedigree of hereditary X-linked RSA. Two point parametric Linkage was performed using Superlink v 1.6 program. Evidence of linkage was observed to markers at Xq23, DXS7133 and at Xq22.1 DXS101, with LOD score of 3.12 and 1.60, respectively. Identified locus in this study may carry a responsible gene in RSA. Narrowing down of this region may leads to identification of this gene

Mutation screening in the mitochondrial d-loop region of tumoral and non-tumoral breast cancer in Iranian patients

The mitochondrial DNA [mtDNA] mutations in mitochondrial coding and non coding regions seem to be important in carcinogenesis. The aim of this investigation was to evaluate coding region [mttRNA[Phe] and tRNA[Pro]] and non-coding sequence, mitochondrial displacement loop [mtDNA D-loop], in the cancerous and non-cancerous lesions of Iranian patients with breast cancer [BC]. Genomic DNA was extracted from 50 breast tumors and surrounding normal tissue pairs as well as from 50 unrelated normal breast tissues from Iranian Kurdish population. Subsequently, PCR amplification was performed using specific primers, and then PCR products were subjected to direct sequencing. 41 genetic variants were identified in mtDNA D-loop among tumoral and non-tumoral tissues but not in tRNA[Phe] and tRNA[Pro] sequences. Our findings indicated that C182T, 194insT, 285insA and 16342delT were just found in BC tumors whereas 302insC, C309T and C16069T found in both tumors and surrounding normal tissues. Although our findings showed that the observed genetic variations were not restricted to breast cancer tissues, some genetic changes were found only in BC tumors. Our results, in agreement with the evidence from earlier studies, confirm that the mtDNA genetic alterations might be implicated in tumor initiation, progression and development.

Lack of association between the C677T single nucleotide polymorphism of the MTHFR gene and glaucoma in Iranian patients

Glaucoma is a major cause of blindness worldwide. A single nucleotide polymorphism of the MTHFR gene [C677T] has been associated with susceptibility to this disease, although this is controversial in the last decade. In this study, the possible association between the MTHFR C677T polymorphism and the risk of developing primary open angle [POAG] and pseudoexfoliation glaucoma [PEXG] was investigated. For this, a prospective study consisting of 73 POAG, 85 PEXG and 90 matched controls was undertaken in an Iranian population. Genomic DNA was extracted from whole blood. Genotyping of all individuals for the MTHFR C677T polymorphism was conducted using the PCR-RFLP technique. Our findings revealed no significant association between the MTHFR C677T polymorphism in POAG and PEXG compared with controls. Consistent with several other studies, our analysis suggests that the MTHFR C677T polymorphism is unlikely to be a factor contributing to the risk of developing specific forms of glaucoma

[Effects of probiotic Lactobacillus acidophilus and Lactobacillus casei on the behavior of colorectal tumor cells]

Probiotic microorganisms are living normal flora of human body that have nutritional value and health benefits when administered in adequate amounts. The health benefits include prevention of bacterial diarrhea, skin eczema and recently understood, prevention and control of various cancers, as well. Different mechanisms such as stimulating the immune system, modifying the composition of gastrointestinal and genitourinary tract normal flora and prevention of the carcinogenic activity of fecal enzymes have been identified for their probiotic activity. Due to the high density of the normal flora in the gut and also preferentially sporadic nature of colorectal cancers, these cancers are among the main candidates of treatment trials with probiotics. In this study, direct effects of probiotic lactobacilli on colon cancer tumor cells were studied. Supernatant fluid and bacterial extracts were prepared and CaCo-2 cells were treated by these materials. Subsequently, the effects of the aforesaid elements were evaluated on cell proliferation, cell necrosis and cell apoptosis by MTT assay, LDH assay and caspase-3 activity. The supernatants of lactobacilli decreased cell proliferation and increased cell apoptosis but they did not have any effect on cell necrosis. In contrast, when cancerous cells were treated by lactobacilli extract, it lead to cell necrosis in addition to reduction in cell proliferation and increase in cell apoptosis. The use of lactobacillus probiotics may reduce proliferation of tumor cells in the early stages of colorectal cancers