Cell Journal (Yakhteh)
Year:2002 | Volume:4 | Issue:13|Papers Count:12

Introduction: Different isolates of human specifies, Giardia intestinalis, also known as Giardia lambia, have been shown to vary in sensitivity to drug, isoenzyme pattern, virulence and infectivity, antigens and restriction enzyme pattern. Material and Methods: Using system that distinguishes sequence differences in the metabolic enzyme tpi, DNA extracted from trophozoite that culture in modified TYI-S-33 was ampilified by using two sets of primers, and ampilification products restricited with NcoI. Results: 683 bp amplification products of (PI) restricted with NcoI yield 2 bands (264+419bp) and 812 bp amplification products of (PII) resreicted with NcoI yield 2 bands (228+583bp). Conclusion: The present study showed that molecular methods such as PCR-RFLP are able to identify different Giardia isolates.

Introduction: Listeria monocytogenes is a gram positive bacteria, frequently found in the environment and is responsible for food-borne disease such as perinatal infections, septicameia and meningeonceohalitis in human and animals.Material and Methods: For this reason, distribution of the ctpA determinate among L. monocytogenes isolated from clinical environment, diry and poultry samples were investigated. Then, ctpA gene was tranfered into E.coil DH5-α.This investigation was carried out in 2 steps (ctpA was found Listeria monocytogenes isolated from different sources, which was kept in culture collection of Adelaiede University, Australia. Then ctpA gene was transferred into E.coil DH5-α). CtpA DNA from Listeria monocytogenes was ampilified by PCR, identified on agarose gel, purified by phenol, and ligated into pGEM-T vector. Then transferred on X-gal plate contaning ampicilin. The sequencing of ctpA DNA in with colonies was determined by using by terminator kit and sequencing machine. Results: Using PCR to identify the homologous DNA in 69 isoltes, 38% of isolates tested contained the ctpA determinate. Our results showed that 90% of clinical and dairy isolates, 85% of environmental isolates and 7% of poultry isolates of L. monocytogenes into E.coil DH5-α was successful.Conclusion: Since, the existence of ctpA in clinical, dairy and environmental samples was 90% and in poultry was 7%, so, the virulence of all of strains of this bacteria are not the same, by introducing of such (ctpA gene) into suitable carrier strains, could be expected to producted to produce a good oral immunogen against L. monocytogenes.

Introduction: Differential diagnosis of potential pathogenic Entamoeba histological from non-pathogenic Entamoeba dispar by a molecular method.Material and Methods: One pair of primers designed based on sequence of genomic DNA coding the 30KD surface antigen of E.histolytica/E.dispar. A 374 base pair PCR product was amplified by using the primers. Electrophoretic patterns of PCR product digested with HinfI was used for differentiation of two species.Results: The RFLP pattern obtained from standard E.histolytica isolate had two fragments (219 bp and 155 bp), but the standard isolate of E.dispar showed three fragments (155, 152, 67bp). Differential diagnosis of 8 isolates from Tehran by using this method indicates that all of them were E.dispar.Conclusion: Differentiation of E.histolytica and E.dispar is of great clinical and epidemiological importance. These two organisms are morphologically indistinguishable but PCR based methods could be used for proper and accurate differentiation of two amoebas.

Introduction: The purpose of this research was to study the effects of two cryoprotectants on viability, motility, morphology and fertilization rate of mouse sperm after freezing-thawing. Material and Methods: Spermatozoa were isolated from caudae epididymis of NMRI male mice (8 to 10 weeks-old) and divided to three groups of control, experimental 1 and 2. Cryoprotectant solution for the first experimental group was made of 18% raffinose (W/V) and 3% skim milk (W/V) in distilled water and for the second experimental group was made using 7% glycerol (V/V), 1% glucose (W/V) and 25% egg yolk (V/V) in PBS. They were added to sperm (1:1). After 2 minutes equilibration at room temperature, the samples were first cooled by introducing to nitrogen vapoure for 10 minutes and plunged directly into liquid nitrogen. For thawing, the samples were removed from nitrogen and diluted quickly. To remove the cryoprotectant from sperm, it was washed twice using T6 medium contaning 5% mg / ml BSA. After incubation, percentage of viability, motility and morphology of sperm at experimental groups examined and compared with control group. To evaluate the fertilization rate of frozen sperm, female NMRI mice, 6-10 weeks old, were superovulated using IP injection of 7.5 IU hMG and 7.5 IU hCG 48 hours later. Ovulated oocytes were collected from the ampullary portion of the oviducts at 12-13 hrs after hCG injection. After that oocytes were inseminated with epididymal sperm and examined pronucleous formation and development to two cell embryos. Results: Results of this research showed that viability rate of spermatozoa at groups of control, experimental 1 and 2 were 80.33%, 35.8%, 20.5% respectively and the observed differences were significant. Their motility 75.4%, 31.8%, 18.6% and normal morphology were 57.2%, 43.2%, 37.8% respectively and again the differences were significant. Fertilization rate of sperm were in control and two experimental groups 87%, 31%, 20%. All differences were significant (between exp. and control groups). Conclusion: Conservation of mouse sperm is still in research, however raffinose and skim milk is a suitable cryoprotectant and preserve sperm against freezing damages better than glycerol-glucose, but more studies to improve freezing techniques are required.

