Enhancing secondary embryogenesis in Brassica napus by selecting hypocotyl-derived embryos and using plant-derived smoke extract in culture medium

Induction of secondary embryogenesis on transformed androgenic microspore-derived embryos (MDEs) is a convenient approach to avoid chimerism and hemizygosis for the introduced transgene. In this work, we improved two aspects related to secondary embryogenesis in rapeseed (Brassica napus L. cv. Topas) MDEs: the identification of the best source of secondary embryos in the germinated MDEs and the increase in the production of secondary embryos (SEs). We performed a ploidy analysis of the different organs of MDEs-derived plantlets by flow cytometry. Our results showed that 60?% of the MDEs-derived plantlets were mixoploid, with 60?% of them having different ploidies for different organs. We concluded that hypocotyl-derived SEs present in general higher levels of genome duplication, which makes them a source of SEs better than cotyledons in terms of genetic stability and avoidance of hemizygosis. In order to increase production of SEs, we used plant-derived aqueous smoke extracts. The aim was to verify whether these extracts have a positive effect on secondary embryogenesis and if so, to identify the most efficient conditions of use. We tested smoke extracts at different incubation times, concentrations and methods of application to MDEs. The use of smoke extract, either prior to or during germination of MDEs, markedly enhances secondary embryogenesis. The best results were obtained with the use of smoke extract as a pretreatment, incubating MDEs for no longer than 15?min with a 1:250 extract concentration.     

Detection of phlD gene in some fluorescent pseudomonads isolated from Iran and its relative with antifungal activities

Fluorescent pseudomonads that produce antibiotic 2,4-diacetylphloroglocinol (2,4-DAPG) are important group of PGRP that inhibit a broad spectrum of plant pathogenic fungi. Studying on genetic diversity of 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads has been shown with special importance. The first step to investigate the genetic diversity of these bacteria is detecting of the genes required for the biosynthesis of this antibiotic. The objectives of the current study were detection of phlD gene within fluorescent pseudomonads by a PCR-based assay, and comparison of phenotypic and genotypic characteristics of fluorescent pseudomonads with proven biocontrol potential against some soil-borne phytopathogenic fungi. We used a collection of 47 fluorescent Pseudomonas spp. some with known biological control activity against Macrophomina phaseolina, Rhizoctonia solani, Phytophthora nicotianae var. parasitica, Pythium sp. and Fusarium sp. in vitro and the potential to produce known secondary metabolites such as, siderophore, HCN and protease. The results indicated that 66, 40.42, 63.82,48.94 and 27.65% of strains revealed antagonistic activity against R. solani, M. phaseolina, Pythium sp., P. nicotianae and Fusarium sp., respectively. Rhizoctonia solani recognized as the most vulnerable fungus. Among 47 strains, 76.59, 97.87 and 17% of strains produced protease, siderophore and HCN, respectively. We could detect phlD gene in strains P-5, P-32, P-47. Strain CHA0 was used as positive control for the detection this gene. Overall, there was no obvious link between the existence of phlD gene and inhibition of fungal growth or production of the antifungal metabolites in vitro. But in some strains such as CHA0 and P-5, we saw a link between the existence of phlD and antifungal activities. Studying on detection and diversity of phlD provides a fundamental knowledge for developing a rapid genetic screening system to identify a potential biocontrol strains.     

Phenotypic and functional heterogeneity of human bone marrow- and amnion-derived MSC subsets

Bone marrow-derived mesenchymal stromal/stem cells (MSCs) are nonhematopoietic cells that are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In addition, they are known to participate in niche formation for hematopoietic stem cells and to display immunomodulatory properties. Conventionally, these cells are functionally isolated from tissue based on their capacity to adhere to the surface of culture flasks. This isolation procedure is hampered by the unpredictable influence of secreted molecules, the interactions between cocultured hematopoietic and other unrelated cells, and by the arbitrarily selected removal time of nonadherent cells before the expansion of MSCs. Finally, functionally isolated cells do not provide biological information about the starting population. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSCs based on the selective expression, or absence, of surface markers. In this report, we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow- and amnion-derived MSCs.     

