Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/‐ 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ‘electroporation model’ the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.

The function of keratinocyte growth factor (KGF) in normal and wounded skin was assessed by expression of a dominant-negative KGF receptor transgene in basal keratinocytes. The skin of transgenic mice was characterized by epidermal atrophy, abnormalities in the hair follicles, and dermal hyperthickening. Upon skin injury, inhibition of KGF receptor signaling reduced the proliferation rate of epidermal keratinocytes at the wound edge, resulting in substantially delayed reepithelialization of the wound.

The hepatitis B virus (HBV) transactivator protein HBx is enigmatic in that it stimulates a striking variety of promoters which do not share a common cis-regulatory element. As it does not bind to DNA, it has been speculated that HBx acts indirectly through cellular pathways. Under certain conditions HBx can have an oncogenic potential, which may be relevant for HBV-associated liver carcinogenesis, but until now the mechanism for transactivation and cell transformation by HBx was unclear. We report here that HBx uses a complex signal transduction pathway for transactivation. An increase in the endogenous protein kinase C (PKC) activator sn-1,2-diacylglycerol and the subsequent activation of PKC give rise to activation of the transcription factor AP-1 (Jun-Fos). As a result, HBx transactivates through binding sites for AP-1 and other PKC-dependent transcription factors (AP-2, NF-kappa B), thereby explaining the as-yet incomprehensible variety of HBx-inducible genes. As the PKC signal cascade also mediates cell transformation by tumour-promoting agents, the mechanism presented here might account for the oncogenic potential of HBx.

Hepatitis B virus (HBV) is regarded as the main aetiologic factor in the development of human hepatocellular carcinoma (HCC), one of the most frequent fatal malignancies worldwide. Detection of integrated HBV sequences in the cellular DNA of almost all HCCs studied, and the recent finding that the integrated HBV open reading frame (orf) X encodes a transactivating activity, supports the notion that integrated HBV DNA could contribute to liver carcinogenesis by activation of cellular genes in trans. But not all HCCs seem to harbour a functional orf X. We report here that 3'-truncated preS2/S sequences in integrated HBV DNA of liver cell carcinomas encode a so far unidentified transcriptional trans-activation activity. This activity is also produced by an artificially 3'-truncated preS2/S gene of the wild-type HBV genome. Besides the simian virus 40 promoter of the reporter plasmid pSV2CAT, the promoter of the human c-myc oncogene can also be activated. These results, taken together with the fact that preS/S is the only HBV gene found to be integrated in almost every HBV-related HCC analysed so far, indicate that trans-activation by integrated preS2/S sequences is a possible mechanism for HBV-associated oncogenesis.

We have developed an in situ hybridization assay capable of detecting enteroviral RNA in myocardial cells, using molecularly cloned coxsackievirus B3 cDNA as a diagnostic probe. Because of the high degree of nucleic acid sequence homology among the numerous enteroviral serotypes, including the group A and B coxsackieviruses and the echoviruses, detection of these various agents commonly implicated in human viral heart disease is possible in a single hybridization assay. We demonstrate the considerable potential of this method for an unequivocal diagnosis of enteroviral heart disease as well as for pathogenicity studies. Using athymic mice persistently infected with coxsackievirus B3 as a model system, we show that the myocardium is affected in a disseminated, multifocal manner.In North America and Europe, acute myocarditis is most commonly associated with infections by coxsackie B viruses (types 1 to 5). Other members of the human enteroviruses comprising at present over 70 serotypes (e.g., various coxsackie A viruses and echoviruses) are also considered to be relatively frequent causes of human viral heart disease (1-4). These agents appear to be capable of producing dilated cardiomyopathy of acute onset or lead to a variety of cardiac arrhythmias. Some of the acute cases may also evolve into a chronic form of dilated cardiomyopathy.The difficulty of establishing a specific diagnosis of viral heart disease is a major problem in clinical cardiology. Confirmation ofthe clinical suspicion of viral heart disease demands demonstration of replicating virus inside myocardial cells, which is exceedingly difficult by conventional methods (5). In addition, the increasing availability of potential antiviral agents has accentuated the need for methods by which endomyocardial biopsy specimens of patients with suspected viral heart disease can be diagnosed conclusively (6, 7).In order to introduce in situ nucleic acid hybridization (8-10) as a diagnostic tool in suspected enteroviral heart disease, we have recently cloned the single-stranded RNA genome of coxsackievirus B3 (CVB3) (11) that had been propagated in cultured human heart cells (12). Full-length reverse-transcribed cloned viral cDNA generated infectious CVB3 upon transfection into mammalian cells, demonstrating the molecular cloning of a faithful transcript ofthe original viral RNA. We now report the use of radioactively labeled cloned CVB3 cDNA as a diagnostic probe for the in situ detection of viral RNA in infected cultured cells and in myocardial tissue sections of CVB3-infected T-cell-deficient mice. We show that the molecular hybridization approach permits the detection of infected myocardial cells at the single-cell level, thus providing a unique possibility for an unequivocal diagnosis.A further advantage of the nucleic acid hybridization approach is provided by the high degree of genetic homology shared among the different serotypes of the human enterovirus group (11, 13-17), making possible their detection in a single hybridization assay. Detectio...