TK-GFP fusion gene virus vectors as tools for studying the features of HSV-TK/ganciclovir cancer gene therapy in vivo

The fusion gene of herpes simplex virus thymidine kinase and green fluorescent protein (TK-GFP) was shown to be a versatile tool for examining the features of thymidine kinase/ganciclovir gene therapy in vitro. In this study, we used viral vectors carrying the fusion gene to characterize the aspects of this gene therapy form in rodent tumor models. Growth of subcutaneous 9L rat tumors transduced ex vivo with TK-GFP gene was prevented when ganciclovir (GCV) treatment was initiated immediately after tumor inoculation. Established tumors (>100 mm(3)), however, were untreatable despite the initial 55% proportion of TK-GFP positive cells. This was due to a rapid clearance of TK-GFP positive cells, but not GFP positive cells. Propidium iodide staining revealed that TK-GFP lentivirus vector was able to induce apoptosis/necrosis in 9L cells, as opposed to the respective GFP vector. Furthermore, when a subcutaneous nude mouse tumor model was used, the percentage of TK-GFP positive cells in vivo was maintained similarly as in cultured cells, suggesting contribution of a fully functional immune response to the disappearance of fusion gene positive cells. In vivo gene transfer studies: adenovirus TK-GFP vector injections resulted in about 25% gene transfer efficiency to 9L tumors and showed that their growth could be significantly reduced even when the tumor volumes were already >120 mm(3). Part of the effect was shown to be due to cytotoxicity of the vector. In summary, our results demonstrate the utility of TK-GFP fusion gene-carrying viral vectors in animal studies and show that readily detectable therapeutic genes can help us to understand the complicated nature of in vivo cancer gene therapy experiments.     

Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris

A cDNA coding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene lcc1, which encodes a laccase isoenzyme of 498 amino-acid residues preceded by a 22-residue signal peptide. The lcc1 cDNA was cloned into the vector pHIL-D2 for expression in Pichia pastoris under the control of the AOX1 promoter. Transformants were found to secrete active recombinant enzyme after induction with methanol. The use of growth medium buffered to pH 6.0 and control of pH during cultivation were found to be important, or even necessary, for obtaining activity in liquid cultures. The effect of exchanging the native secretion signal for the Saccharomyces cerevisiae α-factor pre-pro secretion signal was studied by cloning the portion encoding the mature enzyme into the vector pPIC9. The activity obtained for the construct encoding the native laccase signal sequence was found to be seven-fold higher than for the construct encoding the α-factor secretion signal. Utilisation of the P. pastoris pep4 mutant strain SMD1168 was found to provide a two-fold higher level of activity compared with P. pastoris GS115. Received: 16 May / 8 August 1997     

Isolation of a Trichoderma reesei cDNA encoding GTP: a-d-mannose-1-phosphate guanyltransferase involved in early steps of protein glycosylation

A cDNA coding for GTP: α-d-mannose-1-phosphate guanyltransferase (MPG1 transferase) (EC 2.7.7.13) was isolated from a cDNA library of the Trichoderma reesei RutC-30 strain by suppression of the yeast Saccharomyces cerevisiae mutation in the DPM1gene encoding mannosylphosphodolichol (MPD) synthase. The nucleotide sequence of the 1.6 kb-long cDNA revealed an ORF which encodes a protein of 364 amino acids. Sequence comparisons demonstrate 70% identity with the S. cerevisiae guanyl transferase gene (MPG1) and 75% identity with the Schizosaccharomyces pombe homologue. No similarity was found with the MPD synthase encoded by the S. cerevisiae DPM1 gene. The possibility that cloned cDNA encodes a product with a MPD synthase activity was also excluded by transforming a heterozygous S. cerevisiae dpm1::LEU2/DPM1 diploid, which did not lead to the restoration of viability of the dpm1 spores. Simultaneously, a significant increase in MPG transferase activity, as compared with the wild-type yeast, was observed in cellular extracts when the mpg1 cDNA from Trichoderma was expressed in the S. cerevisiae dpm1-6 mutant. Received: 21 July 1997 / 24 April 1998     

Primary gastrointestinal stromal tumor of the esophagus in an HIV-positive patient

We describe a rare case of malignant gastrointestinal stromal tumor (GIST) of the esophagus presenting in an HIV-positive man. Not only did the tumor arise from an unusual anatomic site for GIST, namely, the esophagus, but it also had a predominant epithelioid cell morphology that is uncommon and preferentially associated with aggressive behavior. Exhaustive immunohistochemical studies showed strong reactivities to the classic GIST marker, CD34, and to the current more sensitive and more specific GIST marker, CD117/ c-kit protein. This immunophenotype corresponded to that of stromal tumors arising in the more common sites like stomach and small intestine as well as to that of a reported series of esophageal GISTs in the general population. Mutations of the c-kit protein was detected in the tumor, confirming previous observations. This further documents that esophageal GIST and the more common benign esophageal spindle cell lesions are pathologically distinct entities and despite its rarity, esophageal GIST should be recognized by pathologists and clinicians. The occurrence of this tumor in an HIV-positive patient is coincidental, and it resulted in an extremely unusual metastatic site that has not been reported for GISTs.     

Molecular epidemiology of an outbreak caused by methicillin-resistant Staphylococcus aureus in a health care ward and associated nursing home

Our point-prevalence survey followed an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in a long-term care facility and identified five MRSA strains, of which two possessed an outbreak genotype not encountered previously and three had another profile. All of them possessed SCCmec type V. Six methicillin-sensitive S. aureus strains were genotypically related to the epidemic strains.