Transmission of Megatrypanum Trypanosomes to Cervus dama by Tabanidae1

Four fallow deer, Cervus dama, became infected with Trypanosoma (Megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome’s efferent arm) make fragments after less heat than controls. If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the “baggage” (nucleus, granuloplasm)—i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.     

Minocycline attenuates depressive-like behaviors in mice treated with the low dose of intracerebroventricular streptozotocin; the role of mitochondrial function and neuroinflammation

Molecular Biology Reports - Neuroinflammation and mitochondrial dysfunction are suggested as mechanisms which are implicated in the pathophysiology of depression. Streptozotocin (STZ) is known to...     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

Effects of age on antibody affinity maturation

The elderly are more susceptible to infectious diseases. Mortality and morbidity from infections increase sharply over the age of 65 years. At the same time, the efficacy of vaccinations in the elderly is decreased. The elderly also have an increased incidence of cancer and inflammatory diseases. All the above indicate an age-related dysregulation of the immune system. Evidence suggests that the change in the humoral immune response with age is a qualitative rather than a quantitative one, i.e. it is the affinity and specificity of the antibody that changes, rather than the quantity of antibody produced. There are a number of possible causes of this failure, one of which is a defect in the mechanism of hypermutation of immunoglobulin genes. We have studied individual clonal responses within germinal centres of spleen and Peyer's patches in young and old patient groups. Our results indicate that there is no difference in the actual mechanism of hypermutation with age. There are, however, differences that are due either to a change in selection processes or to a change in the founder cells available for activation.     

Deriving quantitative constraints on T cell selection from data on the mature T cell repertoire

The T cell repertoire is shaped in the thymus through positive and negative selection. Thus, data about the mature repertoire may be used to infer information on how TCR generation and selection operate. Assuming that T cell selection is affinity driven, we derive the quantitative constraints that the parameters driving these processes must fulfill to account for the experimentally observed levels of alloreactivity, self MHC restriction and the frequency of cells recognizing a given foreign Ag. We find that affinity-driven selection is compatible with experimental estimates of these latter quantities only if 1) TCRs see more peptide residues than MHC polymorphic residues, 2) the majority of positively selected clones are deleted by negative selection, 3) between 1 and 3.6 clonal divisions occur on average in the thymus after completion of TCR rearrangement, and 4) selection is driven by 103-105 self peptides.     

High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution

Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10⁻². By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.     

Models for antigen receptor gene rearrangement: CDR3 length

Despite the various processing steps involved in V(D)J recombination, which could potentially introduce many biases in the length distribution of complementarity determining region 3 (CDR3) segments, the observed CDR3 length distributions for complete repertoires are very close to a normal-like distribution. This raises the question of whether this distribution is simply a result of the random steps included in the process of gene rearrangement, or has been optimized during evolution. We have addressed this issue by constructing a simulation of gene rearrangement, which takes into account the DNA modification steps included in the process, namely hairpin opening, nucleotide additions, and nucleotide deletions. We found that the near-Gaussian- shape of CDR3 length distribution can only be obtained under a relatively narrow set of parameter values, and thus our model suggests that specific biases govern the rearrangement process. In both B-cell receptor (BCR) heavy chain and T-cell receptor beta chain, we obtained a Gaussian distribution using identical parameters, despite the difference in the number and the lengths of the D segments. Hence our results suggest that these parameters most likely reflect the optimal conditions under which the rearrangement process occurs. We have subsequently used the insights gained in this study to estimate the probability of occurrence of two exactly identical BCRs over the course of a human lifetime. Whereas identical rearrangements of the heavy chain are highly unlikely to occur within one human lifetime, for the light chain we found that this probability is not negligible, and hence the light chain CDR3 alone cannot serve as an indicator of B-cell clonality.