Use of immunostimulatory sequence-containing oligonucleotides as topical therapy for genital herpes simplex virus type 2 infection

Synthetic oligonucleotides containing CpG motifs in specific sequence contexts have been shown to induce potent immune responses. We have evaluated mucosal administration of two immunostimulatory sequence (ISS)-containing phosphorothioate-stabilized oligonucleotides for antiherpetic efficacy in animal models. The ISS oligonucleotides, suspended in phosphate-buffered saline, were tested in mouse and guinea pig vaginal models of herpes simplex virus type 2 (HSV-2) infection. For comparison, groups of untreated, non-ISS oligonucleotide-treated, and acyclovir-treated animals also were monitored. The results indicated that vaginal epithelial application of ISS (up to 6 h after viral inoculation) with mice lethally challenged with HSV-2 delayed disease onset and reduced the number of animals that developed signs of disease (P = 0.003). ISS application significantly increased survival rates over those of controls (P = 0.0014). The ISS also impacted an established infection in the guinea pig model of HSV-2 disease. A single administration of ISS (21 days after viral inoculation) significantly reduced the frequency and severity of HSV-2 lesions compared to results with non-ISS oligonucleotide-treated and untreated guinea pigs (P < 0.01). HSV-2 is shed from the vaginal cavity of the guinea pig in the absence of lesions, similar to the case with humans. As an additional indication of ISS efficacy, the magnitude of viral shedding also was significantly reduced in ISS-treated animals (P < 0.001). These effects appeared to be immunologically mediated, since ISS had no direct effect on HSV-2 replication in vitro using standard plaque assays. These data suggest that ISS may be useful in the treatment and control of genital herpes in humans.     

Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60

SPG13, an autosomal dominant form of pure hereditary spastic paraplegia, was recently mapped to chromosome 2q24-34 in a French family. Here we present genetic data indicating that SPG13 is associated with a mutation, in the gene encoding the human mitochondrial chaperonin Hsp60, that results in the V72I substitution. A complementation assay showed that wild-type HSP60 (also known as "HSPD1"), but not HSP60 (V72I), together with the co-chaperonin HSP10 (also known as "HSPE1"), can support growth of Escherichia coli cells in which the homologous chromosomal groESgroEL chaperonin genes have been deleted. Taken together, our data strongly indicate that the V72I variation is the first disease-causing mutation that has been identified in HSP60.     

Gene discovery and microarray analysis of cacao (Theobroma cacao L.) varieties

The cacao bean harvest from the relatively under developed tropical tree cacao (Theobroma cacao L.) is subject to high losses in potential production due to pests and diseases. To discover and understand the stability of putative natural resistance mechanisms in this commodity crop, essential for chocolate production, we undertook a gene-discovery program and demonstrated its use in gene-expression arrays. Sequencing and assembling bean and leaf cDNA library inserts produced a unique contig set of 1,380 members. High-quality annotation of this gene set using Blast and MetaFam produced annotation for 75% of the contigs and allowed us to identify the types of gene expressed in cacao beans and leaves. Microarrays were constructed using amplified inserts of the uni-gene set and challenged with bean and leaf RNA from five cacao varieties. The microarray performed well across the five randomly chosen cacao genotypes and did not show a bias towards either leaf or bean tissues. This demonstrates that the gene sequences are useful for microarray analysis across cacao genotypes and tissue types. The array results, when compared with real-time PCR results for selected genes, showed a correlation with differential gene-expression patterns.We intend that the resultant DNA sequences and molecular microarray platform will help the cacao community to understand the basis, likely stability and pathotype resistance range of candidate cacao plants.     

A technique for microsecond heating and cooling of a thin (submicron) biological sample

Temperature excursions of short duration are useful in exploring the effects of stress on biological systems. Stress will affect the conformation of biological molecules such as proteins, which will lead to an effect on their function. The feasibility of generating such temperature excursions is demonstrated by solving the heat diffusion equation for an aqueous layer on a silicon wafer. The silicon is rapidly heated by a laser pulse and also acts as a heat sink to quench the temperature rise. An oxide layer was used to limit the maximum temperature attained by the sample. We show that exposures above a 50 degrees C benchmark can be confined to times less than 5 micros.     

Ancient teeth and modern human origins: an expanded comparison of African Plio-Pleistocene and recent world dental samples

Previous research by the first author revealed that, relative to other modern peoples, sub-Saharan Africans exhibit the highest frequencies of ancestral (or plesiomorphic) dental traits and, thus, appear to be least derived dentally from an ancestral hominin state. This determination, in conjunction with various other lines of dental morphological evidence, was interpreted to be supportive of an African origin for modern humans. The present investigation expands upon this work by using: 1) direct observations of fossil hominin teeth, rather than data gleaned from published sources, 2) a single morphological scoring system (the Arizona State University Dental Anthropology System) with consistent trait breakpoints, and 3) data from larger and more varied modern human comparative samples. As before, a multivariate distance statistic, the mean measure of divergence, was used to assess diachronic phenetic affinities among the Plio-Pleistocene hominins and modern humans. The present study also employed principal components analysis on dental trait frequencies across samples. Both methods yielded similar results, which support the previous findings; that is, of all modern human samples, sub-Saharan Africans again exhibit the closest phenetic similarity to various African Plio-Pleistocene hominins-through their shared prevalence of morphologically complex crown and root traits. The fact that sub-Saharan Africans express these apparently plesiomorphic characters, along with additional information on their affinity to other modern populations, evident intra-population heterogeneity, and a world-wide dental cline emanating from the sub-continent, provides further evidence that is consistent with an African origin model.     

Loss of myosin VI reduces secretion and the size of the Golgi in fibroblasts from Snell's waltzer mice

Golgi morphology and function are dependent on an intact microtubule and actin cytoskeleton. Myosin VI, an unusual actin-based motor protein moving towards the minus ends of actin filaments, has been localized to the Golgi complex at the light and electron microscopic level. Myosin VI is present in purified Golgi membranes as a peripheral membrane protein, targeted by its globular tail domain. To investigate the function of myosin VI at the Golgi complex, immortal fibroblastic cell lines of Snell's waltzer mice lacking myosin VI were established. In these cell lines, where myosin VI is absent, the Golgi complex is reduced in size by approximately 40% compared with wild-type cells. Furthermore, protein secretion of a reporter protein from Snell's waltzer cells is also reduced by 40% compared with wild-type cells. Rescue experiments showed that fully functional myosin VI was able to restore Golgi complex morphology and protein secretion in Snell's waltzer cells to the same level as that observed in wild-type cells.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.