Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida B:2 in mice model

The present study was conducted to investigate the role of iron-regulated outer membrane proteins (IROMP) of Pasteurella multocida B:2 in mice as potential immunogens. Outer membrane proteins extracted from P. multocida B:2 grown under normal (OMP) and iron-deficient (IROMP) conditions were subjected to discontinuous SDS-PAGE. Nine polypeptides of MW ranging from 85.1 to 16.7 kDa from OMP preparations and two additional polypeptides of MW 95.4 and 89.1 kDa from IROMP preparations were observed with bands of MW 37.2 and 34.7 kDa as major proteins. Mice were immunized twice with OMP, IROMP-enriched fractions and whole cell lysate (WCL) via subcutaneous route at day 0 and 21. Antibody titers were determined from sera collected at weekly interval and protection was studied against challenge using 10(2) cfu of P. multocida two weeks after secondary immunization via intranasal and subcutaneous routes. IROMP and OMP immunized mice provoked significant antibody responses and IROMP induced higher antibody responses. IROMP and OMP immunized mice showed protection (100%) upon intranasal challenge and a protection (84%) following subcutaneous challenge as compared to high mortality (84%) in control mice. These results indicate that OMP enriched with IROMP fractions can be superior means of immunization.     

Rice lectin receptor-like kinase provides salinity tolerance by ion homeostasis

Sensing stress and activating the downstream signaling pathways is the imperative step for stress response. Lectin receptor-like kinase (LecRLK) is an important family that plays a key role in sensing stress conditions through lectin receptor and activates downstream signaling by kinase domain. We identified the role of OsLecRLK gene for salinity stress tolerance and hypothesized its role in Na⁺ extrusion from cell. OsLecRLK overexpression and downregulation (through artificial miRNA) transgenic lines were developed and its comparison with wild-type (WT) plants were performed overexpression transgenic lines showed better performance, whereas downregulation showed poor performance than WT. Lower accumulation of reactive oxygen species (ROS), malondialdehyde and toxic ion, and a higher level of proline, RWC, ROS scavengers in overexpression lines lead us to the above conclusion. Based on the relative expression of stress-responsive genes, ionic content and interactome protein, working model highlights the role of OsLecRLK in the extrusion of Na⁺ ion from the cell. This extrusion is facilitated by a higher expression of salt overly sensitive 1 (Na⁺/K⁺ channel) in overexpression transgenic line. Altered expression of stress-responsive genes and changed biochemical and physiological properties of cell suggests an extensive reprogramming of the stress-responsive metabolic pathways by OsLecRLK under stress condition, which could be responsible for the salt tolerance capability.     

Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach

Rice inflorescences were inoculated with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pJD4 with application of vacuum infiltration. After co-cultivation, callus was initiated and subjected to hygromycin selection, and plants were regenerated from resistant callus lines. Based on the total number of co-cultivated inflorescences bearing flowers 1 to 3 mm in length, the average frequency for recovering independent transgenic rice plants was at least 30%. Seeds from selfed R0 plants were harvested within 6 months after initiation of the experiments. Genomic DNA blot analysis showed that genes in the T-DNA of the binary plasmid were stably integrated into the rice genome, typically at low copy number. In most, but not all, cases the transgene was transmitted to R1 progeny at a frequency characteristic for Mendelian inheritance of a single dominant trait. For selfed progeny of one two-locus insertion line, reactivation of GUS expression was observed for a single copy locus that segregated from a silenced multicopy locus. For this line and some additional plants, fluorescence in situ hybridization was used to visualize the chromosomal location of the transgene insert.     

Comparative Assessment of ISSR and RAPD Marker Assays for Genetic Diversity Analysis in Jojoba [Simmondsia chinensis (Link) Schneider]

A collection of male and female plants of ten Jojoba [Simmondsia chinensis (Link) Schneider] genotypes was analyzed with 50 RAPID and 55 ISSR markers to compare the efficiency and utility of these techniques for detecting genetic polymorphism. RAPID and ISSR analysis yielded 442 and 566 scorable amplified products, respectively, of which 60.7 and 69.3% were polymorphic. ISSRs revealed efficiency over RAPDs due to high EMR (effective multiplex ratio), DI (diversity index, mean PIC per primer) and MI (marker index) values. Jaccard similarity matrices among male plants, among female plants and between male and female plants of the ten jojoba genotypes varied between 0.705-0.784. Dendrograms generated by cluster analysis (UPGMA, NTSYS-pc) supported by bootstrap values using RAPID and ISSR datasets led to grouping of most of male and females genotypes in separate clusters. While pattern of clustering remained more or less same, the two dendrograms did differ with respect to the grouping of a few male and female genotypes. The value of the Mantel test shows poor correlation (r = 0.41) between ISSR and RAPID marker datasets.     

