Molecular evidence that plastids in the toxin-producing dinoflagellate genus Dinophysis originate from the free-living cryptophyte Teleaulax amphioxeia

Some species of the dinoflagellate genus Dinophysis form red tides and are toxin producers with a great environmental impact. The dinoflagellates as a group display high plastid diversity. Several cases indicate that plastids have been replaced. In the case of the genus Dinophysis, the plastids show characteristics of a plastid originating from a cryptophyte. Recent molecular evidence showed that the plastid indeed originates from a cryptophyte, but the source could not be identified to species or genus level. The data presented here show that both a 799 bp region of the psbA gene and 1,221 bp region of the 16S rRNA gene from Dinophysis spp. are identical to the same loci in Teleaulax amphioxeia SCCAP K434. This strongly indicates that the plastid was acquired recently in Dinophysis and may be a so-called kleptoplastid, specifically originating from a species of Teleaulax.     

Thin-layer cytology findings of small cell carcinoma of the lower female genital tract. Review of three cases with molecular analysis

OBJECTIVE: To describe the thin-layer cytology findings of small cell carcinoma of the low female genital tract, with histologic correlation and human papillomavirus (HPV) genotyping. STUDY DESIGN: The authors reviewed the clinical findings, thin-layer cytology and histologic features of small cell carcinoma of the lower female genital tract (cervix or vagina) occurring in three postmenopausal Chinese women at Pamela Youde Nethersole Eastern Hospital, Hong Kong, over a four-year period, from January 1998 to December 2001. Molecular techniques for HPV screening and genotyping using the polymerase chain reaction and restriction fragment length polymorphism were employed on the cytologic specimens. RESULTS: The thin-layer preparations were of moderate to high cellularity. There were loose aggregates of or isolated small round cells with a high nuclear/cytoplasmic ratio, thin but irregular nuclear membrane, hyperchromatic nuclei, inconspicuous nucleoli and scanty cytoplasm. Tumor cell cannibalism was commonly found. Small groups of tumor cells with nuclear molding were noted. There was also obvious tumor diathesis in the background. The necrotic debris was admixed with isolated small round cells, apoptotic bodies and nuclear dust. Associated koilocytosis or squamous intraepithelial lesions were not seen. Histologic examination of the tumor biopsies showed classic features of small cell carcinoma associated with squashing artifacts and vascularized stroma. Molecular analysis revealed the presence of HPV DNA (either type 18 or 16) in all the three liquid-based cytology samples. CONCLUSION: While the cytomorphologic features of small cell carcinoma of the cervix or vagina in thin-layer preparations are slightly different from those in conventional smears, due mainly to the absence of smearing effect, recognition of the subtle but characteristic appearance can enhance the accuracy of the cytologic diagnosis. The association between HPV and primary small cell carcinoma of the lower female genital tract was confirmed by this study.     

Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease

Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.     

VL position 34 is a key determinant for the engineering of stable antibodies with fast dissociation rates

Predictive engineering of antibodies exhibiting fast kinetic properties could provide reagents for biotechnological applications such as continuous monitoring of compounds or affinity chromatography. Based on covariance analysis of murine germline antibody variable domains, we selected position L34 (Kabat numbering) for mutational studies. This position is located at the VL/VH interface, at the base of the paratope but with limited antigen contacts, thus making it an attractive position for mild alterations of antigen binding properties. We introduced a serine at position L34 in two different antibodies: Fab (fragment antigen binding) 57P (Asn34Ser) and scFv (single chain fragment variable) 1F4 (Gln34Ser), that recognize peptides derived from the coat protein of tobacco mosaic virus and the oncoprotein E6, respectively. Both mutated antibodies exhibited similar properties: (i) expression levels of active fragments in Escherichia coli were markedly improved; (ii) thermostability was enhanced; and (iii) dissociation rate parameters (k(off)) were increased by 2- and at least 57-fold for scFv1F4 and Fab57P, respectively, while their association rate parameters (k(on)) remained unchanged. The L34 Ala and Thr mutants of both antibody fragments did not possess these properties. This first demontration of similar effects observed in two antibodies with different specificities may open the way to the predictive design of molecules with enhanced stability and fast dissociation rates.     

Expression, purification, and crystallization of the Escherichia coli selenomethionyl beta-ketoacyl-acyl carrier protein synthase III

Bacterial beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called FabH) catalyzes the condensation and transacylation of acetyl-CoA with malonyl-ACP. In order to understand the mode of enzyme/substrate interaction and design small molecule inhibitors, we have expressed, purified, and crystallized a selenomethionyl-derivative of E. coli KAS III. Several lines of evidence confirmed that purified selenomethionyl KAS III was homogenous, stably folded, and enzymatically active. Dynamic light scattering, size exclusion chromatography, and mass spectrometry results indicated that selenomethionyl KAS III is a noncovalent homodimer. Diffraction quality crystals of selenomethionyl KAS III/acetyl-CoA complex, which grew overnight to a size of 0.2 mm(3), belonged to the tetragonal space group P4(1)2(1)2.     

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.     

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations

Background and Aims

Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections.

Methods

Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population.

Key Results

Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal σ_g and neighbourhood size N_b were estimated to be 4–12 m [95 % confidence interval (CI): 3–25] and 42–73 plants (95 % CI: 28–322) in Meijendel and 10–31 m (95 % CI: 7–∞) and 57–198 plants (95 % CI: 28–∞) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %.

Conclusions

SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.