Polymorphism of Phaseolus vulgaris var. aborigineus (fabaceae). Evidences of natural hibridation

A polymorphic population of Phaseolus vulgaris var. aborigineus growing at the Northwest of Argentina was studied. In order to know the origin of this polymorphism, some plants belonging to the var. aborigineus, other plants showing floral dimorphism and other individuals with particular characters were collected. Their seeds, obtained after field-work treatments of autogamy and free pollination, were sown in a greenhouse, isolated of the access of pollinators. The growth of each plant was followed until its fructification, and the number of plants that died due to infections was recorded. The number of plants that flowered and fructified was registered in order to study their reproductive success. The floral, fruit and seed qualitative and quantitative characters were documented. With the results obtained, the authors concluded that those individuals that showed floral dimorphism are probably a result of hybridization and introgression between the var. aborigineus and "old cultivars". This hypothesis is supported by the presence of divergent segregation, observed in the offspring of the plants with this segregation. Other crops should allow the genic flow between the parental entities, with the consequence of the establishment of an hybrid population coexistent with their ancestors. Perhaps, as a result of introgression, the stabilized lines exhibit characters different from their parental varieties. The results of autopollination and free pollination in those individuals assigned to var. aborigineus, showed that free pollination brings a great genetic plasticity, because next generations can persist and resist infections. The offspring of the F1 was followed. The plants that belonged to var. aborigineus, product of free pollination, exhibited fast growth and were healthy, while the descendant of the individuals with the floral dimorphism showed characteristics that allowed to conclude the possible existence of degeneration of the hybrid progeny; this characteristics were: curled radicles with cotyledons that never emerge, plantule's apex that soon die with the following development of branches from the cotyledon's axil, and death after some weeks. This degeneration indicates that an unwanted gene flow in the area could lead to a decline in the wild bean population. The vigor, high reproductive success and resistance to illnesses of the individuals corresponding to the var. vulgaris, whose progenitor was treated for free fecundation, and the offspring of the plants with cultivated characteristics, are indicative of the necessity of preserving this germplasm to evaluate its agronomic potential to brief term. The DNA analyses already initiated, will allow the confirmation of the hypotheses outlined in this work.     

Multigene DNA prime-boost vaccines for SHIV89.6P

We assessed four prime-boost vaccine regimens with a Gene Gun component for SHIV89.6P in Macaca nemestrina. A dosing experiment using beta-galactosidase plasmid showed that 30 or 45 shots per dose elicited higher titer antibody than smaller doses. For SHIV89.6P, we administered a six-plasmid vaccine capable of producing non-infectious virions in vivo in combination with either vaccinia recombinants or inactivated virus. DNA prime/vaccinia boost, or the reverse, elicited strong immune responses. The SHIV89.6P challenge virus was grown in M. nemestrina peripheral blood mononuclear cells and titered in vivo intrarectally. As has been observed for SHIV89.6P in M. mulatta, the infected M. nemestrina experienced rapid and severe loss of circulating CD4+ T cells. Vaccinated macaques were challenged three weeks after the last boost. DNA prime/vaccina boost or vaccina prime/DNA boost protected 11/12 animals from acute CD4+ T cell depletion and disease, while other regimens were not effective.     

Transposon mutagenesis of Mycoplasma gallisepticum

There are few systems available for studying the genetics of the important avian respiratory pathogen, Mycoplasma gallisepticum. These techniques are needed to develop a mechanism to study the molecular pathogenesis of M. gallisepticum. Tn916 has the ability to transpose into the M. gallisepticum genome by both transformation and conjugation. In this study, PEG-mediated transformation was employed for the transfer of Tn916 into M. gallisepticum and create a transposon mutant library. Transformants were obtained at a frequency of approximately 5 x 10(-8) per recipient CFU. A total of 424 MG/Tn916 mutants were constructed and sequence data from the transposon junctions of 71 mutants was obtained and used to identify transposon insertion sites. Insertions were found throughout the genome in nearly all of the major gene categories, making this the first extensive characterization of a transposon mutant library of M. gallisepticum. Transposon stability was also examined, and it was determined that for two mutants the element was stably maintained in vivo in the absence of selective pressure.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

Short inverse complementary amino acid sequences generate protein complexity

Inversions of short genomic sequences play a central role in the generation of protein complexity. More than half of the 1300 motifs registered in ProSite have protein inverse complementary sequences (princoms) among proteins registered in SwissProt. The observed number of princoms occurrences exceeds by far the expected number (p < 10(-10)). Princoms often endow their host proteins with a whole new range of biochemical and physiological capabilities, including the possibility of intramolecular and intermolecular disulfide bond formation. These results support the idea that, like the duplications, the inversions of small genomic fragments have been a fundamental mechanism for shaping genomes.     

