Production of transgenic sugarbeet (Beta vulgaris L.) plants of O-type using Agrobacterium tumefaciens

A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) by vacuum infiltration has been developed. Transgenic sugarbeet plants of Ukraine breeding were selected for their resistance either to the antibiotic kanamycin or to the herbicide glufosinate ammonium. Integration of transgenes was confirmed by PCR and GUS-assay.     

Lytic replication-defective Kaposi's sarcoma-associated herpesvirus: potential role in infection and malignant transformation

Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G(0)/G(1) apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.     

Native β-glucuronidase activity in sugarbeet (Beta vulgaris)

β-Glucuronidase (EC 3.2.1.31) activity, initially thought absent from plants, has been found in a number of plant families. During an analysis of Agrobacterium -mediated transformation of sugarbeet ( Beta vulgaris L.), significant glucuronidase activity was observed in control (non-transformed) tissues when the fluorogenic substrates 4-methylumbelliferyl-β- d -glucuronic acid, resorufin glucuronic acid and 3-carboxyum-belliferyl-β- d -glucuronic acid were used to quantify β-glucuronidase activity under standard protocol conditions. Similarly, the colorigenic substrate p -nitrophenyl-β- d -glucuronide was hydrolyzed by this sugarbeet-derived glucuronidase. Biochemical and immunological data are presented to indicate significant differences between sugarbeet-derived glucuronidase and that from Escherichia coli (EC 3.2.1.31) encoded by gusA . These differences provide means of distinguishing between the two activities in extracts that contain a mixture of both. Use of X-glue, the substrate utilized in histochemical localizations of glucuronidase activity, gave no reaction product (i.e., indigo precipitate) at pH 7.0. However, at pH 3.0, 4.0 and 5.0 formation of the indigo precipitate was evident within 1 h at 37°C in sugarbeet callus and by 4 h in leaves and petioles. The specific activity of sugarbeet glucuronidase was observed to be strongly pH dependent, with an optimum near pH 4.0. The use of various β-glucuronidase assay techniques as applied to transformation of sugarbeet is discussed.     

Intracranial germ cell tumors: association with Klinefelter syndrome and sex chromosome aneuploidies

Intracranial germ cell tumors (ICGCTs) occur mainly in male children and adolescents. Polyploidy of the X chromosome and X hypomethylation have been suggested as mechanisms of malignant transformation independently of the histological tumor type. On the other hand, several reports associate these tumors with Klinefelter's syndrome (KS). Recent reports indicate that KS patients have an increased relative risk for development of malignant mediastinal germ cell tumors and also around 8% of male patients with primary mediastinal tumors have KS. In an attempt to explore the frequency of KS amongst patients with ICGCTs and to confirm the presence of X chromosome polyploidies in these tumors, we studied 13 young male patients with ICGCTs. Paraffin-embedded tumoral and normal tissues were studied by FISH. KS was found in 15% of the cases, demonstrating that this constitutive aneuploidy may be related to carcinogenesis. When tumor and non-tumor tissues were compared, statistically significant X and Y chromosome polyploidies in tumors were revealed. These results emphasize that aneuploidies are involved in ICGCT tumorigenesis.     

Differential immunogold localisation of sulphated and unsulphated keratan sulphate proteoglycans in normal and macular dystrophy cornea using sulphation motif-specific antibodies

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the corneal stroma for tissue organisation and transparency. Macular corneal dystrophy (MCD) is a rare, autosomal recessive disease characterised by disturbances in KS expression. MCD is caused by mutations in CHST6, a gene encoding the enzyme responsible for KS sulphation. Sulphated KS is absent in type I disease causing corneal opacity and loss of vision. Genetic studies have highlighted the mutational heterogeneity in MCD, but supportive immunohistochemical studies on corneal KS have previously been limited by the availability of antibodies mostly reactive only with highly sulphated KS epitopes. In this study, we employed four antibodies against specific KS sulphation patterns, including one against unsulphated KS, to investigate their reactivity in a case of MCD compared with normal cornea using high-resolution immunogold electron microscopy. Mutation analysis indicated type I MCD with deletion of the entire open reading frame of CHST6. Contrast enhanced fixation revealed larger PG structures in MCD than normal. Unlike normal cornea, MCD cornea showed positive labelling with antibody to unsulphated KSPG, but was negative with antibodies to sulphated KSPG. These antibodies will thus facilitate high-resolution investigations of phenotypic heterogeneity in support of genetic studies in this disease.     

