<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

Geographical determinants and environmental implications of livestock production intensification in Asia

Under growing and urbanizing demand, livestock production is rapidly evolving in South, East and South-east Asia, with both an increase of production and a shift to intensive production systems. These changes infer impacts on the environment, on public health and on rural development. Environmental impacts are mainly associated with a mismanagement of animal excreta, leading to pollution of surface water, ground water and soils by nutrients, organic matter, and heavy metals. In the framework of the Livestock Environment and Development Initiative, this research aims at assessing, on a regional scale, the impacts of livestock production on nutrient fluxes. Phosphate (P(2)O(5)) mass balances were chosen as an indicator and were calculated on the basis of spatially modelled livestock densities, estimated excretion values and crop uptake. The results show a strong West--East gradient regarding the distribution of monogastrics, with clear concentration in densely populated areas and around urban centres. P(2)O(5) overloads are estimated on 23.6% of the study area's agricultural land, mainly located in eastern China, the Ganges basin and around urban centres such as Bangkok, Ho Chi Minh City and Manila. On average, livestock manure is estimated to account for 39.4% of the agricultural P(2)O(5) supply (the remaining share being supplied by chemical fertilisers). Livestock is the dominant agricultural source of P(2)O(5) around urban centres and in livestock specialised areas (southern and north-eastern China), while chemical fertilisers are dominant in crop (rice) intensive areas.     

Identification of a domain in the alpha-subunit of the oxaloacetate decarboxylase Na+ pump that accomplishes complex formation with the gamma-subunit

The oxaloacetate decarboxylase Na+ pumps OAD-1 and OAD-2 of Vibrio cholerae are composed of a peripheral alpha-subunit associated with two integral membrane-bound subunits (beta and gamma). The alpha-subunit contains the carboxyltransferase domain in its N-terminal portion and the biotin-binding domain in its C-terminal portion. The gamma-subunit plays a profound role in the assembly of the complex. It interacts with the beta-subunit through its N-terminal membrane-spanning region and with the alpha-subunit through its hydrophilic C-terminal domain. The biochemical and structural requirements for the latter interaction were analysed with OAD-2 expression clones for subunit alpha-2 and the C-terminal domain of gamma-2, termed gamma'-2. If the two proteins were synthesized together in Escherichia coli they formed a complex that was stable at neutral pH and dissociated at pH<5.0. An internal stretch of 40 amino acids of alpha-2 was identified by deletion mutagenesis to be essential for the binding with gamma'-2. This portion of the alpha-subunit is connected via flexible linker peptides to the carboxyltransferase domain at its N terminus and to the biotin-binding domain at its C terminus. Results of site-directed mutagenesis indicated that a conserved tyrosine (491) and threonine 494 of this peptide contributed significantly to the stability of the complex with gamma'-2. This peptide therefore represents a newly identified, separate domain of the alpha-subunit and has been called the 'association domain'.     

Hormonal regulation of chemosignal-stimulated precopulatory behaviors in male housemice (Mus musculus)

Five experiments examined the hormonal regulation of the precopulatory reproductive behavior of male housemice of two genotypes (DBA/2J inbreds and C57BL/6J X AKR/J hybrids). The two precopulatory behaviors examined were preferences for female urinary odors and ultrasonic courtship vocalizations to anesthetized females. The preferences were then used to make inferences about odor attractiveness. Gonadally intact hybrid males were highly attracted to the airborne urinary odors of female mice but were either indifferent to, or exhibited less attraction to, male urinary odors. Castration decreased male attraction to female odor such that castrated males were equally attracted to male and female odors. Normal levels of attraction could be maintained in castrated hybrid males by Silastic implants of either testosterone or estradiol. While Silastic implants of dihydrotestosterone (DHT) were also effective in maintaining attraction in hybrids, this hormone was ineffective in inbreds. The effectiveness of estradiol, DHT, and testosterone in maintaining attraction following castration was paralleled in castrated hybrids by the effects of these hormones in maintaining courtship vocalizations to females. In contrast to the genotype-specific effects of DHT upon behavior, DHT was effective in both genotypes in maintaining seminal vesicle weight. Estradiol, on the other hand, which was quite effective in maintaining both precopulatory behaviors in hybrids, had little effect upon seminal vesicle weight. Thus these experiments dissociate the behavioral effects of steroids from their effects upon peripheral morphology. We suggest that testosterone can activate precopulatory behaviors following either aromatization or 5-alpha reduction but that genetic variability somehow gives rise to strain differences in DHT responsiveness.     

Differential Morphogenetic Responses of Cotyledonary Explants of Vigna mungo

The morphogenetic responses of cotyledonary nodal explants of Vigna mungo (L.) Hepper cv. VBN1 cultured on the same Murashige and Skoog's medium, B5 vitamins, and 13.31 µM N⁶-benzylaminopurine showed variations in the pattern of multiple shooting and morphology of leaves in dependence on initial explants (presence/absence of cotyledons). The regenerated shoots elongated in the initial medium and most of them rooted in the presence of 2.41 µM indole-3-butyric acid, and flowered in vitro. Rooted plants could be transferred to the field after hardening.     

Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double‐deficient mice

The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double‐deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double‐deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose‐binding protein and Cre (MBP‐Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks. genesis 47:545–558, 2009. © 2009 Wiley‐Liss, Inc.     

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[¹⁸F]fluoro-glucose (FDG) and 3′-deoxy-3′-[¹⁸F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC₅₀ values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 µM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.     

Structure-Function Relations in Oxaloacetate Decarboxylase Complex. Fluorescence and Infrared Approaches to Monitor Oxomalonate and Na+ Binding Effect

Background

Oxaloacetate decarboxylase (OAD) is a member of the Na⁺ transport decarboxylase enzyme family found exclusively in anaerobic bacteria. OAD of Vibrio cholerae catalyses a key step in citrate fermentation, converting the chemical energy of the decarboxylation reaction into an electrochemical gradient of Na⁺ ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of α, β and γ subunits. The α subunit contains the carboxyltransferase catalytic site.

Methodology/Principal Findings

In this report, spectroscopic techniques were used to probe oxomalonate (a competitive inhibitor of OAD with respect to oxaloacetate) and Na⁺ effects on the enzyme tryptophan environment and on the secondary structure of the OAD complex, as well as the importance of each subunit in the catalytic mechanism. An intrinsic fluorescence approach, Red Edge Excitation Shift (REES), indicated that solvent molecule mobility in the vicinity of OAD tryptophans was more restricted in the presence of oxomalonate. It also demonstrated that, although the structure of OAD is sensitive to the presence of NaCl, oxomalonate was able to bind to the enzyme even in the absence of Na⁺. REES changes due to oxomalonate binding were also observed with the αγ and α subunits. Infrared spectra showed that OAD, αγ and α subunits have a main component band centered between 1655 and 1650 cm⁻¹ characteristic of a high content of α helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of β sheet structures and a concomitant increase of α helix structures. Oxomalonate binding to αγand α subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex.

Conclusion

Oxomalonate binding affects the tryptophan environment of the carboxyltransferase subunit, whereas Na⁺ alters the tryptophan environment of the β subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the α-helical as well as β-strand secondary structure elements.     

Blood gene expression markers to detect and distinguish target organ toxicity

The purpose of this study was to investigate whether the expression of specific genes in peripheral blood can be used as surrogate marker(s) to detect and distinguish target organ toxicity induced by chemicals in rats. Rats were intraperitoneally administered a single, acute dose of a well-established hepatotoxic (acetaminophen) or a neurotoxic (methyl parathion) chemical. Administration of acetaminophen (AP) in the rats resulted in hepatotoxicity as evidenced from elevated blood transaminase activities. Similarly, administration of methyl parathion (MP) resulted in neurotoxicity in the rats as evidenced from the inhibition of acetyl cholinesterase activity in their blood. Administration of either chemical also resulted in mild hematotoxicity in the rats. Microarray analysis of the global gene expression profile of rat blood identified distinct gene expression markers capable of detecting and distinguishing hepatotoxicity and neurotoxicity induced by AP and MP, respectively. Differential expressions of the marker genes for hepatotoxicity and neurotoxicity were detectable in the blood earlier than the appearance of the commonly used clinical markers (serum transaminases and acetyl cholinesterase). The ability of the marker genes to detect hepatotoxicity and neurotoxicity was further confirmed using the blood samples of rats administered additional hepatotoxic (thioacetamide, dimethylnitrobenzene, and carbon tetrachloride) or neurotoxic (ethyl parathion and malathion) chemicals. In summary, our results demonstrated that blood gene expression markers can detect and distinguish target organ toxicity non-invasively.