Genetic engineering approaches to enhance oil content in oilseed crops

Oilseed crops play an important role in the agricultural economy. Apart from being an integral component of human diet and industrial applications, they are also gaining importance as replacement to fossil fuels for meeting the energy needs. The last two decades have been marked by several important events in genetic engineering and identification of gene targets for enhancing seed oil content in oilseed crops, and will aid the successful development of new generation high yielding oil crops. Specifically, genetic engineering has shown real breakthrough in enhancing oil content in oilseed rape, camelina, soybean and maize. Moreover, ongoing research efforts to decipher the possibilities of genetic modifications of key regulators of oil accumulation along with physiological and biochemical studies to understand lipid biosynthesis will set a platform to produce transgenic oilseed crops with enhanced oil content. In this review, we briefly describe different genetic engineering approaches explored by different researchers for enhancing oil content. Further, we discuss a few promising and potential approaches and challenges for engineering oil content in oilseed crops.     

Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens

The structure and infectivity of the oocysts of a new species of Cryptosporidium from the feces of humans are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts. and measure 4.4-5.4 microm (mean = 4.86) x 4.4-5.9 microm (mean = 5.2 microm) with a length to width ratio 1.0-1.09 (mean 1.07) (n = 100). Oocysts were not infectious for ARC Swiss mice, nude mice. Wistar rat pups, puppies, kittens or calves, but were infectious to neonatal gnotobiotic pigs. Pathogenicity studies in the gnotobiotic pig model revealed significant differences in parasite-associated lesion distribution (P = 0.005 to P = 0.02) and intensity of infection (P = 0.04) between C. parvum and this newly described species from humans. In vitro cultivation studies have also revealed growth differences between the two species. Multi-locus analysis of numerous unlinked loci, including a preliminary sequence scan of the entire genome demonstrated this species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further support for its species status. Based on biological and molecular data, this Cryptosporidium infecting the intestine of humans is proposed to be a new species Cryptosporidium hominis n. sp.     

Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

Effect of Withania somnifera Dunal in ethanol-induced anxiolysis and withdrawal anxiety in rats

Withania somnifera (WS) or its psychotropic preparation is known to play a critical role in morphine, alcohol and benzodiazepines addiction. This study investigates the role of WS in acute ethanol and withdrawal from chronic ethanol consumption using elevated plus maze paradigm in rats. Acute administration of ethanol (1.5-2 g/kg, ip) triggered anxiolytic effect and withdrawal from prolonged ethanol (9% v/v ethanol, 15 days) consumption elicited enhanced behavioral despair (anxiety). Acute administration of WS (50 mg/kg, oral) potentiated the anxiolytic action of subeffective dose of ethanol (0.5 or 1 g/kg, ip). Moreover, the ethanol withdrawal anxiety was markedly antagonized in dose dependent manner by WS at 200 and 500 mg/kg or higher dose of ethanol (2.5 g/kg). However, co-administration of subeffective doses of WS (50 mg/kg, oral) and ethanol also attenuated withdrawal-induced anxiety due to chronic ethanol (9% v/v ethanol, 15 days) consumption. The results suggest the protective effect of WS in the management of ethanol withdrawal reactions.     

Cytotoxic and genotoxic effects of methotrexate in germ cells of male Swiss mice

Methotrexate (MTX) is an anti-metabolite drug widely used in the treatment of neoplastic disorders, rheumatoid arthritis and psoriasis. Developed as an analogue of folic acid, it inhibits purine and pyrimidine synthesis that accounts for its therapeutic efficacy as well as for its toxicities. MTX has narrow therapeutic index and its toxicity has been reported in various organ systems including gastrointestinal, haematologic and central nervous system. The objective of the present study is to investigate the germ cell toxicity induced by MTX in male Swiss mice. MTX was administered intraperitoneally (ip) at the doses of 5, 10, 20 and 40mg/kg to mice (20-25g) weekly once (wk) for 5 and 10 weeks. The animals were sacrificed 1 week after receiving the last treatment of MTX. The germ cell toxicity was evaluated using testes weight (wt), sperm count, sperm head morphology, sperm comet assay, histology, TUNEL and halo assay in testis. MTX treatment significantly reduced the sperm count and increased the occurrence of sperm head abnormalities in a dose dependent manner. It induced the testicular toxicity as evident from the histology of testis. Sperm comet, TUNEL and halo assay in testis also revealed significant DNA damage after MTX treatment. On the basis of the present study, it can be concluded that MTX induced germ cell toxicity in mice.     

