Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: detection of translocations in embryos by fluorescence in situ hybridization

Robertsonian translocation rob(16;20) in the heterozygous state was discovered in a subfertile bull of the Czech Siemmental breed. A chromosomal analysis of its family has shown that this dicentric fusion is formed de novo. The present experiments were designed to detect rob(16;20) and determine its incidence for in vitro produced embryos, using fluorescence in situ hybridization (FISH) and rob(1;29) as a detection control. To characterize semen of both bulls with the rob translocations, their sperm was examined for DNA integrity by the sperm chromatin structure assay (SCSA). For in vitro fertilization of oocytes, spermatozoa from a rob(16;20) bull carrier (Czech Siemmental breed) and those from a rob(1;29) bull carrier (Charolais breed) were used. Embryos at the 6- to 8-cell stage were cultured in a vinblastine-supplemented medium for 17 h, and embryos at the blastocyst stage were cultured in a colcemide-supplemented medium for 4 h. The embryos were fixed in methanol and acetic acid with Tween-20. Painting probes for chromosomes 16 (Spectrum Green) and 20 (Spectrum Orange) and chromosomes 1 (Spectrum Orange) and 29 (Spectrum Green) were simultaneously hybridized. In the embryos derived from the rob(16;20) bull, the presence of this translocation was not detected. On the other hand, 52.5% of the embryos derived from the rob(1;29) bull were translocation carriers. There was no significant difference in the frequency of this translocation between early and advanced embryos.     

Mechanistic study of proton transfer and hysteresis in catalytic antibody 16E7 by site-directed mutagenesis and homology modeling

Antibody 16E7 catalyzes the carbon protonation of enol ether 2 to hemiacetal 3, and the carbon deprotonation of benzisoxazole 7 to phenol 8. This antibody shows an extreme case of hysteresis, requiring several hours to reach full activity. Antibody 16E7 was expressed as recombinant chimeric Fab in Escherichia coli. A model for the three-dimensional structure was produced by homology modeling and used for a docking procedure to obtain models for antibody-ligand complexes. Site-direct mutagenesis of GluL39, identified as a possible catalytic residue by the model, to either glutamine or alanine abolished catalysis, showing that both the protonation reaction of enol ether 2 and the deprotonation of benzisoxazole 7 are promoted by the same residue. The model furthermore suggested that substrate access to the catalytic site might be hindered by a flexible HCDR3 loop held in closed position by a hydrogen bond between SerH99 and GluL39, which could explain the observed hysteresis effect. In agreement with this model, mutagenesis of SerH99 to alanine, or deletion of this residue, was found to reduce hysteresis by approximately 50%.     

Bystander effect of normal fibroblasts for macrophages co-cultured with susceptible transformed target cells

Macrophages attack and kill pathologically changed, transformed and tumor cells. However, in some cases they may also support tumor growth, modulate the action of anticancer drugs, and even facilitate the development of drug resistance in tumor cells. Here we present data that bystander fibroblasts NIH3T3 were not only resistant to murine macrophages J774.2 but also blocked their killing action towards murine transformed fibroblasts L929. Macrophages were isolated from mixed cultures by means of CD11b specific immunomagnetic beads, and changes induced by their former co-culturing were studied using DNA microarray technology and other tests. An expression of candidate genes coding for cytokines and for signal transduction pathway proteins was estimated in macrophages in different variants of their co-culture with target cells. Changes in expression of mRNA for interleukin 1beta, NFkappaB, IkappaBalpha, gadd45, and CD5 were detected as the most prominent in the macrophages co-cultured with the transformed cells. Bystander NIH3T3 fibroblasts abolished these changes in the macrophages J774.2, and the level of expression of the above mentioned genes was close to the level seen in the macrophages which did not exert cytotoxicity towards the target fibroblasts. Potential implications and research perspectives of using the macrophage-target cell co-cultures with different bystander cellular partners are discussed.     

The gene ENHANCER OF PINOID controls cotyledon development in the Arabidopsis embryo

During Arabidopsis embryo development, cotyledon primordia are generated at transition stage from precursor cells that are not derived from the embryonic shoot apical meristem (SAM). To date, it is not known which genes specifically instruct these precursor cells to elaborate cotyledons, nor is the role of auxin in cotyledon development clear. In laterne mutants, the cotyledons are precisely deleted, yet the hypocotyl and root are unaffected. The laterne phenotype is caused by a combination of two mutations: one in the PINOID (PID) gene and another mutation in a novel locus designated ENHANCER OF PINOID (ENP). The expression domains of shoot apex organising genes such as SHOOT MERISTEMLESS (STM) extend along the entire apical region of laterne embryos. However, analysis of pid enp stm triple mutants shows that ectopic activity of STM does not appear to cause cotyledon obliteration. This is exclusively caused by enp in concert with pid. In pinoid embryos, reversal of polarity of the PIN1 auxin transport facilitator in the apex is only occasional, explaining irregular auxin maxima in the cotyledon tips. By contrast, polarity of PIN1:GFP is completely reversed to basal position in the epidermal layer of the laterne embryo. Consequently auxin, which is believed to be essential for organ formation, fails to accumulate in the apex. This strongly suggests that ENP specifically regulates cotyledon development through control of PIN1 polarity in concert with PID.     

