Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-beta gene. The resultant virus (VSV-IFNbeta) was successfully propagated in cells not receptive to murine IFN-alpha/beta and expressed high levels of functional heterologous IFN-beta. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNbeta was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-beta, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNbeta grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-beta gene (VSV-hIFNbeta) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNbeta was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNbeta retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.     

Three-dimensional reconstruction of a light-harvesting complex I-photosystem I (LHCI-PSI) supercomplex from the green alga Chlamydomonas reinhardtii. Insights into light harvesting for PSI

A supercomplex containing the photosystem I (PSI) and chlorophyll a/b light-harvesting complex I (LHCI) has been isolated using a His-tagged mutant of Chlamydomonas reinhardtii. This LHCI-PSI supercomplex contained approximately 215 chlorophyll molecules of which 175 were estimated to be chlorophyll a and 40 to be chlorophyll b, based on P700 oxidation and chlorophyll a/b ratio measurements. Its room temperature long wavelength absorption peak was at 680 nm, and it emitted chlorophyll fluorescence maximally at 715 nm (77 K). The LHCI was composed of four or more different types of Lhca polypeptides including Lhca3. No LHCII proteins or other phosphoproteins were detected in the LHCI-PSI supercomplexes suggesting that the cells from which they were isolated were in State 1. Electron microscopy of negatively stained samples followed by image analysis revealed the LHCI-PSI supercomplex to have maximal dimensions of 220 A by 180 A and to be approximately 105 A thick. An averaged top view was used to model in x-ray and electron crystallographic data for PSI and Lhca proteins respectively. We conclude that the supercomplex consists of a PSI reaction center monomer with 11 Lhca proteins arranged along the side where the PSI proteins, PsaK, PsaJ, PsaF, and PsaG are located. The estimated molecular mass for the complex is 700 kDa including the bound chlorophyll molecules. The assignment of 11 Lhca proteins is consistent with a total chlorophyll level of 215 assuming that the PSI reaction center core binds approximately 100 chlorophylls and that each Lhca subunit binds 10 chlorophylls. There was no evidence for oligomerization of Chlamydomonas PSI in contrast to the trimerization of PSI in cyanobacteria.     

Detecting and quantifying colocalization of cell surface molecules by single particle fluorescence imaging

Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function. We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules. This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging. The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules. Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules. A method of correcting for positional shifts that result from chromatic aberration is presented. The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters. We have obtained preliminary data for colocalization of molecules on intact cells. Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described.     

A function for subtelomeric DNA in Saccharomyces cerevisiae

The subtelomeric DNA sequences from chromosome I of Saccharomyces cerevisiae are shown to be inherently poor substrates for meiotic recombination. On the basis of these results and prior observations that crossovers near telomeres do not promote efficient meiosis I segregation, we suggest that subtelomeric sequences evolved to prevent recombination from occurring where it cannot promote efficient segregation.     

Optimizing nitrogen management in food and energy production and environmental protection: summary statement from the Second International Nitrogen Conference

Human efforts to produce food and energy are changing the nitrogen (N) cycle of the Earth. Many of these changes are highly beneficial for humans, while others are detrimental to people and the environment. These changes transcend scientific disciplines, geographical boundaries, and political structures. They challenge the creative minds of natural and social scientists, economists, engineers, business leaders, and decision makers. The Second International Nitrogen Conference was designed to facilitate communications among all stakeholders in the "nitrogen community" of the world. The Conference participants" goal in the years and decades ahead is to encourage every country to make optimal choices about N management in food production and consumption, energy production and use, and environmental protection. Scientific findings and recommendations for decision makers that emerged from the Conference are presented.     

Breakdown of CTL tolerance to self HLA-B*2705 induced by exposure to Chlamydia trachomatis

There is a strong association between seronegative arthritis and HLA B27, but it is still unresolved whether the contribution of B27 to disease pathogenesis is solely as a restriction element for an arthritogenic peptide, or whether B27 itself serves as an autoantigen. This study uses transgenic rats to address the question as to whether exposure to an arthritogenic pathogen can alter tolerance to B27. Unlike their nontransgenic counterparts, B27-transgenic rats are tolerant of B27 immunization using either B27(+) splenocytes or plasmid DNA and do not develop anti-B27 CTL. However, if splenocytes from such immunized animals are exposed to Chlamydia in vitro, CTL are generated that lyse B27(+) targets. No killing was seen with targets transfected with control B7, B14, B40, or B44. This phenomenon was not observed with immunization by nontransgenic splenocytes, or HLA-A2 DNA alone. Using targets expressing mutated B27, we show that the epitope for autoreactive CTL recognition of B27 involves the Lys(70) amino acid residue in the alpha(1) domain of the MHC class I molecule. The generation of CTL with specificity for B27 under these conditions demonstrates that tolerance to B27 can be subverted by CHLAMYDIA: This indicates a dynamic interrelationship between the pathogen and B27, which may have important implications for B27-related spondyloarthropathies triggered by intracellular bacteria.     