Transformation of Sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens

A method has been developed for the regeneration of transformedplants of the commercially important crop sugarbeet (Beta vulgarisL.), using Agrobacterium tumefaciens. Binary vectors were used,carrying both screenable and selectable genes. Plant regenerationfrom shoot-base tissues was found to be relatively rapid andfrequent compared with petioles or leaf tissue. Inoculationof cultured shoot-base tissues resulted in the production oftransformed plants, as determined by (1) introduced resistanceto kanamycin, (2) introduced CAT or GUS activity, and (3) Southernblot analysis to show the integration of foreign DNA. The transformationfrequency was found to be dependent upon explant source, plantgenotype and selection conditions used. Key words: Agrobacterium tumefaciens, sugarbeet (Beta vulgaris L.), transformation.     

Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

Kaposi’s sarcoma (KS) is an angioproliferative and invasive tumor caused by Kaposi’s sarcoma-associated herpesvirus (KSHV). The cellular origin of KS tumor cells remains contentious. Recently, evidence has accrued indicating that KS may arise from KSHV-infected mesenchymal stem cells (MSCs) through mesenchymal-to-endothelial transition (MEndT), but the transformation process has been largely unknown. In this study, we investigated the KSHV-mediated MEndT process and found that KSHV infection rendered MSCs incomplete endothelial lineage differentiation and formed hybrid mesenchymal/endothelial (M/E) state cells characterized by simultaneous expression of mesenchymal markers Nestin/PDGFRA/α-SAM and endothelial markers CD31/PDPN/VEGFR2. The hybrid M/E cells have acquired tumorigenic phenotypes in vitro and the potential to form KS-like lesions after being transplanted in mice under renal capsules. These results suggest a homology of KSHV-infected MSCs with Kaposi’s sarcoma where proliferating KS spindle-shaped cells and the cells that line KS-specific aberrant vessels were also found to exhibit the hybrid M/E state. Furthermore, the genetic analysis identified KSHV-encoded FLICE inhibitory protein (vFLIP) as a crucial regulator controlling KSHV-induced MEndT and generating hybrid M/E state cells for tumorigenesis. Overall, KSHV-mediated MEndT that transforms MSCs to tumorigenic hybrid M/E state cells driven by vFLIP is an essential event in Kaposi’s sarcomagenesis.     

Diversity and distribution of microbial long-chain fatty acid biosynthetic genes in the marine environment

Bacterial production of long-chain fatty acids via a polyketide synthase-related mechanism has thus far only been investigated in isolate-based studies. Here, the genetic capacity for production of long-chain fatty acids was investigated using a culture-independent approach. PCR primers targeting the keto-acyl synthase (KS) domain of the pfaA gene involved in omega-3 polyunsaturated fatty acid (PUFA) biosynthesis were used to construct clone libraries to investigate KS sequence diversity in disparate marine habitats. Of the 446 sequences recovered, 123 (27.6%) clustered with KS sequences involved in the synthesis of eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6). The remaining 72.4% of clones formed environmental-only groups or grouped with the KS domains of pfaA homologues from organisms producing unidentified products. In total, 17 groups were recovered - four known and 13 newly identified. A query of metagenomic data sets revealed sequences related to EPA KS domains, as well as sequences related to four environmental-only groups discovered in the clone libraries. The phylogenetic affiliation and end product of these environmental-only KS clusters is unknown. These findings reveal a widespread capacity for long-chain fatty acid production in marine microorganisms, including biosynthetic pathways not yet characterized.     

Isochromosome X mosaicism in a child with Kabuki syndrome phenotype: A rare cytogenetic association

Isochromosome is a structurally unbalanced chromosome consisting of two short arms or two long arms, which are derived by abnormal centromere division or sister-chromatid exchange. Most autosomal isochromosomes are unusual, while those involving sex chromosomes are common. Kabuki syndrome (KS, OMIM 147920) is a multiple malformation/mental retardation syndrome of unknown etiology. A conventional cytogenetic study on lymphocytes from a 4-year-old girl with physical features suggestive of KS was found to have mosaicism for isochromosome for the long arm of the X. Although most manifestations present in this patient have been described before, this report is a rare association of clinical and cytogenetic findings in this syndrome. A genome-wide analysis and a larger number of patient groups studied could improve our understanding of the genetic basis of KS.     

Deletion of KDM6A, a histone demethylase interacting with MLL2, in three patients with Kabuki syndrome

Kabuki syndrome (KS) is a rare genetic disease that causes developmental delay and congenital anomalies. Since the identification of MLL2 mutations as the primary cause of KS, such mutations have been identified in 56%-76% of affected individuals, suggesting that there may be additional genes associated with KS. Here, we describe three KS individuals with de novo partial or complete deletions of an X chromosome gene, KDM6A, that encodes a histone demethylase that interacts with MLL2. Although KDM6A escapes X inactivation, we found a skewed X inactivation pattern, in which the deleted X chromosome was inactivated in the majority of the cells. This study identifies KDM6A mutations as another cause of KS and highlights the growing role of histone methylases and histone demethylases in multiple-congenital-anomaly and intellectual-disability syndromes.     