Evaluation of the effect of GHRH(1-44)NH2 on the secretion of interleukin-2 (IL-2) and soluble IL-2 receptor alpha (sIL-2Ralpha) from human peripheral blood mononuclear cells in vitro

OBJECTIVE: The bidirectional communication between the neuroendocrine and the immune systems is now a subject of an intensive investigation. Somatoliberin (GHRH) is a hypothalamic hormone that enhances the synthesis and the release of growth hormone (GH) from the anterior pituitary cells. Few recent reports demonstrate also role of the neuropeptide in the regulation of the immune system function. AIM: The aim of the study was to examine the influence of GHRH on IL -2 as well as sIL -2Ralpha secretion from mitogen-stimulated peripheral blood mononuclear cells. MATERIAL AND METHOD: Mononuclear cells (PBMC) were isolated from the peripheral blood of healthy adults according to the technique described by B?yum. Cells, cultured 24 hours at 37 degrees C in humidified atmosphere of 95% air and 5% CO2, were stimulated with phytohemaglutinin (PHA; 10 microg/ml), and then GHRH(1-44)NH2 at the final concentrations 10(-12), 10(-10), 10(-8) and 10(-6) M was added to appropriate wells. ELISA kits were used to measure IL-2 and sIL-2Ralpha concentrations in the supernatants of cultured cells. Comparisons between tested groups were made by U Mann-Whitney test. The differences were considered significant if p < 0.05. RESULTS: GHRH stimulated the secretion of IL -2 into the supernatants acting significantly at the concentration of 10(-12) M (p < 0.001). Moreover, GHRH at concentrations 10(-10) M and 10(-8) M significantly increased the secretion of sIL-2Ralpha as well (p < 0.001). Strong positive correlation between tested GHRH concentrations and sIL-2Ralpha levels in the supernatants was demonstrated (r = 0.8664; p <0.001). CONCLUSIONS: The results demonstrate the potential involvement of GHRH in the regulation of T lymphocytes secretory function.     

Origin of the sequence-dependent polyproline II structure in unfolded peptides

Recent studies have indicated that the unfolded states of polypeptides contain a substantial amount of polyproline type II (P(II)) structure. This energetically and structurally preorganized state may contribute to the reduction of the folding search, as well as to the recognition of intrinsically unstructured proteins and polyproline ligands. Using Monte Carlo simulations of natively unfolded peptides in the presence of explicit aqueous solvation, we observe that residue-specific P(II) conformational propensity is the result of the modulation of polypeptide backbone hydration by a proximal side-chain. Such a mechanism may be unique among those that contribute to the modulation of secondary structures in proteins. The calculated conformational propensities should prove useful for the development of a configurational P(II) scale necessary for the prediction and design of natural-like polypeptides.     

Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes

The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a beta-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute beta-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.     

A major histocompatibility complex-peptide-restricted antibody and t cell receptor molecules recognize their target by distinct binding modes: crystal structure of human leukocyte antigen (HLA)-A1-MAGE-A1 in complex with FAB-HYB3

Antibodies with T cell receptor-like specificity possess a considerable diagnostic and therapeutic potential, but the structural basis of the interaction between an antibody and an histocompatibility antigen has so far not been determined. We present here the crystal structure (at 2.15 A resolution) of the recombinant, affinity-matured human antibody fragment Fab-Hyb3 bound to the tumor-associated human leukocyte antigen (HLA)/peptide complex HLA-A1.MAGE-A1. Fab-Hyb3 employs a diagonal docking mode resembling that of T cell receptors. However, other than these natural ligands, the antibody uses only four of its six complementarity-determining regions for direct interactions with the target. It recognizes the C-terminal half of the MAGE-A1 peptide, the HLA-A1 alpha1-helix, and N-terminal residues of the alpha2-helix, accompanied by a large tilting angle between the two types of molecules within the complex. Interestingly, only a single hydrogen bond between a peptide side chain and Fab-Hyb3 contributes to the interaction, but large buried surface areas with pronounced shape complementarity assure high affinity and specificity for MAGE-A1. The HLA-A1.MAGE-A1.antibody structure is discussed in comparison with those of natural ligands recognizing HLA.peptide complexes.     

gamma-Glutamyl dipeptides in Petiveria alliacea

Three gamma-glutamyl dipeptides have been isolated from Petiveria alliacea L. roots. These dipeptides include (S(C2)R(C7))-gamma-glutamyl-S-benzylcysteine together with two diastereomeric sulfoxides, namely (S(C2)R(C7)R(S))- and (S(C2)R(C7)R(S))-gamma-glutamyl-S-benzylcysteine S-oxides (gamma-glutamyl-petiveriins A and B, respectively). Their structures and absolute configurations have been determined by NMR, MALDI-HRMS, IR and CD spectroscopy, and confirmed by comparison with authentic compounds obtained by synthesis.     

A novel imidazole nucleoside containing a diaminodihydro-S-triazine as a substituent: inhibitory activity against the West Nile virus NTPase/helicase

The attempted synthesis of a ring-expanded guanosine (1) containing the imidazo[4,5-e][1,3]diazepine ring system by condensation of 1-(2'-deoxy-beta-D-erythropentofuranosyl)-4-ethoxycarbonylimidazole-5-carbaldehyde (2) with guanidine resulted in the formation of an unexpected product, 1-(2'-deoxy-beta-D-erythropentofuranosyl)-5-(2, 4-diamino-3, 6-dihydro-1,3, 5-triazin-6-yl)imidazole-4-carboxamide (7). The structure as well as the pathway of formation of 7 was corroborated by isolation of the intermediate, followed by its conversion to the product. Nucleoside 7 showed promising in vitro anti-helicase activity against the West Nile virus NTPase/helicase with an IC50 of 3-10 microg/mL.