Serum markers in pre-eclampsia

Preeclampsia occurs approximately in 10% of pregnancies and remains a leading cause of maternal and neonatal mortality and morbidity worldwide, particularly in developing countries. The condition is usually diagnosed in late pregnancy by the presence of hypertension with proteinuria and/ or edema. Prevention of any disease process requires knowledge of its etiology and pathogenesis, as well as the availability of methods for prediction of those at high risk for this disorder. Numerous clinical, biophysical, and biochemical tests have been proposed for prediction or early detection of preeclampsia. This review will explore the current tests available in the evaluation of hypertensive complications of pregnancy.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

Serum Fluoride and Sialic Acid Levels in Osteosarcoma

Osteosarcoma is a rare malignant bone tumor most commonly occurring in children and young adults presenting with painful swelling. Various etiological factors for osteosarcoma are ionizing radiation, family history of bone disorders and cancer, chemicals (fluoride, beryllium, and vinyl chloride), and viruses. Status of fluoride levels in serum of osteosarcoma is still not clear. Recent reports have indicated that there is a link between fluoride exposure and osteosarcoma. Glycoproteins and glycosaminoglycans are an integral part of bone and prolonged exposure to fluoride for long duration has been shown to cause degradation of collagen and ground substance in bones. The present study was planned to analyze serum fluoride, sialic acid, calcium, phosphorus, and alkaline phosphatase levels in 25 patients of osteosarcoma and age- and sex-matched subjects with bone-forming tumours other than osteosarcoma and musculo-skeletal pain (controls, 25 each). Fluoride levels were analyzed by ISE and sialic acid was analyzed by Warren’s method. Mean serum fluoride concentration was found to be significantly higher in patients with osteosarcoma as compared to the other two groups. The mean value of flouride in patients with other bone-forming tumors was approximately 50% of the group of osteosarcoma; however, it was significantly higher when compared with patients of group I. Serum sialic acid concentration was found to be significantly raised in patients with osteosarcoma as well as in the group with other bone-forming tumors as compared to the group of controls. There was, however, no significant difference in the group of patients of osteosarcoma when compared with group of patients with other bone-forming tumors. These results showing higher level of fluoride with osteosarcoma compared to others suggesting a role of fluoride in the disease.     

Salivary vitamins E and C in oral cancer

Lipid peroxidation may be involved in cancer and essential nutrients that can scavenge free radicals, such as vitamins E and C, operate in concert. Levels of antioxidant vitamins E and C were estimated in 50 patients with oral cancer and 24 healthy persons served as control. Significantly lower levels of vitamins E and C were observed in oral cancer patients as compared to controls (P < 0.011). Antioxidant nutrients may be utilized to a greater extent in oral cancer patients to counteract free radical-mediated cell disturbances, resulting in a reduction in salivary antioxidant levels.     

Development of male-specific SCAR marker in date palm (Phoenix dactylifera L.)

Genetics of control mechanisms that underlies sex differentiation in date palm is not known. Sex of the plants becomes known only at the time of first flowering, which takes around 5 years. In comparison, molecular diagnosis (if available/feasible) promises quick and reliable identification of sex types very early when plantlets are growing in seedbeds. To develop such an assay, genomic DNA from 45 individual plants (25 female and 20 male) belonging to different varieties of date palm was subjected to PCR amplification using 100 random amplified polymorphic DNA (RAPD) and 104 intersimple sequence repeat (ISSR) primers. Initially, two bulk genomic DNA samples (each made by pooling DNA from ten male and female plants, separately) were used. A primer showing sex-specific band in bulked samples was further used for amplification of the genomic DNA of the individual samples of that bulk. Only one RAPD primer, OPA-02, amplified a fragment of ~1.0 kb in all the individual samples of male genotypes, whereas this fragment was absent in all the female genotypes. This male-specific fragment was cloned and sequenced (GenBank accession no. JN123357), and a sequence-characterized amplified region (SCAR) primer pair was designed that amplified a 406-bp fragment in both female and male genotypes and a unique fragment of 354 bp in only male genotypes. The SCAR marker was further validated using 25 female and ten male date palm plants belonging to different varieties collected from different locations.