Changes in proteasome expression and activity during differentiation of neuronal precursor NTera 2 clone D1 cells

The effect of differentiation of the human neuronal progenitor cell line NTera 2 clone D1 (NT2/D1) by retinoic acid on components of the proteasome system was studied. The chymotrypsin-like and peptidylglutamyl peptide bond hydrolyzing activities of the proteasome increased five weeks after retinoic acid, and following treatment with mitotic inhibitors returned to levels detected in non-differentiated cells. A selective induction of the MHC class II region encoded LMP7 and LMP2 proteasome subunits occurred during differentiation, whereas there were no changes in the expression of the constitutive LMP2 counterpart (delta-subunit) or the constitutive C2 subunit. Immunofluorescence revealed marked LMP7 accumulation in fully differentiated cells, with no changes in the labeling pattern of the constitutive proteasome antigens. The expression of the alpha-subunit of the PA28 proteasome activator was down-regulated in fully differentiated neurons, but was not correlated with changes in enzymatic activity. Changes in proteasome activity and composition may contribute to the processes leading to differentiation of human neurons in vitro and to the properties of fully differentiated neurons.     

Bone marrow angiogenesis and proliferation in B-cell chronic lymphocytic leukemia

OBJECTIVE: To assess angiogenesis and the proliferative activity of bone marrow in patients with chronic lymphocytic leukemia (CLL) in relation to the bone marrow infiltration pattern. STUDY DESIGN: Bone marrow samples were obtained by trephine biopsy from 46 patients with B-cell CLL (B-CLL). Infiltration pattern was diffuse in 20 patients and nondiffuse--i.e., nodular, interstitial or mixed--in the remaining 26 patients. Ten normal bone marrow samples were used as a control group. Studies were carried out by immunohistochemical staining of paraffin-embedded bone marrow samples. Angiogenesis was assessed in the zones of highest vascular density (hot spots), visualized by the expression of endothelial antigen CD34 and expressed as a number of microvessels per high-powerfield (hpf) (final magnification, 400x). Proliferative activity was estimated by the expression of nuclear protein Ki-67, cyclin A and mean number of nucleolar organizer regions (AgNORs). RESULTS: Microvessel density was higher in B-CLL marrow than in normal marrow (30.1 and 16.44 per hpf, respectively) and was higher in the diffuse than nondiffuse pattern (33.6 and 27.5 per hpf, respectively). B-CLL bone marrow also showed higher proliferative activity, as assessed by mean number of AgNORs, than did normal marrow (1.52 and 1.25, respectively) and a higher mean percentage of cyclin A-positive cells (7.5 and 6.8, respectively). In contrast, mean Ki-67 expression was similar in B-CLL and the control group. Mean AgNORs number, Ki-67 and cyclin A-positive cell percentage were significantly higher in B-CLL marrow with a diffuse as compared to nondiffuse involvement pattern (AgNORs, 1.75 and 1.35; cyclin A, 9.27% and 3.95%; Ki-67, 34.9% and 23.3%, respectively). CONCLUSION: Our results indicate enhancement of bone marrow angiogenesis in B-CLL and a relationship between microvessel density and the bone marrow infiltration pattern. The study points also to a possible relationship between the bone marrow infiltration pattern and proliferative activity of bone marrow cells.     

Effects of melatonin on luteinizing hormone secretion in anestrous ewes following dopamine and opiate receptor blockade