Molecular mechanisms of pre-T cell receptor-induced survival

En route to maturing as T cell receptor (TCR) alphabeta-expressing cells, the development of thymocytes is contingent on expression of a pre-TCR complex comprising a TCRbeta chain paired with a surrogate TCRalpha chain, pre-Talpha (pTalpha). The pre-TCR has been proposed to promote cell survival, proliferation, differentiation, and lineage commitment. However, the precise molecular mechanisms governing this variety of effects remain elusive. Here, we present a cellular system designed to biochemically dissect signals elicited upon pre-TCR expression. Using the T cell line 4G4 stably transfected with one of the two known pTalpha isoforms or selective pTalpha deletion mutants and TCRbeta, we were able to observe that expression of a functional pre-TCR complex is sufficient to control the levels of surface Fas protein, the stimulation of mitogen-activated and stress-regulated kinases, and the activation status of the p53 antioncogene. We demonstrate that this regulation has a major impact on the expression of important regulators of apoptosis, such as Bcl-2 family members, and the cell cycle, such as p21(WAF). Furthermore, we show here that cells expressing a functional pre-TCR are more resistant to different types of DNA damage-induced apoptosis and that these effects are contingent on an intact cytoplasmic tail of pTalpha. We finally propose that the presence of a functional pre-TCR complex triggers many intracellular pathways capable of driving and ensuring thymocyte survival in the presence of DNA damage.     

Electron crystallographic study of photosystem II of the cyanobacterium Synechococcus elongatus

The determination of the structure of PSII at high resolution is required in order to fully understand its reaction mechanisms. Two-dimensional crystals of purified highly active Synechococcus elongatus PSII dimers were obtained by in vitro reconstitution. Images of these crystals were recorded by electron cryo-microscopy, and their analysis revealed they belong to the two-sided plane group p22(1)2(1), with unit cell parameters a = 121 A, b = 333 A, and alpha = 90 degrees. From these crystals, a projection map was calculated to a resolution of approximately 16 A. The reliability of this projection map is confirmed by its close agreement with the recently presented three-dimensional model of the same complex obtained by X-ray crystallography. Comparison of the projection map of the Synechococcus elongatus PSII complex with data obtained by electron crystallography of the spinach PSII core dimer reveals a similar organization of the main transmembrane subunits. However, some differences in density distribution between the cyanobacterial and higher plant PSII complexes exist, especially in the outer region of the complex between CP43 and cytochrome b(559) and adjacent to the B-helix of the D1 protein. These differences are discussed in terms of the number and organization of some of the PSII low molecular weight subunits.     

Heterozygous submicroscopic inversions involving olfactory receptor-gene clusters mediate the recurrent t(4;8)(p16;p23) translocation

The t(4;8)(p16;p23) translocation, in either the balanced form or the unbalanced form, has been reported several times. Taking into consideration the fact that this translocation may be undetected in routine cytogenetics, we find that it may be the most frequent translocation after t(11q;22q), which is the most common reciprocal translocation in humans. Case subjects with der(4) have the Wolf-Hirschhorn syndrome, whereas case subjects with der(8) show a milder spectrum of dysmorphic features. Two pairs of the many olfactory receptor (OR)-gene clusters are located close to each other, on both 4p16 and 8p23. Previously, we demonstrated that an inversion polymorphism of the OR region at 8p23 plays a crucial role in the generation of chromosomal imbalances through unusual meiotic exchanges. These findings prompted us to investigate whether OR-related inversion polymorphisms at 4p16 and 8p23 might also be involved in the origin of the t(4;8)(p16;p23) translocation. In seven case subjects (five of whom both represented de novo cases and were of maternal origin), including individuals with unbalanced and balanced translocations, we demonstrated that the breakpoints fell within the 4p and 8p OR-gene clusters. FISH experiments with appropriate bacterial-artificial-chromosome probes detected heterozygous submicroscopic inversions of both 4p and 8p regions in all the five mothers of the de novo case subjects. Heterozygous inversions on 4p16 and 8p23 were detected in 12.5% and 26% of control subjects, respectively, whereas 2.5% of them were scored as doubly heterozygous. These novel data emphasize the importance of segmental duplications and large-scale genomic polymorphisms in the evolution and pathology of the human genome.