The Effect of Social Stress on Chronic Pain Perception in Female and Male Mice

The current investigations on social stress primarily point to the negative health consequences of being in a stressful social hierarchy. The repetitive nature of such stressors seems to affect behavioral response to pain both in rodents and humans. Moreover, a large discrepancy in the possibility of social stresses affecting pain perception in the two genders exists. The present study examined the effect of chronic social stress on nociceptive responses of both sexes by implementing of food deprivation, food intake inequality and unstable social status (cage-mate change every 3 days) for a period of 14 days in 96 Balb/c mice. In this regard we injected 20 µl formalin 2% into the plantar surface of hind paw at the end of stress period and scored pain behaviors of all subjects, then serum concentrations of proinflammatory cytokines were measured. Our results showed that there was significant difference in chronic phase of formalin test following implementation of food deprivation and inequality (P<0.05) as compared to control group, so that pain perception was decreased considerably and this decline in inequality exposed subjects was well above isolated ones (P<0.05); whereas unstable social situation did not affect pain perception. Moreover, IL-1 and IL-6 concentrations in serum of stressed mice of both genders were well above control group (p<0.05). Finally, despite chronic pain perception in control and unstable male subjects was larger than females; the decrease of chronic pain perception in male stressed animals (poverty and inequality experienced subjects) was much more than stressed females. These results revealed that although food deprivation and social inequality can induce hypoalgesia, some socioeconomic situations like social instability don''t affect pain sensation, whereas there were similar increases of proinflammatory cytokines level in all socially stressed subjects. In addition, males display larger hypoalgesic responses to inequality as compared with females.     

Study of antifungal activities of seven essential oils from some Iranian medicinal plants against various postharvest phytopathogenic fungi

The aim of this study was to find the antifungal activities of seven essential oils from some Iranian medicinal plants that have maximum (100%) inhibition effect on the mycelium growth of postharvest phytopathogenic fungi. Among 20 examined species belonging to three families, only 7 species could stop the mycelium growth of phytopathogenic fungi. The selected plants include Trachyspermum ammi, Zataria multiflora Boiss., Satureia hortensis, Caryophillum aromaticus, Menthe piperita, Cuminum cyminum L. and Carum carvi, and fungi include Aspergillus flavus, Botrytis cinerea, Penicillium italicum, Penicillium expansum, Penicillium commune, Rhizopus stolonifer and Rhizopus lyococcus. The results showed that the essential oil of these plants could stop the mycelium growth at 500 ppm, but could not completely inhibit the spore germination, however reduced the spore germination to 80–90%. Among the fungi Rhizopus stolonifer and Rhizopus lyococcus are more resistant to the inhibition effects of essential oils. Among the plants, Cuminum cyminum L. and Carum carvi were slightly weaker than other plants. Also except for Cuminum cyminum L. and Carum carvi, the essential oils of other plants had fungicide effect while these two plants in most cases had fugistatic effect. The results showed that these essential oils can be used as an effective alternative control method.     

Two pools of beta-endorphin-like immunoreactivity in blood: plasma and erythrocytes

While attempting to delineate the reason for the reported extreme variability of beta-endorphin-like immunoreactivity (beta-ir) in human plasma (eg., nondetectable to 1 ng/ml) by standard radioimmunoassay, we noted that a substantial portion of circulating beta-ir was associated with erythrocytes. That erythrocyte associated beta-ir is authentic beta-endorphin (beta-EP) was confirmed by high performance liquid chromatography (HPLC). Analysis of blood samples from rabbits, rats and mice revealed the presence of beta-ir in erythrocytes from these species as well. These results suggest that there are two pools of beta-endorphin-like immunoreactivity in blood: plasma and erythrocytes.     

The delayed effect of mustard gas on housekeeping gene expression in lung biopsy of chemical injuries

ObjectiveSulfur mustard (SM) was used as a chemical weapon in Iraq-Iran war. Exposed people have major complications in important organs such as pulmonary system. Some studies have shown that SM could affect the expression of endogenous genes and non-housekeeping genes, time dependently. To understand the accurate molecular mechanism of the delayed effect of SM, the identification of the gene expression pattern in these patients is essential. Hence, we have evaluated mRNA expression of four common housekeeping genes (ACTIN, PGK1, β2m, GAPDH) in SM-exposed and non-exposed (control) formalin-fixed, paraffin-embedded (FFPE) human lung tissues.MethodParaffin block of lung biopsy of SM-exposed people (11 cases) and people without exposure to SM as control group (9 cases) have been selected. The mRNA expression of four endogenous control genes has been evaluated by qRT-PCR. The stability value of each gene was calculated by different methods.ResultIt was found that ACTIN mRNA has the highest expression (30.26±2.87) and PGK1 has the lowest standard deviation (SD) (30.885±2.215) between pooled groups. The best correlation was between ACTIN and PGK1 expressions. The M value has shown that ACTIN and then PGK1 are the most stable housekeeping genes among. The results obtained from the GeNorm and NormFinder have indicated that the pair ACTIN- PGK1 is the most suitable choice for endogenous control genes.ConclusionACTIN and PGK1 genes are stable in studied lung tissues and are the better than two other housekeeping genes. In addition, mustard gas does not affect their expression in long term.     

