Comparative Assessment on the Expression Level of Recombinant Human Follicle-Stimulating Hormone (FSH) in Serum-Containing Versus Protein-Free Culture Media

Production of recombinant pharmaceutical proteins has made a great contribution to modern biotechnology. At present, quick advances in protein expression lead to the enhancement of product quantity and quality as well as reduction in timescale processing. In the current study, we assessed the expression level of recombinant human follicle-stimulating hormone (rhFSH) in adherent and suspension Chinese hamster ovary (CHO) cell lines by cultivation in serum-containing and chemically defined, protein-free media. The expression cassette entailing FSH subunits was transfected to CHO/dhfr- and CHO DG44 cell lines, and gene amplification was achieved using dihydrofolate reductase (DHFR)/methotrexate (MTX) system. Afterward, the expression level of rhFSH was studied using real-time PCR, Western blotting and ELISA. Our achievements revealed that stepwise increase in MTX [up to 2000 nano-molar (nM)] leads to boost the expression level of rhFSH mRNA in both cell lines, although DG44 have better results, as mRNA expression level reached 124.8- and 168.3-fold in alpha and beta subunits, respectively. DG44 cells have also the best protein production in 2000 nM MTX, which reached 1.7-fold in comparison with that of the mock group. According to the above results and many advantages of protein-free media, DG44 is preferable cell line for future steps.     

Description of Boleodorus Bushehrensis n. Sp. (Rhabditida: Tylenchidae) from Southern Iran,and Observations on a Commonly Known Species

A new species of the genus Boleodorus, recovered from southern Iran, is described and illustrated based upon morphological and molecular data. B. bushehrensis n. sp. is mainly characterized by having a wide and low cephalic region (which is continuous with the adjacent body), the oral aperture in a depression in side view under a light microscope, four lines in the lateral field, weak metacorpus with a vestigial-to-invisible valve, and short (26–38 mm long) hooked tail with rounded tip. The females are 334–464 mm long and have a spherical spermatheca filled with spheroid sperm; males have 11.5- to 12.0-mm-long spicules and weakly developed bursa. The new species has an annulated low cephalic region, four large cephalic papillae, and small crescent-shaped amphidial openings when observed by scanning electron microscopy (SEM). Its morphological and morphometric differences with seven known species are discussed. The phylogenetic relationships of the new species with other relevant genera and species have been studied using partial sequences of small and large subunit ribosomal DNA (SSU and LSU rDNA). In both the SSU and LSU phylogenies, the sequences of B. bushehrensis n. sp. and other Boleodorus spp. formed a clade. A second species, B. thylactus, when studied under SEM, has a raised, smooth cephalic region, four large cephalic papillae, and oblique amphidial slits, with the oral opening in a depression.     

Genetic Diversity of Persian Ecotypes of Indian Walnut (<Emphasis Type="Italic">Aeluropus littoralis</Emphasis> (Gouan) Pari.) by AFLP and ISSR Markers

Indian walnut Aeluropus littoralis (Gouan) Pari, well known for its salt tolerance and forage quality, is a perennial monocot grass from Chlorideae tribe that shows a high potential to become an important genetic model. Although, Aeluropus germplasm is very rich in Iran; but is still unexplored. In this study, 20 ecotypes of A. littoralis were gathered from different geographic zones and a set 16 primers (ISSRs-AFLPs combined) was used for genetic diversity evaluation. A total of 635 fragments and 594 polymorphic fragments were produced (93.5%) and modest levels of genetic similarity were found (ranging from 0.38 to 0.71) among ecotypes. The results showed an equal competition between the two markers on polymorphism detection, so both molecular techniques were able to distinguish the A. littoralis ecotypes. Furthermore, Dendrogram based on both the Bayesian and PCoA analysis showed a clear discrimination and significant variation among ecotypes; however, molecular clustering indicated a weak concordance with geographical grouping. The amount of molecular diversity revealed by molecular markers for A. littoralis in this study can be exploited for pasture conservation programs and new forage crop development.     

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.