Synthesis of the tobacco mosaic virus in blocked spreading of infection

The possibility of infection of tobacco upper leaves with tobacco mosaic virus (TMV) was examined in experiments where the inoculum was imbibed through the cut stem. The inoculum used were: a) a preparation of a virus-specific informosome-like ribonucleoproteins (vRNP) isolated from TMV-infected plants; b) a TMV preparation; or c) a mixture of TMV and vRNP. Multiplication of TMV in upper leaves was observed in neither of the variants; nevertheless in the vascular tissue and/or probably in adjoining parenchymal cells, two kinds of RNA were synthesized: of mol. w. (1.1--1.3) X 10(6) and (0.6--0.8) X 10(6). These RNA were not found in healthy plants in the presence of actinomycin D. The synthesis of genomic TMV RNA is suppressed under these conditions. Thus, some kind of abortive TMV infection takes place under the condition of experimental inoculation of plants through a cut stem. Molecular hybridization with the DNA of recombinant plasmid containing a nucleotide sequence complementary to the 3'-portion of genomic TMV RNA proves that short RNAs synthesized under the abortive infection conditions are TMV-specific. The experiments with differential temperature treatment of N-gene-containing plants under abortive infection conditions suggest that necrotization is not necessarily induced by genomic TMV RNA synthesis.     

Increase in duration of paradoxical sleep after carotid occlusion in rats

Nonlinear rats, which survived after occlusion of one or two common carotid arteries (N = 30, about 30% survived) were examined. Sham-operated animals formed a control group. In animals with ischemia, the total sleep duration in a three-hour period was substantially increased as compared to control group at the expense of a substantial growth (8-9 times) of paradoxical sleep (PS) phase. In the animals with ischemia, a sharp increase in PS was observed on the first postoperation day, and then PS gradually decreased from the first to the fortieth days. The results suggest the PS involvement in the brain intrinsic reparation functions.     

Use of RAPD and ITE molecular markers in studying the genetic structure of the Crimean population of T. boeoticum Boiss

The influence of ecological factors on variation of random PCR markers (RAPD and inter-MITE polymorphism (IMP) primers) was evaluated in two wild Triticum boeoticum populations with contrasting climatic conditions in Crimea. The proportion of variation that undergoes natural selection was compared for these two types of molecular markers. The Sapun Mountain and Baidar Valley populations differed significantly in 24.7% of the analyzed loci, with a low Nei distance between these populations (0.0324). The number of significantly different RAPD and IMP markers in the two populations was nearly equal. Testing for evolutionary neutrality showed that 13 loci (17.8%) were influenced by natural selection. The Sapun Mountain population was more dependent on natural selection (ten loci, 13.7%) than the Baidar Valley population (four loci, 5.5%). Eleven out of the 13 loci undergoing selection were amplified with IMP primers.     

Investigation of genetically modified soybean biosafety in the center of the origin and diversity of Russian Far East

Wild soybean (Glycine soja Sieb. & Zucc.) is the nearest relative of a soybean crop (Glycine max (L.) Merr.). Study of population genetic structure of wild-growing relatives ofgenetically modified (GM) plants in the centers of their origin is one of the main procedures before introduction of GM crops in these areas. We have studied genetic variability of nine wild growing soya populations of Primorye Territory using RAPD analysis. The level of G. soja genetic variability was considerably higher than that of G. max. We have analyzed phylogenetic relationships in the genus Glycine subgenus Soja using RAPD markers. Our data confirm validity of allocation G. gracilis in a rank of a species.     

Study of genetic diversity and spatial structure of the wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &; Zucc.) population from the neighborhood of the settlement of Ekaterinovka in the south of Primorskii krai

Data are presented on the genetic diversity and spatial structure of the natural wild soybean population from the neighborhood of the settlement of Ekaterinovka in Primorskii krai and on the relationship between the genetic structure of this population and its spatial organization. These data are discussed in comparison with the results of studies of wild soybean populations in the Far East region of the Russian Federation and China. Recommendations are given concerning the collection of genetic wild soybean resources.     

Plant-made trastuzumab (herceptin) inhibits HER2/Neu+ cell proliferation and retards tumor growth

Background

Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (mAb), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the mAb. Here we used an Agrobacterium-mediated transient expression system to produce trastuzumab in plant cells.

Methodology/Principal Findings

We describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron-optimized Tobacco mosaic virus- and Potato virus X-based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full-size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK-BR-3, in fluorescence-activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER-2-expressing cancer cell proliferation. We show that plant-made trastuzumab (PMT) bound to the Her2/neu oncoprotein of SK-BR-3 cells and efficiently inhibited SK-BR-3 cell proliferation. Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her2+ cells.

Conclusions/Significance

We conclude that PMT is active in suppression of cell proliferation and tumor growth.     

Increase of histidine content in Brassica rapa subsp. oleifera by over-expression of histidine-rich fusion proteins

An approach that enables the increase of the quantity of a specific amino acid in crop plants is reported. Oleosin gene from Arabidopsis thaliana or 30K movement protein gene of Tobacco mosaic virus (TMV; genus Tobamovirus) were cloned under the control of napin or hybrid promoters, and in fusion to synthetic poly-histidine (poly-His) sequences for transformation into spring turnip rape (Brassica rapa subsp. oleifera; synonym to B. campestris). The most stable expression cassettes for the poly-His production prior to the plant transformation were selected by analyzing the protein expression in in vitro translation and in transient plant expression systems using GFP as marker. Expression of the poly-His-constructs in transgenic Brassica rapa plants was analyzed using dot and western blotting and PCR. The constructs were stably expressed in the third generation of the transgenic plant lines. Histidine content was measured from the seeds of the transgenic plants, and some plant lines had more than 20% increase in histidine content compared to wild type. The methodology may be widely applicable to increase the content of any amino acid in crop plants including those encoded by rare codons.     

Role of the leader sequence in tobacco pectin methylesterase secretion

We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.