In the present investigation we have examined the ability of melatonin to modify the pulsatile LH secretion induced by treatment with a DA antagonist (sulpiride, SULP) or opioid antagonist (naloxone, NAL) in intact mid-anestrous ewes. The experimental design comprised the following treatments-in experiment 1: (1) intracerebroventricular (i.c.v.) infusion of vehicle (control I); (2) pretreatment with SULP (0.6 mg/kg subcutaneously) and then i.c.v. infusion of vehicle (SULP + veh); (3) pretreatment with SULP and then i.c.v. infusion of melatonin (SULP + MLT, 100 microg per 100 microl/h, total 400 microg). In experiment 2: (4) i.c.v. infusion of vehicle (control II); (5) i.c.v. infusion of NAL (NAL-alone, 100 microg per 100 microl/h, total 300 microg); (6) i.c.v. infusion of NAL in combination with MLT (NAL + MLT, 100 microg + 100 microg per 100 microl/h). All infusions were performed during the afternoon hours. Pretreatment with SULP induced a significant (P < 0.01) increase in LH pulse frequency, but not in mean LH concentration, compared with control I. In SULP + MLT-treated animals, the LH concentration was significantly (P < 0.01) higher during MLT infusion, but due to highly increased LH secretion in only one ewe. The significant changes in the SULP + MLT group occurred in LH pulse frequency. A few LH pulses were noted after melatonin administration compared with the number during the infusion (P < 0.05) and after vehicle infusion in the SULP + MLT group (P < 0.05). The i.c.v. infusion of NAL evoked a significant increase in the mean LH concentration (P < 0.001) and amplitude of LH pulses (P < 0.01) compared with these before the infusion. The enhanced secretion of LH was also maintained after i.c.v. infusion of NAL (P < 0.01) with a concomitant decrease in LH pulse frequency (P < 0.05). In NAL + MLT-treated ewes, mean plasma LH concentrations increased significantly during and after the infusion compared with that noted before ( P < 0.001). No difference in the amplitude of LH pulses was found in the NAL + MLT group, but this parameter was significantly higher in ewes during infusion of both drugs than during infusion of the vehicle (P < 0.01). The LH pulse frequency differed significantly (p < 0.05), increasing slightly during NAL + MLT administration and decreasing after the infusion. In conclusion, these results demonstrate that: (1) in mid-anestrous ewes EOPs, besides DA, are involved in the inhibition of the GnRH/LH axis; (2) brief administration of melatonin in long-photoperiod-inhibited ewes suppresses LH pulse frequency after the elimination of the inhibitory DA input, but seems to not affect LH release following opiate receptor blockade.     

A fragment of chloroplast DNA was transferred horizontally,probably from non-eudicots,to mitochondrial genome of <Emphasis Type="Italic">Phaseolus</Emphasis>

The mitochondrial genomes of some Phaseolus species contain a fragment of chloroplast trnA gene intron, named pvs-trnA for its location within the Phaseolus vulgaris sterility sequence (pvs). The purpose of this study was to determine the type of transfer (intracellular or horizontal) that gave rise to pvs-trnA. Using a PCR approach we could not find the respective portion of the trnA gene as a part of pvs outside the Phaseolus genus. However, a BLAST search revealed longer fragments of trnA present in the mitochondrial genomes of some Citrus species, Helianthus annuus and Zea mays. Basing on the identity or near-identity between these mitochondrial sequences and their chloroplast counterparts we concluded that they had relocated from chloroplasts to mitochondria via recent, independent, intracellular DNA transfers. In contrast, pvs-trnA displayed a relatively higher sequence divergence when compared with its chloroplast counterpart from Phaseolus vulgaris. Alignment of pvs-trnA with corresponding trnAfragments from 35 plant species as well as phylogenetic analysis revealed that pvs-trnA grouped with non-eudicot sequences and was well separated from all Fabalessequences. In conclusion, we propose that pvs-trnA arose via horizontal transfer of a trnA intron fragment from chloroplast of a non-eudicot plant to Phaseolus mitochondria. This is the first example of horizontal transfer of a chloroplast sequence to the mitochondrial genome in higher plants.     