Biochemical and genetic characterization of mundticin KS,an antilisterial peptide produced by Enterococcus mundtii NFRI 7393

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.     

Metabolic control of seed germination

We have used proteomics to better characterize germination and early seedling vigor in sugarbeet. Our strategy includes (1) construction of proteome reference maps for dry and germinating seeds of a high-vigor reference seed lot; (2) investigation of the specific tissue accumulation of proteins (root, cotyledon, perisperm); (3) investigation of changes in protein expression profiles detected in the reference seed lot subjected to different vigor-modifying treatments, e.g. aging and/or priming. More than 1 000 sugarbeet seed proteins have been identified by LC/MS-MS mass spectrometry (albumins, globulins and glutelins have been analyzed separately). Due to the conservation of protein sequences and the quality of MS sequencing (more than 10 000 peptide sequences have been obtained), the success rate of protein identification was on the average of 80%. This is to our knowledge the best detailed proteome analysis ever carried out in seeds. The data allowed us to build a detailed metabolic chart of the sugarbeet seed, generating new insights into the molecular mechanisms determining the development of a new seedling. Also, the proteome of a seed-storage tissue as the perisperm is described for the first time.     

Human herpesvirus-8 encoded Kaposin: subcellular localization using immunofluorescence and biochemical approaches

Human herpesvirus-8 (HHV-8) has been causally linked to the development of Kaposi's sarcoma (KS). DNA sequence analysis of the viral genome revealed a total of 81 open reading frames (ORF). Interestingly, only a small subset of these ORFs has been shown to be transcribed in cells latently infected with HHV-8 and in cells of the KS lesions. Among the genes active during latency, kaposin, is noted for its abundance and ability to transform cells in culture, thus implicating a potential role in KS pathogenesis. This has prompted us to undertake an investigation on elucidating the mechanism(s) by which Kaposin brings about transformation of cells. Towards this goal, we have generated an eukaryotic expression plasmid encoding Kaposin (Kap). As Kaposin is predicted to be a type II membrane protein, several strategies were utilized to address this, including the generation of Kaposin with the Flag (FL) epitope (DYKDDDDK) at the C-terminus of the protein (Kap-C-FL). Antibodies specific for Kaposin (kap-2), recognized both Kaposin and Kaposin-Flag, while antibodies against the Flag epitope recognized only Kaposin-Flag. Transfection of Kap and Kap-C-FL expression plasmid DNA into NIH3T3 cells resulted in cellular clones that exhibited a phenotypic property of transformation by forming large, multiclustered cells, when grown on soft agar. Because there is controversial data regarding the localization of Kaposin in cells, we examined the subcellular localization of Kaposin using confocal microscopy. We observed that Kaposin and Kaposin-Flag showed an intense staining surrounding the nucleus. Although there was no staining at the cell membrane of transfected cells, FACS analysis using kap-2 or Flag antibodies, under nonpermeable conditions, showed positivity. Cell fractionation studies further showed that the majority of Kaposin was detected in the nuclear fraction by Western blot analysis. The cytoplasmic and detergent soluble membrane fractions did not show Kaposin protein; however, a small amount was detected in the detergent insoluble membrane fraction. Taken together, these results suggest that Kaposin exhibits multicompartmental localization in cells.     

Production of transgenic sugarbeet plants (Beta vulgaris L.) using Agrobacterium rhizogenes

Normal phenotype sugarbeet plants transformed with Agrobacterium rhizogenes were produced using direct regeneration from explants without hairy root phase. Kanamycin resistant plants and Ri-roots carrying the genes of neomycin phosphotransferase II and b-glucuronidase have been obtained. Integration of transgenes into sugarbeet genome was confirmed with GUS-assay and PCR using primers for the introduced genes.     

