Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj‐Apis362 for high gamma‐aminobutyric acid production

Gamma‐aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full‐length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA‐production, the GAD gene was cloned into pMG36e‐LbGAD, and then expressed in Lactobacillus plantarum Taj‐Apis362 cells. The overexpression was confirmed by SDS‐PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone‐back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA‐rich products.     

Pinpointing phosphotyrosine-dependent interactions downstream of the collagen receptor DDR1

Activation of the receptor tyrosine kinase DDR1 by collagen results in robust and sustained phosphorylation, however little is known about its downstream mediators. Using phosphopeptide mapping and site-directed mutagenesis, we here identified multiple tyrosine phosphorylation sites within DDR1. We found that Nck2 and Shp-2, two SH2 domain-containing proteins, bind to DDR1 in a collagen-dependent manner. The binding site of Shp-2 was mapped to tyrosine-740 of DDR1 within an ITIM-consensus sequence. Lastly, ablation of DDR1 in the mouse mammary gland resulted in delocalized expression of Nck2, suggesting that defects observed during alveologenesis are caused by the lack of the DDR1-Nck2 interaction.     

In situ release of coral mucus by <Emphasis Type="Italic">Acropora</Emphasis> and its influence on the heterotrophic bacteria

In situ mucus release by Acropora nobilis and degradation of mucus from A. nobilis and Acropora formosa, by heterotrophic bacteria were investigated at Bidong and Tioman Island, Malaysia. Mucus release rate for A. nobilis was on average 38.7 ± 35.2 mg C m⁻² h⁻¹, of which ca. 70% consisted of dissolved organic carbon (DOC) and 30% particulate organic carbon (POC). In the mucus degradation experiment, seawater-mucus mixtures were incubated and compared with control runs for 24 h. Bacterial abundance in the seawater-mucus mixture increased significantly and coincided with a decline in DOC concentration. In controls, bacteria and DOC did not significantly change. The coral mucus had a high content of inorganic phosphate. It is suggested that the coral mucus rich in DOC and phosphate can induce the high bacterial growth.     

Occasional intraguild predation structuring small mammal assemblages: the marsupial Didelphis aurita in the Atlantic Forest of Brazil

The didelphid marsupial, Didelphis aurita, is suggested as an intraguild predator and as key‐species in small mammal assemblages of the Atlantic Forest of Brazil. The field experiments required to test this hypothesis are complex to implement, but the recent revival of regression methods offers a viable alternative. Here we use the dynamic and static regression methods to determine the importance of D. aurita as a competitor and intraguild predator. Capture–recapture data from two localities in the Rio de Janeiro State were used, Garrafão (municipality of Guapimirim), a coastal forest of the Serra do Mar, and Barra de Maricá, a costal sand dune vegetation. Population and microhabitat variables were monitored from April 1997 to April 2003 in Garrafão, and from January 1986 to July 1990 in Barra de Maricá. Microhabitat variables were related to Canopy, Plant, Litter and Rock covers, Obstruction from 0 to 1.5 m, and Number of logs. Exploitation competition was tested by the dynamic method, which models the effects of D. aurita on the per capita growth rate of a species. Interference by predation or competition was tested by the static method, where the abundance of D. aurita at trap stations was regressed against the abundance of other small mammals, after removal of any variation associated with microhabitat factors. Exploitation competition was not detected, but the interference of D. aurita was pervasive, affecting all small mammals studied in the two localities. The clear avoidance of D. aurita by all small mammals tested in two localities of different physiognomies indicates that it functions as an intraguild predator, even if actual predation by D. aurita is an occasional event.     

In utero gene transfer to the mouse nervous system

The cellular and molecular environment present in the fetus and early newborn provides an excellent opportunity for effective gene transfer. Innate and pre-existing anti-vector immunity may be attenuated or absent and the adaptive immune system predisposed to tolerance towards xenoproteins. Stem cell and progenitor cell populations are abundant, active and accessible. In addition, for treatment of early lethal genetic diseases of the nervous system, the overarching advantage may be that early gene supplementation prevents the onset of irreversible pathological changes. Gene transfer to the fetal mouse nervous system was achieved, albeit inefficiently, as far back as the mid-1980s. Recently, improvements in vector design and production have culminated in near-complete correction of a mouse model of spinal muscular atrophy. In the present article, we review perinatal gene transfer from both a therapeutic and technological perspective.     

Terpenoid,Benzenoid, and Phenylpropanoid Compounds in the Floral Scent of <Emphasis Type="Italic">Vanda</Emphasis> Mimi Palmer

Vanda Mimi Palmer is the product of a cross between Vanda Tan Chay Yan and Vanda tessellata. The flower of this hybrid produces a sweet-smelling fragrance during day time at the open-flower stage. This study aimed to investigate the floral scent constituents in Vanda Mimi Palmer. Scent emission analysis of this orchid was carried out at different time points in a 24-h cycle and also at different floral developmental stages. A comparison was also made on the volatiles emitted by Vanda Mimi Palmer and both of its parents. Gas chromatography-mass spectrometry (GC-MS) analysis showed that the scent of Vanda Mimi Palmer was dominated by terpenoid, benzenoid, and phenylpropanoid compounds. The identified terpenoids were ocimene, linalool oxide, linalool, and nerolidol; while the benzenoid and phenylpropanoid compounds were methylbenzoate, benzyl acetate, phenylethanol, and phenylethyl acetate. The emission of terpenoid, benzenoid, and phenylpropanoid compounds was developmentally and temporally regulated. Comparison of the volatiles emitted by both of its parents showed that the scent of Vanda Mimi Palmer is dissimilar to that of its fragrant parent, V. tessellata.     

Spatial and acoustic pressure dependence of microbubble-mediated gene delivery targeted using focused ultrasound

BACKGROUND: Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. METHODS: Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. RESULTS: In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. CONCLUSIONS: Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.     

toxin-mediated insect resistance in plants

We are currently in an interesting phase of plant biotechnology releases, both for the scientists responsible for these innovations who are beginning to see their ideas realized, and for the biotechnology companies that are starting to see a return on their investment. One of the most notable examples, is the introduction of transgenic crops that are engineered to express a Bacillus thuringiensis toxin that confers resistance to insect predation. However, the picture is not altogether positive - there is concern that the introduction of this technology was premature or should not have happened at all, and that the valuable insecticidal properties of Bacillus thuringiensis will be lost.