Isolation, cloning and characterisation of motifs containing (GA/TC)n repeats isolated from vetch, Vicia bithynica

Microsatellites are widely distributed in plant genomes and comprise unstable regions that undergo mutational changes at rates much greater than that observed for non-repetitive sequences. They demonstrate intrinsic genetic instability, manifested as frequent length changes due to insertions or deletions of repeat units. Detailed analysis of 1600 clones containing genomic sequences of Vicia bithynica revealed the presence of microsatellite repeats in its genome. Based on the screening of a partial DNA library of plasmids, 13 clones harbouring (GA/TC)n tracts of various lengths of repeated motif were identified for further analysis of their internal sequence organization. Sequence analyses revealed the precise length, number of repeats, interruptions within tracts, as well as sequence composition flanking the repeat motifs. Representative plasmids containing different lengths of (GA/TC)n embedded in their original flanking sequence were used to investigate the genetic stability of the repeats. In the study presented herein, we employed a well characterised and tractable bacterial genetic system. Recultivations of Escherichia coli harbouring plasmids containing (GA/TC)n inserts demonstrated that the genetic instability of (GA/TC)n microsatellites depends highly on their length (number of repeats). These observations are in agreement with similar studies performed on repetitive sequences from humans and other organisms.     

Methods of embryo scoring in in vitro fertilization

It has been 25 years since the introduction of in vitro fertilization (IVF) for treatment of infertility. During this time very dynamic advances have taken place in all aspects of assisted reproductive technology (ART). The rapid improvement in embryological methods, especially these related to preimplantation embryo evaluation are of great importance. This article is a review of embryo classification systems utilized in ART programs. The most widely used scoring systems of zygotes and embryos (including blastocysts) are described. Additionally, the advantages of advanced embryo classifications in relation to ART success rates are presented.     

Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.     

Signaling pathways of the F11 receptor (F11R; a.k.a. JAM-1, JAM-A) in human platelets: F11R dimerization, phosphorylation and complex formation with the integrin GPIIIa

The F11 receptor (F11R) (a.k.a. Junctional Adhesion Molecule, JAM) was first identified in human platelets as a 32/35 kDa protein duplex that serves as receptor for a functional monoclonal antibody that activates platelets. We have sequenced and cloned the F11R and determined that it is a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules. The signaling pathways involved in F11R-induced platelet activation were examined in this investigation. The binding of M.Ab.F11 to the platelet F11R resulted in granule secretion and aggregation. These processes were found to be dependent on the crosslinking of F11R with the Fc gammaRII by M.Ab.F11. This crosslinking induced actin filament assembly with the conversion of discoidal platelets to activated shapes, leading to the formation of platelet aggregates. We demonstrate that platelet secretion and aggregation through the F11R involves actin filament assembly that is dependent on phosphoinositide-3 kinase activation, and inhibitable by wortmannin. Furthermore, such activation results in an increase in the level of free intracellular calcium, phosphorylation of the 32 and 35 kDa forms of the F11R, F11R dimerization coincident with a decrease in monomeric F11R, and association of the F11R with the integrin GPIIIa and with CD9. On the other hand, F11R-mediated events resulting from the binding of platelets to an immobilized surface of M.Ab.F11 lead to platelet adhesion and spreading through the development of filopodia and lammelipodia. These adhesive processes are induced directly by interaction of M.Ab.F11 with the platelet F11R and are not dependent on the Fc gammaRII. We also report here that the stimulation of the F11R in the presence of nonaggregating (subthreshold) concentrations of the physiological agonists thrombin and collagen, results in supersensitivity of platelets to natural agonists by a F11R-mediated process independent of the Fc gammaRII. The delineation of the two separate F11R-mediated pathways is anticipated to reveal significant information on the role of this cell adhesion molecule in platelet adhesion, aggregation and secretion, and F11R-dependent potentiation of agonist-induced platelet aggregation. The participation of F11R in the formation and growth of platelet aggregates and plaques in cardiovascular disorders, resulting in enhanced platelet adhesiveness and hyperaggregability, may serve in the generation of novel therapies in the treatment of inflammatory thrombosis, heart attack and stroke, and other cardiovascular disorders.