Effect of immunization with lipid associated polysaccharide antigen and anti CD-2 antibodies on class II MHC expression and cellular immune response in BALB/C mice infected with Leishmania donovani

In a bid to characterize the antigens and immunization mechanisms which may be used to produce a protective response against L. donovani, role of lipid associated polysaccharide (LPS) antigen and whole antigen was evaluated. BALB/C mice were immunized with whole or LPS antigen in combination with one of three putative adjuvents (anti CD-2 antibody/FIA/0.85% Saline). LPS antigen emulsified in anti CD-2 antibody was found to induce significant antibodies in mice on day 28 against challenge with lethal dose of L. donovani. Immunoprophylactic properties of LPS and whole antigen was investigated on day 40 through cytokine elicitation (IL-2), MIF) in culture supernatants of spleen cells, but before that MHC-II expressed on macrophage was studied. The LPS antigen in combination with anti CD-2 antibody was found to be most immuno-reactive inducing higher MHC-II expression on macrophages which was associated with substantial rise in the level of MIF and IL-2. It coincided with decline in antibody titre in 100% mice immunized with LPS antigen while Leishmania injected as whole antigen failed to induce specific macrophage and T-cell response with all the above formulations. We surmise from our data that lipid associated polysaccharide antigen linked to anti CD-2 antibody has potential for eliciting protective immunity against Leishmania.     

Molecular and phylogenetic characterisation of Cryptosporidium from birds

Avian isolates of Cryptosporidium species from different geographic locations were sequenced at two loci, the 18S rRNA gene and the heat shock gene (HSP-70). Phylogenetic analysis of the sequence data provided support for the existence of a new avian species of Cryptosporidium infecting finches and a second species infecting a black duck. The identity of Cryptosporidium baileyi and Cryptosporidium meleagridis as valid species was confirmed. Also, C. baileyi was identified in a number of isolates from the brown quail extending the host range of this species.     

Crude oil degradation efficiency of a recombinant Acinetobacter baumannii strain and its survival in crude oil-contaminated soil microcosm

A hydrocarbon degrading Acinetobacter baumannii S30 strain, isolated from crude oil-contaminated soil, was inserted with the lux gene from the luciferase gene cassette luxCDABE. Soil microcosms were designed to study the degradation efficacy for total petroleum hydrocarbon (TPH) of crude oil by lux-tagged A. baumannii S30 pJES. Bioaugmentation of a TPH-contaminated microcosm with A baumannii S30 pJES showed that TPH levels were reduced from 89.3 to 53.9 g/kg soil in 90 days. Biodegradation of TPH by A baumannii S30 pJES was also monitored in shake flask conditions, which showed a reduction of initial TPH levels by over 50% at the end of 120 h. A lux-PCR-based approach along with the standard dilution plating with selective antibiotics was successfully utilized to monitor the survivability of the lux-tagged strain A. baumannii S30 pJES in soil microcosms and stability of the lux insert in the host strain A. baumannii S30. The selective plating technique indicated the population of A. baumannii S30 pJES to be 6.5+/-0.13 x 10(8) CFU/g at day zero (just after bioaugmentation) and 2.09+/-0.08 x 10(8) CFU/g of soil after 90 days of incubation. lux-PCR confirmed the stability of the insert in all the randomly selected colonies of A. baumannii strains from the antibiotic plates. The lux insert was stable after 50 generations in Luria Bertini broth and storage at -70 degrees C as glycerol stocks for over a year. These results revealed that the lux insert was stable and lux-tagged A. baumannii S30 strain could survive in a TPH-contaminated soil microcosm and could degrade TPH in the soil microcosm conditions. It can be used as an effective marker to monitor the survival of augmented strains at a bioremediation site.     

