Roles for Kisspeptin in proliferation and differentiation of spermatogonial cells isolated from mice offspring when the cells are cocultured with somatic cells

Kisspeptin (Kp) expression in testis has caused most of the recent research surveying its functional role in this organ. This peptide influences spermatogenesis and sperm capacitation, so it is considered as a regulator of reproduction. Kp roles exert through hypothalamic/pituitary/gonadal axis. We aimed to evaluate direct roles for Kp on proliferation and differentiation of spermatogonial cells (SCs) when the cells are cocultured with somatic cells. Somatic cells and SCs were isolated from adult azoospermic and newborn mice and then enriched using a differential attachment technique. After the evaluation of identity and colonization for SCs, the cells were cocultured with somatic cells, and three doses of Kp (10⁻⁸-10⁻⁶ M) was assessed on proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c-Kit and SCP₃, TP_1, TP₂, and, Prm₁ markers) of the coculture system. Investigations were continued for four succeeding weeks. At the end of each level of testosterone in the culture media was also evaluated. We found positive influence from Kp on proliferative and differentiative markers in SCs cocultured with somatic cells. These effects were dose-dependent. There was no effect for Kp on testosterone level. From our findings, we simply conclude that Kp as a neuropeptide for influencing central part of reproductive axis could also positively affect peripheral processes related to spermatogenesis without having an effect on steroidogenesis.     

Low representation of Fc receptor-like 1–5 molecules in leukemic cells from Iranian patients with acute lymphoblastic leukemia

Recent studies have demonstrated expression of Fc receptor-like (FCRL) molecules, a newly identified family with preferential B-cell lineage expression, in some chronic B-cell leukemias with possible implication for classification and/or targeted immunotherapy. In this study, the expression pattern of FCRL1-5 genes was studied in 73 Iranian ALL patients and 35 normal subjects using semi-quantitative RT-PCR method. FCRL protein expression was also investigated by flow cytometry. Our results indicate significant down-regulation of all FCRL genes in ALL compared to normal subjects. Although, FCRL mRNA expression was almost exclusively confined to normal isolated B-cells compared to T-cells, but these genes were similarly expressed in B-ALL, T-ALL and different B-ALL immunophenotypic subtypes. Surface protein expression of FCRL1, 2, 4, and 5 molecules in 10 ALL and 5 normal samples confirmed the PCR results. Expression profile of FCRL molecules in different subtypes of ALL argues against their potential implication as suitable targets for classification and/or immunotherapy of ALL. T. Kazemi, H. Asgarian-Omran and A. Memarian have contributed equally to this study.     

Assessment of the Potential Role of Muscle Spindle Mechanoreceptor Afferents in Chronic Muscle Pain in the Rat Masseter Muscle

Background

The phenotype of large diameter sensory afferent neurons changes in several models of neuropathic pain. We asked if similar changes also occur in “functional” pain syndromes.

Methodology/Principal Findings

Acidic saline (AS, pH 4.0) injections into the masseter muscle were used to induce persistent myalgia. Controls received saline at pH 7.2. Nocifensive responses of Experimental rats to applications of Von Frey Filaments to the masseters were above control levels 1–38 days post-injection. This effect was bilateral. Expression of c-Fos in the Trigeminal Mesencephalic Nucleus (NVmes), which contains the somata of masseter muscle spindle afferents (MSA), was above baseline levels 1 and 4 days after AS. The resting membrane potentials of neurons exposed to AS (n = 167) were hyperpolarized when compared to their control counterparts (n = 141), as were their thresholds for firing, high frequency membrane oscillations (HFMO), bursting, inward and outward rectification. The amplitude of HFMO was increased and spontaneous ectopic firing occurred in 10% of acid-exposed neurons, but never in Controls. These changes appeared within the same time frame as the observed nocifensive behaviour. Ectopic action potentials can travel centrally, but also antidromically to the peripheral terminals of MSA where they could cause neurotransmitter release and activation of adjacent fibre terminals. Using immunohistochemistry, we confirmed that annulospiral endings of masseter MSA express the glutamate vesicular transporter VGLUT1, indicating that they can release glutamate. Many capsules also contained fine fibers that were labelled by markers associated with nociceptors (calcitonin gene-related peptide, Substance P, P2X3 receptors and TRPV1 receptors) and that expressed the metabotropic glutamate receptor, mGluR5. Antagonists of glutamatergic receptors given together with the 2^nd injection of AS prevented the hypersensitivity observed bilaterally but were ineffective if given contralaterally.

Conclusions/Significance

Low pH leads to changes in several electrical properties of MSA, including initiation of ectopic action potentials which could propagate centrally but could also invade the peripheral endings causing glutamate release and activation of nearby nociceptors within the spindle capsule. This peripheral drive could contribute both to the transition to, and maintenance of, persistent muscle pain as seen in some “functional” pain syndromes.     

Single molecule force spectroscopy of salt-dependent bacteriophage T7 gene 2.5 protein binding to single-stranded DNA

The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage lambda DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-Delta26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to single-stranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 +/- 0.6) x 10(5) m(-1) in a 50 mm Na(+) solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 +/- 0.9) x 10(7) m(-1) under the same solution conditions. T7 gp2.5-Delta26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.     

