Transformation of maize by 2,4-dihydroxy-7-methoxy-2H-1,4-benzo- xazin-3(4H)-one resistant Agrobacterium strains

One of the important factors responsible for recalcitrance of maize tissue towards Agrobacterium-mediated transformation is the presence of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), an inhibitory metabolite found in maize cells. DIMBOA-resistant strains of Agrobacterium tumefaciens were used to transfer genes coding for GUS (-glucuronidase) and NPTII (neomycine phosphotransferase II) in maize shoot apical meristems derived from 20 day-old seedlings and immature embryos. GUS expression was higher (21–34%) in the apical meristem and was dependent on the type of infecting strain and explant-age. The PCR analysis of selected tissues confirmed the presence of GUS gene in the transformed cells.     

A cytosolic form of aminopeptidase P from Drosophila melanogaster: molecular cloning and characterization

Using a functional genomic approach, we have identified and characterized a cytosolic form of aminopeptidase P from Drosophila melanogaster. This study represents the first characterization of an insect aminopeptidase P. The complete sequence of a 12.5 kbp genomic clone from D. melanogaster showed the presence of a 1,839 bp ORF, encoding a protein of 613 amino acids with a calculated molecular mass of 68.5 kDa. The deduced amino acid sequence was 48% identical and 66% similar to rat and human cytosolic aminopeptidase P. Amino acids important for catalytic activity and the metal binding ligands were found to be conserved between Drosophila AP-P and its mammalian homologues. The recombinant enzyme expressed in Escherichia coli hydrolyzed the amino terminal Xaa-Pro bond of substance P and bradykinin, revealing its functional identity. Further enzyme characterization showed the enzyme to be a manganese-dependent metallopeptidase. Immunoblot analysis showed that DAP-P is located exclusively in the cytosol and is temporally regulated during Drosophila development. In the adult fly, the protein could be detected in gut, testis and ovary, with a high level of expression in brain.     

Rapamycin treatment results in GATA factor-independent hyperphosphorylation of the proline utilization pathway activator in Saccharomyces cerevisiae

Treatment of Saccharomyces cerevisiae cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation. Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes. The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin. Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment. Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p. The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene PUT1 were also examined. We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced PUT1 expression. Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased PUT1 expression. Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation. A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source.     

Central Mucoepidermoid carcinoma of mandible – A case report and review of the literature

BACKGROUND: Primary central mucoepidermoid carcinoma of jaws is a rare lesion comprising 2-3% of all mucoepidermoid carcinomas reported in literature. CASE PRESENTATION: The case presented here illustrates the hypothesis that its specific pathogenesis is unknown. CONCLUSIONS: Mucoepidermoid carcinoma of the jaw is a rare tumour of unknown aetiology. Although about a 100 cases has been reported in literature, the speculation on its aetiopathogenesis has mainly centred on the pluripotential capabilities of the epithelial lining of odontogenic cysts.     

New haplotypes in the Bedlington terrier indicate complexity in copper toxicosis

Copper toxicosis (CT) is an autosomal recessive disorder common in Bedlington terriers. Previously, the CT locus was mapped to canine Chromosome (Chr) 10q26 through linkage to marker C04107. Diagnosis, traditionally based on liver biopsy, has recently shifted to interpretation of the C04107 microsatellite alleles where allele 2 segregates with the disease with 90–95% accuracy. Recently, CT has been attributed to a deletion of exon 2 in the MURR1 gene. We also identified a deletion of exon 2 of MURR1 in our collection of 2-2 homozygous affected terriers. However, our collection also included affected 1-1 homozygotes and 1-2 heterozygotes, and these dogs did not have the homozygous deletion. In addition to C04107, we analyzed an adjacent microsatellite (C04107B), and two novel SNPs, all within intron 1 of MURR1, and sequenced all exons and their intronic boundaries. Pedigree analysis indicates that there are two typical haplotypes, one normal and one affected, maintaining complete linkage disequilibrium between C04107 allele 2 and the deletion in most pedigrees. Most importantly, we identified a recombinant haplotype present in a North American pedigree, where allele 2 is not linked with the deletion, and a fourth haplotype containing a splice site variant. Although the splice site alteration appears to be a normal variant, it is present in two affected dogs, which do not carry homozygous deletions of MURR1.     