Muscle specific kinase: organiser of synaptic membrane domains

Muscle Specific Kinase (MuSK) is a transmembrane tyrosine kinase vital for forming and maintaining the mammalian neuromuscular junction (NMJ: the synapse between motor nerve and skeletal muscle). MuSK expression switches on during skeletal muscle differentiation. MuSK then becomes restricted to the postsynaptic membrane of the NMJ, where it functions to cluster acetylcholine receptors (AChRs). The expression, activation and turnover of MuSK are each regulated by signals from the motor nerve terminal. MuSK forms the core of an emerging signalling complex that can be acutely activated by neural agrin (N-agrin), a heparin sulfate proteoglycan secreted from the nerve terminal. MuSK activation initiates complex intracellular signalling events that coordinate the local synthesis and assembly of synaptic proteins. The importance of MuSK as a synapse organiser is highlighted by cases of autoimmune myasthenia gravis in which MuSK autoantibodies can deplete MuSK from the postsynaptic membrane, leading to complete disassembly of the adult NMJ.     

Analysis and Frequency of Bovine Lymphocyte Antigen (BoLA-DRB3) Alleles in Iranian Holstein Cattle

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11, and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2, and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4 and 4.4%, respectively. Thus, in the Iranian Holstein cows studied alleles associated with resistance to various diseases are found. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.__________From Genetika, Vol. 41, No. 6, 2005, pp. 817–822.Original English Text Copyright © 2005 by Nassiry, Eftekhar Shahroodi, Mosafer, Mohammadi, Manshad, Ghazanfari, Mohammad Abadi, Sulimova.The article was submitted by the authors in English.     

A new diagnostic tool for rapid and accurate detection of Mycobacterium tuberculosis

Mycobacterium tuberculosis, acid fast bacilli from the family of Mycobacteriaceae, is the causative agent of most cases of tuberculosis. Tuberculosis, as a communicable disease, remains a serious public health threat, killing more than one million people globally every year. Primary diagnosis of tuberculosis bacilli (TB) relies mainly on microscopic detection of acid fast bacilli (AFB), but the method suffers from low sensitivity and the results largely depend on the technician’s skill. New diagnostic tools are necessary to be introduced for rapid and accurate detection of the bacilli in sputum samples. We, in collaboration with Anda Biologicals, have developed a new platform, named as “Patho-tb”, for rapid detection of AFB with high sensitivity and with low dependence on human skills. Evaluation of Patho-tb test performance was done in two settings: (1) primary field study conducted using 38 sputa from high TB prevalence area of Iran (Zabol city near to the Afghanistan border), and (2) main study conducted using 476 sputa from Tehran, capital of Iran. Patho-tb was applied for processed sputum samples in parallel with routine diagnostic methods (including AFB microscopy, culture and PCR). All test results were compared to final clinical diagnostic state of an individual and diagnostic sensitivity (DSe), specificity, positive predictive value, negative predictive value and accuracy of each test results were calculated using standard formulations. Analytical sensitivity and specificity of the Patho-tb test were also determined. Calculated values for five above mentioned parameters are as follows: for field study: AFB (DSe: 29.6, DSp: 81.8, PPV: 80, NPV: 23.1, AC: 44.7), Patho-tb (DSe: 63, DSp: 72.7, PPV: 85, NPV: 44.4, AC: 65.8), and for main study: AFB (DSe: 86.1, DSp: 99.4, PPV: 98.5, NPV: 93.9, AC: 95.2), Patho-tb (DSe: 97.4, DSp: 92.9, PPV: 86.5, NPV: 98.7, AC: 94.3). Reproducibility of Patho-tb test results were near to 100% (Cohen’s kappa value between 0.85 and 1). The detection limit of Patho-tb test with 100% positivity rate was 3 × 10³ cells/ml of sputum. In the field study, Patho-tb test was 33.4% more sensitive than AFB microscopy, while the improvement was only 11.3% during the main study. Patho-tb results are easy to interpret and the test can be merged with other screening tests, like AFB. Totally, Patho-tb test alone or in conjunction with AFB microscopy is a useful screening tool for TB detection especially in poor geographical lab conditions.