Tolerance induction in composite facial allograft transplantation in the rat model

Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. The authors introduce a hemifacial allograft transplant model to investigate the rationale for the development of functional tolerance across the major histocompatibility complex barrier. Eighteen rats in three groups were studied. The composite hemifacial allotransplantations including the ear and scalp were performed between Lewis-Brown Norway (RT1l+n) and Lewis (RT1l) rats and isotransplantations were performed between Lewis rats. Isograft controls (n = 6) and allograft controls (n = 6) did not receive treatment. Allografts in treatment group (n = 6) were treated with cyclosporine A 16 mg/kg/day during the first week; this dose was tapered to 2 mg/kg/day over 4 weeks and maintained at this level thereafter. Functional tolerance to face allografts was evaluated clinically and histologically. Donor-specific chimerism was assessed at days 21 and 63 by flow cytometry. In vitro evaluation of donor-specific tolerance was performed by mixed lymphocyte reaction at day 160 after transplantation. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 7 days after transplantation, as confirmed by histopathologic analysis. Five of six face allografts under the cyclosporine A protocol showed no signs of rejection for up to 240 days and remained alive and under evaluation, whereas one animal showed signs of rejection at day 140. This was reversed by adjustment of the cyclosporine A dose. At day 21 after transplantation, flow cytometric analysis of the donor-specific chimerism showed 1.11 percent of double-positive CD4FITC/RT1Ac-Cy7 and 1.43 percent of double-positive CD8PE/RT1Ac-Cy7 T-cell populations in the peripheral blood of hemiface allotransplant recipients. The chimerism level of double-positive CD4FITC/RT1Ac-Cy7 T cells increased to 3.39 percent, whereas it remained stable for the double-positive CD8PE/RT1Ac-Cy7 T-cell population at day 63 after transplantation (1.00 percent). The mixed lymphocyte reaction assay at day 160 after transplantation revealed donor-specific tolerance to donor (Lewis-Brown Norway) antigens and strong reactivity to the third-party (ACI) alloantigens. In this study, donor-specific chimerism and functional tolerance were induced in hemifacial allograft transplants across the major histocompatibility complex barrier under cyclosporine A monotherapy protocol. This model will allow further studies on tolerance induction protocols.     

Cholecystokinin pathways modulate sensations induced by gastric distension in humans

Ingested fat releases CCK, causes gastric relaxation, delays gastric emptying, and limits meal size; however, the mechanistic link among these actions has not been established. Fatty acid release of CCK is chain-length sensitive; dodecanoic acid (C12) induces greater CCK release than decanoic acid (C10). The effect of C12 or C10 on tolerance to subsequent intragastric infusion of liquid was determined in healthy subjects, with and without the CCK(1) receptor antagonist dexloxiglumide. Gastric wall relaxation after either fatty acid was assessed by graded volume distension and by barostat; gastric emptying was measured by gastric aspiration and by a [(13)C]octanoic acid breath technique. C12 released more CCK (mean plasma CCK after vehicle, 4.7 +/- 0.8 pM; C10, 4.8 +/- 0.3 pM; C12, 8 +/- 1.2 pM; P < 0.05 C12 vs. C10 or vehicle) and reduced the volume of water (and of 5 and 25% glucose solutions) delivered at maximum tolerance compared with C10 or vehicle (volume of water tolerated after vehicle, 1,535 +/- 164 ml; C10, 1,335 +/- 160 ml; C12, 842 +/- 103 ml; P < 0.05 C12 vs. C10 or vehicle); this effect was abolished by dexloxiglumide. Intragastric volumes were always similar at the limit of tolerance, and, whereas gastric relaxation occurred to similar degrees after the fatty acids, its duration was longer after C12, which also induced a longer delay in half-gastric emptying [t(1/2)(min) after vehicle, 53 +/- 2; C10, 67 +/- 3; C12, 88 +/- 7; P < 0.05 C12 vs. C10 or vehicle]. In conclusion, ingestion of a CCK-releasing fatty acid reduces the tolerated volume of liquid delivered into the stomach, primarily via a CCK(1) receptor-mediated delay in gastric emptying.