Generation of marker-free transgenic maize by regular two-border <Emphasis Type="Italic">Agrobacterium</Emphasis> transformation vectors

By introducing additional T-DNA borders into a binary plasmid used in Agrobacterium-mediated plant transformation, previous studies have demonstrated that the marker gene and the gene of interest (GOI) can be carried by independent T-strands, which sometimes integrate in unlinked loci in the plant genome. This allows the recovery of marker-free transgenic plants through genetic segregation in the next generation. In this study, we have found that by repositioning the selectable marker gene in the backbone and leaving only the GOI in the T-DNA region, a regular two-border binary plasmid was able to generate marker-free transgenic maize plants more efficiently than a conventional single binary plasmid with multiple T-DNA borders. These results also provide evidence that both the right and left borders can initiate and terminate T-strands. Such non-canonical initiation and termination of T-strands may be the basis for the elevated frequencies of cotransformation and unlinked insertions.     

Association of MAD2 expression with mitotic checkpoint competence in hepatoma cells

Chromosomal instability (CIN) refers to high rates of chromosomal gains and losses and is a major cause of genomic instability of cells. It is thought that CIN caused by loss of mitotic checkpoint contributes to carcinogenesis. In this study, we evaluated the competence of mitotic checkpoint in hepatoma cells and investigated the cause of mitotic checkpoint defects. We found that 6 (54.5%) of the 11 hepatoma cell lines were defective in mitotic checkpoint control as monitored by mitotic indices and flow-cytometric analysis after treatment with microtubule toxins. Interestingly, all 6 hepatoma cell lines with defective mitotic checkpoint showed significant underexpression of mitotic arrest deficient 2 (MAD2), a key mitotic checkpoint protein. The level of MAD2 underexpression was significantly associated with defective mitotic checkpoint response (p<0.001). In addition, no mutations were found in the coding sequences of MAD2 in all 11 hepatoma cell lines. Our findings suggest that MAD2 deficiency may cause a mitotic checkpoint defect in hepatoma cells.     

Effects of 'casoparan', a peptide isolated from casein hydrolysates with mastoparan-like properties

Casein, a protein found in milk of several species, is divided into different chains from 19 to 25 kDa. Casein is also considered as a source of amino acids and generating peptides with biological activities such as opiate, immunostimulating, antibacterial, peptidase inhibitors, among others. In this work, Sephadex G-10 chromatography followed by high-performance liquid chromatography isolation purified NZCase TT, an industrial culture media for tetanus toxin production. In the first step, four pools were isolated and tested in different assays: isolated smooth muscle assay (guinea pig ileum, rat uterus), phagocytosis in vitro of opsonized sheep red blood cells, and hydrogen peroxide (H2O2) release from mouse peritoneal macrophages. Pool III was the main active pool being able to potentiate bradykinin action in guinea pig ileum, stimulating phagocitic activity by resident macrophages and increasing H2O2 release from macrophages previously activated with bacille Calmette Guérin. Using mass spectra the primary structure of the main peptide from pool III was obtained--INKKI, which corresponds to beta-casein fragment 26-30. The immunostimulating action is probably related to a direct action in macrophage cells.     

Seasonal changes in social behaviour and spatial structure of<Emphasis Type="Italic">Crocidura leucodon</Emphasis> in north-western Bulgaria

The patterns of social behaviour and spatial structure ofCrocidura leucodon (Hermann, 1780) were studied against the background of the seasonal changes in its density and demographic features. Field and lab research were conducted. Field investigations (with CMR method) were conducted from 1997 to 1999 in north-western Bulgaria, in a grassland with patches of tree-shrub vegetation. In the lab, the social interactions were studied in male-male, female-female and male-female dyadic encounters during summer and autumn. A total of 26 shrews were tested. The number ofC. leucodon was lowest in spring and greatest in autumn. In early summer the sex ratio was balanced, the home range of one male overlapped that of one female, and aggressiveness and marking activity was increased. So, it can be assumed that in this period males and females form pairs, which possess defended territories. Higher aggressiveness and marking activity displayed by males suggest their greater role in defending the territories. In autumn the home ranges overlapped greatly and amicable interactions prevailed over agonistic ones probably due to shifting to a gregarious way of life. So, the patterns of spatial structure and social behaviour undergo the seasonal changes inC. leucodon life-cycle and its density dynamics.     

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides to define substrate specificity of carboxydipeptidases: assays with human cathepsin B

We have developed positional scanning synthetic combinatorial libraries to define the substrate specificity of carboxydipeptidases. The library Abz-GXXZXK(Dnp)-OH, where Abz is ortho-aminobenzoic acid, K(Dnp) is N^ε-2,4-dinitrophenyl-lysine with free carboxyl group, the Z position was successively occupied with 1 of 19 amino acids (cysteine was omitted), and X represents randomly incorporated residues, was assayed initially with human cathepsin B, and arginine was defined as one of the best residues at the P₁ position. To examine the selectivity of , S₂, and S₃ subsites, the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH were then synthesized. The peptide Abz-GIVRAK(Dnp)-OH, which contains the most favorable residues in the positions identified by screening of the libraries with cathepsin B, was hydrolyzed by this enzyme with k_cat/K_m = 7288 mM⁻¹ s⁻¹. This peptide is the most efficient substrate described for cathepsin B to this point, and it is highly selective for the enzyme among the lysosomal cysteine proteases.     