Introduction: The objective of this study was to assess developmental potential of mouse morulae, early and laic blastocysts after vitrification in R2 and R2+Vero mediums. Material and Methods: Morulae, early and late blastocysts were obtained from superovulated NMRI female mice. Embryos in experimental group were suspended in a solution of Ethylene glycol, ficoll and sucrose (EFS40) for 2 minutes and were then loaded into 0.25 ml straws, holding them on liquid nitrogen vapour for 3-5 minutes, finally were immersed into liquid nitrogen. Embryos were thawed and equilibrated in 0.5 mol sucrose solution for 5 minutes, were cultured in R2 or R2+Vero mediums for I 20 hours. Results: Results indicate high survival rate or embryos after vitrification (95, 85, 76% for morulae, early and late blastocysts respectively). Sixty three, 70 and 75% in R2 and 67, 73, 80% in R2+Vero reached to hatching and hatched blastocyst alter 4.S hours Respectively for morulae, early and late blastocyst of control groups. While 24, 19 and 12% in R2, and 18, 22 and 16% in R2+Vero of vitrified embryos were reached to Hatching and hatched blastocyst after 4S hours. Sixty five, 58 and 44% in R2 and 70, 63 and 56% in R2+Vero of vitrified embryos following a delay of 48 hours on the 4th of cultivation day reached lo hatching and hatched blastocyst. Conclusion: Our results indicate that R2 and R2+Vero have the identical property lo support development of vitrified and non-vitrified embryos. Development of vitrified embryos show the same development competence comparing to control group alter a delay of 48 hours, from this statement we conclude that although vitrification damage the embryos Inn most of these damage are reversible in a suitable medium is less expensive and more convenient than R2+Vero.

Introduction: The ionic currents show nonlinear behavior and electrical stimuli can influence their activation and inactivation kinetics. The time constant of depolarizing or hyperpolarizing current pulses has an important role in the firing behavior of neurons. In the present study, the effects of quasitrpezoidal command potentials on L-type calcium channel of somata of F1 neuron in Helix aspesa were investigated.Material and Methods: In Na+ -K+ free solution, 8 F1 neurons were clamped from holding potentials of -90 or -40 mV to +90 mV by two forms of command potentials, rectangular and quasitrapezodial command potentials. Then F1 neurons in the presence of nifedipine (1μM), anorganic blocker of L-type calcium channel, were clamped by two forms command potentials.Results: In Na+ -K+ free solution, the quasitrapezoidal command potentials in compared with rectangular command potentials caused a 36% decerment in peak of Ca2+ currents and shift threshold voltage toward positive potentials. After application of nifedipine, while the quasitrapezoidal command potentials caused the threshold voltage of nifedipine – resistance Ca2+ currents became more positive but it caused no considerable change in peak current.Conclusion: The results show that slowly hyperpolarization of membrane potential through the quasitrapezoidal command potentials can influence kinetics of ionic channels.

Introduction: Animal models of Parkinson’s disease (PD) with partial damage of nigrostriatal dopaminergic system are very suitable for development of treatment strategies. Although drug-induced rotational behavior has been conventionally used for analysis of lesioned animals, but bcause of the development of sensitization, it is necessary to invent a pure behavioral test that can evaluate such animals in a drug-free state which may better reflect a more natural response of the animal following lesion. This study was conducted to evaluate the efficiency of elevated-body swing test in analysis of unilaterally lesioned animals in an early model of Parkinson’s disease. Material and Methods: Thirty male rats were randomly divided into sham-operated and lesion groups. An early model of PD was developed by interstitial injection of 12.2 μg of 6-hydroxydopamine (6-OHDA) into the left striatum. Apomorphine-induced rotational and drug-free elevated body swing behaviors were evaluated. Results: Results of behavioral tests revealed that apomorphine caused a very significant contralateral turning in the rats of the lesion group (L+V) compared to sham-operated group (SH) (P<0.0001). In addition, there existed a significant difference between +V and SH groups in the second (P<0.01) and fourth weeks (P<0.05) after surgery. Further analysis of correlation for not number of rotations versus net number of rotations versus net number swings revealed a significant and positive correlation (r=0.52) in the second week in L+V group, but no such correlation was observed in the fourth week (r=0.24). Conclusion: Taken together, it is concluded that elevated body swing test may be of less value in evaluation of motor asymmetry in an early model of Parkinson’s disease compared to drug-induced rotational behavior. Therefore, it may be used as a complementary test to drug-induced rotational test for alternate evaluation of motor asymmetry in unilateral model of early PD in rats.

Introduction: Suspension of isolated hepatocyes is now being used in an increasing number of biochemical investigations, including drug metabolism and toxicity studies. Isolated hepatocyes, retain many of the essential properties of the intact tissue including permeability charactreristics. Material and Methods: In this method at first liver is subjected to a nonrecirculating perfusion with calcium free containing a calcium chelator like EGTA which causes irreversible separation of gap, tight and desmosomal junctions. In the second step, perfusion is continued with a buffer containing collagenase in order to dissolve extracellular matrix. In this step calcium is added back to the medium to ensure maximal enzyme activity. Results: In situ perfusion of rat liver using collagenase very high recovery of intact isolated hepatocyes (more than %90), Approximately 500 millions of intact cells have been obtained from each rat weighing 200g. Conclusion: The availability of these methods to isolate intact viable hepacoyes has provided a system that can be utilized in biochemistry, pharmacology, and toxicology. Suspension of isolated hepatocyes is now frequently used in biochemical studies. This experimental tool has been successfully employed in studies on gluconeogenesis, glycolysis, protein, lipid, fatty acid, and urea syntheses, keton body production, protein metabolism, ethanol oxidation, membrane transport, and response to hormones.