Modulation by antiidiotypic monoclonal antibodies of immune lysis mediated by anti-HLA monoclonal antibodies

The mAb R18-9 recognizes a cross-reacting idiotope outside the Ag-combining site of the syngeneic anti HLA-DQw3 mAb KS13, whereas the mAb R1-38, KO3-34, KO3-256, and KO3-335 recognize spatially close private idiotopes within the Ag-combining site of mAb KS13. All the analyzed Id require the association of the H and L chain of mAb KS13 for their expression. The mAb R1-38 and R18-9 were shown to markedly differ in their ability to modulate immune lysis of target cells mediated by mAb KS13. mAb R18-9 did not affect C-dependent lysis of cultured B lymphoid cells WALK mediated by mAb KS13, but enhanced cell-dependent mAb KS13-mediated lysis. mAb R1-38 inhibited both C and cell-dependent lysis mediated by mAb KS13. The effect was influenced by the incubation conditions. mAb R1-38 completely inhibited lysis when it was preincubated with mAb KS13 before being added to target cells, inhibited it partially when it was added simultaneously with mAb KS13 to target cells and did not affect it when added to target cells which had been preincubated with mAb KS13. Neither mAb R1-38 nor R18-9 in combination with mAb KS13 modulated T cell proliferation induced by allogeneic HLA mismatched lymphocytes. The system we have described may represent a useful in vitro model to investigate the mechanism(s) by which antiidiotypic antibodies may influence the outcome of organs transplanted in recipients with a history of humoral presensitization to donor's HLA Ag.     

Non-AIDS associated Kaposi's sarcoma: clinical features and treatment outcome

Background

Kaposi''s sarcoma (KS) in HIV negative patients is rare and has to be distinguished from AIDS associated KS. Two groups are at risk to develop non-AIDS related KS: elderly men mainly of Mediterranean origin and persons with iatrogenic immunosuppression.

Patients and Methods

In order to define risk-groups and major clinical features we retrospectively evaluated clinical data of all patients with non-AIDS associated KS presenting to the Department of Dermatology, University Hospital Tuebingen between 1987 and 2009. Data were extracted from the tumor registry of the Comprehensive Cancer Center Tuebingen and from patient records.

Results

20 patients with non-AIDS KS have been identified. The average age at KS onset was 66.6 years; the male-to-female-ratio was 3∶1. Most of the patients were immigrants from Mediterranean or Eastern European countries (60%). 15 cases of classic KS versus 5 cases of iatrogenic KS were observed. In 95% of the cases, KS was limited to the skin, without mucosal, lymph node or visceral manifestation. KS lesions were in all cases multiple and mostly bilateral, the most common localization was the skin of the lower extremities. Tumor control was achieved in nearly all cases by the use of local or systemic therapy. No patient died from KS.

Conclusions

Unlike KS in AIDS patients, non-AIDS associated KS is a rather localized process which rarely involves lymph nodes or organs. It is mostly seen in elderly males from Mediterranean or Eastern European countries and in most cases responsive on local or systemic therapeutic strategies.     

Clinical manifestations of impaired GnRH neuron development and function

Gonadotropin-releasing hormone (GnRH) and olfactory neurons migrate together in embryologic development, and disruption of this process causes idiopathic hypogonadotropic hypogonadism (IHH) with anosmia (Kallmann syndrome (KS)). Patients with IHH/KS generally manifest irreversible pubertal delay and subsequent infertility due to deficient pituitary gonadotropins or GnRH. The molecular basis of IHH/KS includes genes that: (1) regulate GnRH and olfactory neuron migration; (2) control the synthesis or secretion of GnRH; (3) disrupt GnRH action upon pituitary gonadotropes, or (4) interfere with pituitary gonadotropin synthesis or secretion. KS patients may also have midline facial defects indicating the diverse developmental functions of genes involved. Most causative genes cause either normosmic IHH or KS except FGFR1, which may cause either phenotype. Recently, several balanced chromosomal translocations have been identified in IHH/KS patients, which could lead to the identification of new disease-producing genes. Although there are two cases reported who have digenic disease, this awaits confirmation in future larger studies. The challenge will be to determine the importance of these genes in the 10-15% of couples with normal puberty who have infertility.     

Keratan sulfate: structure, biosynthesis, and function

The last 5 years have seen a marked increase in research on keratan sulfate (KS) and a concomitant increase in our understanding of the range of molecules that carry this adaptable polysaccharide. More than 15 KS-linked proteins have been identified and many of the genes encoding these have been cloned. KS-containing molecules have been identified in numerous epithelial and neural tissues in which KS expression responds to embryonic development, physiological variations, and to wound healing. A corneal cell culture system has been developed in which long-term KS biosynthesis is maintained. Progress has been made toward identification of the glycosyl- and sulfotransferases responsible for KS biosynthesis. A mouse knockout of a corneal KS-proteoglycan has provided the first experimental support for the role of KS in corneal transparency. Evidence has also been presented supporting functional roles of KS in cellular recognition of protein ligands, axonal guidance, cell motility, and in embryo implantation. These findings have served to expand the concept of what keratan sulfate is and the potential roles it may play in the cellular biology of diverse tissues.     

Development of Efficient Plant Regeneration and Transformation System for Impatiens Using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>and Multiple Bud Cultures as Explants

Background  

Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop.