Chromosomal integration of transduced recombinant baculovirus DNA in mammalian cells

Our group and others have recently demonstrated the ability of recombinant baculoviruses to transduce mammalian cells at high frequency. To further characterize the use of baculovirus as a mammalian gene delivery system, we examined the status of transduced DNA stably maintained in Chinese hamster ovary (CHO) cells. Four independent clones carrying two introduced markers, the genes for neomycin resistance (Neo) and green fluorescent protein (GFP), were selected. PCR analysis, Southern blotting, and DNA sequencing showed that discrete portions of the 148-kb baculovirus DNA were present as single-copy fragments ranging in size from 5 to 18 kb. Integration into the CHO cell genome was confirmed by fluorescent in situ hybridization (FISH) analysis. For one clone, the left and right viral/chromosomal junctions were determined by DNA sequencing of inverse PCR products. Similarly, for a different clone, the left viral/chromosomal junction was determined; however, the right junction sequence revealed the joining to another viral fragment by a short homology (microhomology), a hallmark of illegitimate recombination. The random viral breakpoints and the lack of homology between the virus and flanking chromosomal sequences are also suggestive of an illegitimate integration mechanism. To examine the long-term stability of reporter gene expression, all four clones were grown continuously for 36 passages in either the presence or absence of selection for Neo. Periodic assays over a 5-month period showed no loss of GFP expression for at least two of the clones. This report represents the first detailed analysis of baculovirus integrants within mammalian cells. The potential advantages of the baculovirus system for the stable integration of genetic material into mammalian genomes are discussed.     

Effects of pCIneo and pCEP4 expression vectors on transient and stable protein production in human and simian cell lines

To support and meet the demand for recombinant proteins early in the drug discovery process, much work has been directed toward improving the methods used for transient gene transfection and expression. A factor which could potentially affect the outcome of experiments is the choice of the expression vector. Conventional vectors such as pCIneo and pcDNA3 have been used frequently. Each of these places the gene of interest under the control of the CMV promoter. An interesting alternative is provided by episomal vectors. For example, the pCEP4 vector contains the gene coding for the Epstein Barr nuclear antigen as well as the EBNA ori P sequence. This combination allows for the episomal replication of the plasmid. In preliminary experiments, we compared transient secreted placental alkaline phosphatase production in 8 cell lines from 3 different species using the pCIneo vs. pCEP4 vectors and found the utility of the pCEP4 vector to be limited to the human 293 EBNA cell line. In this paper, we have compared the two vectors in six cell lines of simian and human origin, measuring the transient production of secreted placental alkaline phosphatase and human hepatocyte growth factor. In general, the pCEP4 vector produced higher amounts of both proteins in transient transfections. Results were particularly pronounced in the HEK 293 and 293 EBNA cell lines. Stable pools of cells (uncloned) expressing human hepatocyte growth factor were isolated using pCIneo and pCEP4 and protein production levels were compared to those seen in transient transfections. Stable expression with pCEP4 was found to produce the highest levels of human hepatocyte growth factor in 3 of 4 cell lines. Finally, electroporation and FuGENE^TM6(Roche, Indianapolis IN) as transfection methods were compared measuring transient production of secreted placental alkaline phosphatase, human hepatocyte growth factor, and green fluorescent protein. FuGENE produced higher protein concentrations in less time than electroporation for all 3 proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nanotube-mediated cross-feeding couples the metabolism of interacting bacterial cells

Bacteria frequently engage in cross-feeding interactions that involve an exchange of metabolites with other micro- or macroorganisms. The often obligate nature of these associations, however, hampers manipulative experiments, thus limiting our mechanistic understanding of the ecophysiological consequences that result for the organisms involved. Here we address this issue by taking advantage of a well-characterized experimental model system, in which auxotrophic genotypes of E. coli derive essential amino acids from prototrophic donor cells using intercellular nanotubes. Surprisingly, donor–recipient cocultures revealed that the mere presence of auxotrophic genotypes was sufficient to increase amino acid production levels of several prototrophic donor genotypes. Our work is consistent with a scenario, in which interconnected auxotrophs withdraw amino acids from the cytoplasm of donor cells, which delays feedback inhibition of the corresponding amino acid biosynthetic pathway and, in this way, increases amino acid production levels. Our findings indicate that in newly established mutualistic associations, an intercellular regulation of exchanged metabolites can simply emerge from the architecture of the underlying biosynthetic pathways, rather than requiring the evolution of new regulatory mechanisms.     

Tissue Specificity of Human Angiotensin I-Converting Enzyme

Background

Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood.

Methods/Principal Findings

We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests.

Conclusions

Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs.     

The evolutionary emergence of stochastic phenotype switching in bacteria

Stochastic phenotype switching - or bet hedging - is a pervasive feature of living systems and common in bacteria that experience fluctuating (unpredictable) environmental conditions. Under such conditions, the capacity to generate variable offspring spreads the risk of being maladapted in the present environment, against offspring likely to have some chance of survival in the future. While a rich subject for theoretical studies, little is known about the selective causes responsible for the evolutionary emergence of stochastic phenotype switching. Here we review recent work - both theoretical and experimental - that sheds light on ecological factors that favour switching types over non-switching types. Of particular relevance is an experiment that provided evidence for an adaptive origin of stochastic phenotype switching by subjecting bacterial populations to a selective regime that mimicked essential features of the host immune response. Central to the emergence of switching types was frequent imposition of 'exclusion rules' and 'population bottlenecks' - two complementary faces of frequency dependent selection. While features of the immune response, exclusion rules and bottlenecks are likely to operate in many natural environments. Together these factors define a set of selective conditions relevant to the evolution of stochastic switching, including antigenic variation and bacterial persistence.     

Multiple component system of sugars and polyols in the overwintering spruce bark beetle, Ips typographus

Overwintering adults of the spruce bark beetle, Ips typographus (L.) showed an unusually complex sugar/polyol cryoprotectant system. The major components of the multiple system were: glucose (177.6 mmolL(-1), March); trehalose (175.0 mmolL(-1), December); sorbitol (147.9 mmolL(-1), January); mannitol (81.2 mmolL(-1), March); and erythritol (40.7mmolL(-1), March) (in the parentheses, the maximum concentrations are shown and the month when they were reached). Other minor components were glycerol, fructose, threitol, myo-inositol, arabinitol and ribitol. Distinct seasonal patterns of accumulation/depletion in various components were found. Glycerol, trehalose and glucose started to accumulate first, during early autumn, when the air temperatures fluctuated between 20 and 0 degrees C, and diapause beetles continued in feeding. Glycerol was depleted, glucose remained stable and trehalose continued in accumulation during late autumn when the temperatures oscillated around 0 degrees C. During early winter severe frosts reaching -20 degrees C came, the beetles terminated their diapause and trehalose was partially depleted, while mannitol, sorbitol, fructose, threitol and erythritol started to accumulate. Cold weather continued also during late winter when the beetles remained quiescent. During this period, trehalose was re-accumulated, threitol and erythritol continued to increase, mannitol remained stable and sorbitol, fructose decreased. All cryoprotectans were finally cleared in the beetles which were spontaneously leaving bark during early spring. The seasonal maximum of total concentration of all cryoprotectants (578.2 mOsmol L(-1)) was reached in March. Such a concentration results in colligative depression of melting point of body fluids down by 1.08 degrees C only. It suggests that the potential cryoprotective effect of accumulated sugars and polyols was related rather to their non-colligative functions.