Depletion of polyubiquitin encoded by the UBI4 gene confers pleiotropic phenotype to Candida albicans cells

We have studied the roles of polyubiquitin in Candida albicans physiology. Heterologous expression of the C. albicans polyubiquitin (UBI4) gene in a ubi4 Saccharomyces cerevisiae strain suppressed the mutant phenotype (hypersensitivity to heat shock). A heterozygous strain UBI4/Deltaubi4::hisG, obtained following the ura-blaster procedure, was used to construct a conditional mutant using a pCaDis derivative plasmid. By serendipity we isolated the UBI4 conditional mutant as well as a UBI4 mutant containing a non-functional MET3 promoter. Depletion of polyubiquitin conferred pleiotropic effects to mutant cells: (i) a limited increased sensitivity to mild heat shock; (ii) increased formation of colony morphology variants; and (iii) induction of hyphal and pseudohypal development. These results indicate that polyubiquitin in C. albicans is involved in the negative control of switching, as well as in maintaining the yeast cell morphology, probably by silencing mechanisms triggering the hyphal and pseudohyphal development in the absence of environmental inducers.     

Acute cold exposure,leptin, and somatostatin analog (octreotide) modulate thyroid 5'-deiodinase activity

We investigated the effect of acute cold exposure, leptin, and the somatostatin analog octreotide (OCT) on thyroid type I (D1) and II (D2) deiodinase activities. Microsomal D1 and D2 activities were measured by the release of (125)I from (125)I-reverse triiodothyronine (rT(3)) under different assay conditions. Rats exposed to 4 degrees C (15, 30, 60, and 120 min) showed progressive reduction in thyroidal D1 and D2, reaching approximately 40% at 2 h (P < 0.05) despite increased circulating TSH (P < 0,05) associated with the higher thyroid D1 and D2 in hypothyroid rats. A single injection of leptin (8 microg/100 g body wt sc) induced increased thyroid and liver D1 (P < 0.05), but not thyroid D2, activities at 30 and 120 min, independently of the serum TSH rise shown only at 2 h. OCT (1 microg/kg body wt sc) increased D1 and D2 activity significantly 24 h after a single injection, with no changes in serum TSH. Therefore, leptin and somatostatin are potential physiological upregulators of thyroid deiodinases, and their low secretion during acute cold exposure may be a potential mechanism contributing to cold-induced reduction in thyroid deiodinase activity.     

Detection of HBD1 peptide in peripheral blood mononuclear cell subpopulations by intracellular flow cytometry

Production of human beta-defensin1 (HBD1) in response to LPS in monocytes, myeloid dendritic cells and plasmacytoid dendritic cells (PDC) was examined. Since PDC make up only 0.1-0.5% of the peripheral blood mononuclear cell population, we developed a method to determine HBD1 peptide levels using four-color flow cytometry, which can examine several cell surface or intracellular markers at once. Coupled with intracellular flow cytometry, we determined that PDC and monocytes only made significant amounts of HBD1 when exposed to >50ng/ml LPS for 2h. This response was limited to monocytes when ultrapure LPS was used, and was inhibited in PDC by chloroquine treatment.     

Novel application of a fibrin cell block method to evaluate melanocytic cell populations

Confirming melanocytic lineage and purity is important for experiments using cultured human melanocytes. The objective of this study was to develop a simple, reliable method to evaluate and archive cultured melanocytic cells. Melanocytes were isolated from adult skin biopsies or from neonatal foreskins using standard culturing methods. Fibrin cell blocks (FCBs) were prepared from cultured cells at passages two and six. Fibrin blocks were paraffin-embedded and sectioned for immunohistochemical (CD68, Melan-A, and HMB-45) and H & E staining. Flow cytometry was performed (Melan-A) at passage six. A mixing experiment with cultured melanocytes and fibroblasts was performed and cell population purity was determined by manual counts of positively staining cells in the FCBs and by flow cytometry. The FCB method of evaluating population purity was validated experimentally and by correlation with flow cytometry results. Preparation of a FCB followed by immunohistochemical staining is an easy and inexpensive way to confirm melanocytic lineage, estimate population purity, and provide a permanent archive of cultured cells.