Anthocyanin production in a callus line of <Emphasis Type="Italic">Panax sikkimensis</Emphasis> Ban

A root-derived callus line of Panax sikkimensis that stably accumulates anthocyanins was established by small cell aggregate selection method. The selected line showed a growth index of 221.36 and an anthocyanin content of 2.76 mg/g fw (7.076% dw) in 50–60 d of growth on a modified MS medium containing 4.5 μM 2,4-dichlorophenoxy acetic acid and 1.2 μM kinetin under 16-h light and 8-h dark photoperiodic conditions. Incubation under continuous light increased the growth index to 435.57 but led to a marginal dilution of anthocyanin content to 2.192 mg/g fw (6.928% dw). The purple-red pigment had absorption maximum at 528 nm. The selected callus line has shown sustained growth and productivity for more than 6 yr now. Interestingly, pigment accumulation in the selected line did not hinder the ginsenoside production in the callus tissue (0.9–1.2% fw).     

Growth kinetics and ginsenosides production in transformed hairy roots of American ginseng—Panax quinquefolium L

A thin, profusely branched, fast growing hairy root line of Panax quinquefolium (American ginseng) was established by co-culturing epicotyl explants with a wild type strain of Agrobacterium rhizogenes. The transformed roots grew by over 10-fold from the initial inoculum within 8 weeks. The crude ginsenosides content in the roots was about 0.2 g/g dry wt level up to the 10th week of culture. Ginsenosides Rb2, Rd, Re, Rf and Rg1 constituted 47–49% of the crude saponin fraction between 6 and 8 weeks of growth whereas, Rc ginsenoside was accumulated only after 9th weeks when the biomass started receding. PCR amplification analysis of the hairy roots confirmed their transgenic nature by showing the presence of Ri-TL DNA with rolA, rolB and rolC genes in their genome.     

A comparative evaluation of EPR and OxyLite oximetry using a random sampling of pO(2) in a murine tumor

Methods currently available for the measurement of oxygen concentrations (oximetry) in viable tissues differ widely from each other in their methodological basis and applicability. The goal of this study was to compare two novel methods, particulate-based electron paramagnetic resonance (EPR) and OxyLite oximetry, in an experimental tumor model. EPR oximetry uses implantable paramagnetic particulates, whereas OxyLite uses fluorescent probes affixed on a fiber-optic cable. C3H mice were transplanted with radiation-induced fibrosarcoma (RIF-1) tumors in their hind limbs. Lithium phthalocyanine (LiPc) microcrystals were used as EPR probes. The pO(2) measurements were taken from random locations at a depth of approximately 3 mm within the tumor either immediately or 48 h after implantation of LiPc. Both methods revealed significant hypoxia in the tumor. However, there were striking differences between the EPR and OxyLite readings. The differences were attributed to the volume of tissue under examination and the effect of needle invasion at the site of measurement. This study recognizes the unique benefits of EPR oximetry in terms of robustness, repeatability and minimal invasiveness.     

Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by STAT1-dependent interferon responses

Human noroviruses are the major cause of nonbacterial epidemic gastroenteritis worldwide. However, little is known regarding their pathogenesis or the immune responses that control them because until recently there has been no small animal model or cell culture system of norovirus infection. We recently reported the discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and its cultivation in macrophages and dendritic cells in vitro. We further defined interferon receptors and the STAT-1 molecule as critical in both resistance to MNV-1-induced disease in vivo and control of virus growth in vitro. To date, neither histopathological changes upon infection nor viral replication in wild-type mice has been shown. Here we extend our studies to demonstrate that MNV-1 replicates and rapidly disseminates to various tissues in immunocompetent mice and that infection is restricted by STAT1-dependent interferon responses at the levels of viral replication and virus dissemination. Infection of wild-type mice is associated with histopathological alterations in the intestine (mild inflammation) and the spleen (red pulp hypertrophy and white pulp activation); viral dissemination to the spleen, liver, lung, and lymph nodes; and low-level persistent infection in the spleen. STAT-1 inhibits viral replication in the intestine, prevents virus-induced apoptosis of intestinal cells and splenocytes, and limits viral dissemination to peripheral tissues. These findings demonstrate that murine norovirus infection of wild-type mice is associated with initial enteric seeding and subsequent extraintestinal spread, and they provide mechanistic evidence of the role of STAT-1 in controlling clinical norovirus-induced disease.