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 micro g/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.     

Expanding the armamentarium for the spondyloarthropathies

Ankylosing spondylitis (AS) is a member of the family of spondyloarthropathies, which are inflammatory arthritides largely involving the axial skeleton and commonly accompanied by peripheral arthritis. Genetic factors, particularly the presence of HLA-B27, are major contributors to the susceptibility for AS. Despite some therapeutic advances, the treatment options for patients with AS and related disorders have been limited. Several lines of evidence have led to the hypothesis that patients with AS might benefit from treatment with tumor necrosis factor (TNF). Specifically, TNF concentrations are known to be significantly elevated in the synovium of patients with rheumatoid arthritis (RA), in the inflamed gut of patients with inflammatory bowel disease, and in the inflamed sacroiliac joints of patients with AS. The anti-TNF agents have been shown to be of benefit in, and currently have indications for, RA (etanercept, infliximab, adalimumab), Crohn's disease (infliximab), and psoriatic arthritis (etanercept). Because the spondyloarthropathies share pathogenetic mechanisms with the above-specified disease states, studies have been conducted to evaluate the effectiveness of anti-TNF agents in several disorders, including AS. Data from clinical trials so far with infliximab and etanercept show that patients with AS and related disorders achieve significant improvement in clinical signs and symptoms based on validated outcomes measures. Computed tomography and magnetic resonance imaging (MRI) can facilitate the early diagnosis of AS. Studies with infliximab using MRI together with updated scoring methods demonstrated significant decreases in associated spinal inflammation. TNF antagonist therapy is well tolerated in patients with AS, with a side effect profile consistent with the prior experience of patients with RA.     

Does safety make a difference in selecting the right TNF antagonist?

Tumor necrosis factor (TNF) antagonists are biologic response modifiers that have significantly improved the outcomes in patients with rheumatoid arthritis (RA). At this report, safety data were collected on approximately 271,000 patients administered infliximab (as of February 2002), 121,000 patients administered etanercept (as of December 2001), and on 2400 patients who received adalimumab in trials in connection with the regulatory approval process (approval granted December 2002 in the US and September 2003 in European Union). Infliximab and etanercept have predictable and manageable safety profiles, and preliminary data suggest that the profile of adalimumab is comparable. Safety issues involving the anti-TNF agents as a class include the risk of injection-site reactions or infusion-related reactions, infection (for example, serious, opportunistic, or tubercular), malignancy, autoimmunity, and demyelinating and neurologic disorders. Injection-site and infusion-related reactions are most often easily managed and rarely lead to discontinuation of therapy. Infections can be minimized or prevented by screening and careful monitoring and follow-up; most infections respond to appropriate medical treatment. More studies are needed to evaluate the occurrence of malignancies in patients with RA to determine the potential risk posed by therapy. Antibody formation can follow the administration of any biologic agent. Although demyelinating disease has been reported with anti-TNF agents, it is not clear whether a causal relationship exists. Overall, the anti-TNF agents are well tolerated and have demonstrated a favorable benefit-to-risk profile in patients with RA.     

Anti-Ri antibodies associated with short-term memory deficits and a mature cystic teratoma of the ovary

BACKGROUND: The IgG autoantibody ANNA-2 (anti-Ri) is a type 2 antineuronal antibody that has been found to bind to highly conserved and widely distributed adult brain proteins encoded by the Nova-1 and Nova-2 genes. Anti-Ri antibodies are typically detected in the serum and cerebrospinal fluids of patients with neurological disorders such as opsoclonus/myoclonus and cerebellar ataxia and in association with gynecologic and breast malignancies. CASE PRESENTATION: This report describes an unusual example of a 33-year-old female patient who developed short-term memory deficits over a 3-month period. An extensive neurological work-up, including a panel of paraneoplastic markers was negative with the exception of a high titer serum Anti-Ri (1:15,3600). A large left ovarian mass was palpated, surgically resected and eventually diagnosed as a mature cystic teratoma. Post-operatively, memory deficits had disappeared within 1 month and serum Anti-Ri titers had decreased significantly to 1:256. An extensive diagnostic work-up for other malignancies was negative. CONCLUSION: Although, Anti-Ri antibodies are typically associated with malignancies, this case illustrates the potential association between benign tumors and this autoantibody.     

A method for rapidly screening functionality of actin mutants and tagged actins

Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three β-actin mutants that have been associated with diseases. Published: October 25, 2004.