The efficient isolation of murine splenic dendritic cells and their cytochemical features

Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.     

Antioxidative enzymes activities and accumulation of steroids in hairy roots of Trigonella

Steroidal sapogenins and phytosterols are a group of secondary metabolites which are very considerable in the pharmaceutical industry. Fenugreek (Trigonella foenum-graecum L.) is the good source of these compounds. In recent decades, there is a great interest to production of these compounds by cultivation of transformed roots. In present study, hairy roots induction in two Trigonella species (T. foenum-graeceum, T. monantha) with three strains of Agrobacterium rhizogenes (15,834, A4 and wt) was investigated. Transgenic status of roots was confirmed by PCR using rolB specific primers. Virulence of these strains was examined on explants of leaf, leaf cotyledone and hypocotyle in both species. The best strain was wt for hairy root induction in hypocotyle and leaf explants of T. foenum-graeceum and T. monantha. Significant quantitative differences were showed between shoot, root and hairy roots in both species. Protein content in root and hairy root of both species was significantly lower in comparison with shoot. Superoxide dismutase (SOD) and peroxidase (POX) activities in hairy roots of both species were higher as compared to other organs. The hairy roots of both species showed an ability to synthesize steroidal sapogenins. These results presented that hairy roots could be a suitable procedure for producing sapogenins compounds that have medicinal value in Trigonella.     

Evaluation of satellite data efficiency in identification of plant groups

Application of remote sensing (RS) and geographical information system (GIS) techniques has been increased in natural sciences. In fact, it is inevitable applying of these techniques in vegetation studies due to the existence of some problems in traditional methods (e.g. sampling, calculation, analysis and so on). On this scope, scientists must have sufficient information about the efficiency of these techniques as a useful tool in their studies. This study aims to evaluate the IRS-P6 LISS III and Landsat ETM⁺ efficiency in plant groups’ identification. In order to this purpose, 143 training samples were collected from areas that showed homogenous composition of plant species in at least area of 3600 m² (60 × 60 m). Coordinates of these training samples were recorded using a GPS device and transferred to a GIS database. Also, ENVI 4.2 package has used to process and analyze the satellites data. Several methods of processing such as; spectral separability, supervised classification and assessment of classification accuracy were used in order to gain a satisfy evaluation of the data efficiency. The results indicated that net farming of alfalfa and Juniperus polycarpus–Artemisia kopetdaghensisi community have the most separability on the satellite images (1.99 for Landsat and 2 for IRS). Against, the least separabilities on the Landsat data were between Ju. polycarpus–Onobrychis cornuta and Ju. polycarpus–Ar. kopetdaghensis communities (1.57) and between Ju. polycarpus–Ar. kopetdaghensis and Ju. polycarpus–Agropyron intermedium (1.53) on the IRS data. According to these results, it is concluded that the satellite data are somedeal able to identify plant groups when vegetation communities are sufficiently homogenous, abundant and spectrally and ecologically separable.     

Dosimetric characteristics of the INTRABEAM ® system with spherical applicators in the presence of air gaps and tissue heterogeneities

The main aim of this study was to investigate the dosimetric characteristics of the INTRABEAM ® system in the presence of air gaps between the surface of applicators (APs) and tumor bed. Additionally, the effect of tissue heterogeneities was another focus. Investigating the dosimetric characteristics of the INTRABEAM® system is essential to deliver the required dose to the tumor bed correctly and reduce the delivered dose to the ribs and lung. Choosing the correct AP size and fitting it to the lumpectomy cavity is essential to remove the effect of air gaps and avoid inaccurate dose delivery. Consequently, the Geant4 toolkit was used to simulate the INTRABEAM ® system with spherical APs of various sizes. The wall effect of the ion chamber (IC) PTW 34013 used in the present study was checked. The simulations were validated in comparison with measurements, and then used to calculate any inaccuracies in dose delivery in the presence of 4- and 10-mm air gaps between the surface of the APs and the tumor bed. Also, the doses received due to tissue heterogeneities were characterized. It turned out that measurements and simulations were approximately in agreement (± 2%) for all sizes of APs. The perturbation factor introduced by the IC due to differences in graphite-coated polyethylene and air as compared to the phantom material was approximately equal to one for all AP. The greatest relative dose delivery difference was observed for an AP with a diameter of 1.5 cm, i.e., 44% and 70% in the presence of 4- and 10-mm air gaps, respectively. In contrast, the lowest relative dose delivery difference was observed for an AP with a diameter of 5 cm, i.e., 24% and 42% in the presence of 4- and 10-mm air gaps, respectively. Increasing APs size showed a decrease in relative dose delivery difference due to the presence of air gaps. In addition, the undesired dose received by the ribs turned out to be higher when a treatment site closer to the ribs was assumed. The undesired dose received by the ribs increased as the AP size increased. The lung dose turned out to be decreased due to the shielding effect of the ribs, small lung density, and long separation distance from the AP surface.