Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Multiple mechanisms of target cell disintegration are employed in cytotoxicity reactions mediated by human natural killer cells

The rate of disintegration of target cells subsequent to lytic programming by human peripheral blood natural killer (NK) cells was investigated using a quantitative calcium pulse technique. The rate of this initial calcium-independent target cell disintegration was indicative of a first-order decay process for programmed target cells with a calculated half-life of less than 3 min. This initial, rapid disintegration phase was independent of the overall cytotoxic activity of the lymphocyte preparation tested. Moreover, initial rates of target cell disintegration were comparable for target cell lines that exhibit up to 6-fold differences in overall susceptibility to natural cytotoxicity. In these studies we also consistently observed very slow, calcium-independent disintegration of additional target cells following apparent completion of the rapid disintegration process. Using a 51Cr release assay and K-562 target cells, the kinetics of this slow disintegration process were examined and found to be similar for donors exhibiting up to a 2-fold difference in overall cytotoxic activity and independent of the concentration of programed target cells. Whereas the initial rapid disintegration mechanism was independent of temperature over the range of 10-37 degrees C, the slow disintegration mechanism exhibited a direct dependence on the incubation temperature. Furthermore, we observed that supernatants obtained after the termination of lytic programing by ethylene diaminetetraacetic acid could effect the slow lysis of fresh NK-susceptible target cell lines. These results support the utilization of at least two distinct mechanisms for target cell lysis by human NK cells.     

Congenic Strains Confirm the Pleiotropic Effect of Chromosome 4 QTL on Mouse Femoral Geometry and Biomechanical Performance

A pleiotropic quantitative trait locus (QTL) for bone geometry and mechanical performance in mice was mapped to distal chromosome 4 via an intercross of recombinant congenic mice HcB-8 and HcB-23. To study the QTL in isolation, we have generated C3H.B10-(rs6355453-rs13478087) (C.B.4.3) and C3H.B10-(rs6369860-D4Mit170) (C.B.4.2) congenic strains that harbor ~20 Mb and ~3 Mb, respectively, of chromosome 4 overlapping segments from C57BL/10ScSnA (B10) within the locus on a C3H/DiSnA (C3H) background. Using 3-point bend testing and standard beam equations, we phenotyped these mice for femoral mid-diaphyseal geometry and biomechanical performance. We analyzed the results via 2-way ANOVA, using sex and genotype as factors. In the C.B.4.3 strain, we found that homozygous B10/B10 male mice had smaller cross sectional area (CSA) and reduced total displacement than homozygous C3H/C3H mice. Sex by genotype interaction was also observed for maximum load and stiffness for C3H/C3H and B10/B10 mice, respectively. In C.B.4.2 strain, we found that homozygous B10/B10 mice had lower total displacement, post-yield displacement (PYD), stiffness, yield load and maximum load than mice harboring C3H allele. Sex by genotype interaction was observed in B10/B10 mice for perimeter, outer minor axis (OMA) and CSA. There were no significant differences in tissue level mechanical performance, which suggest that the QTL acts primarily on circumferential bone size. These data confirm the prior QTL mapping data and support other work demonstrating the importance of chromosome 4 QTL on bone modeling and bone responses to mechanical loading.     

Characterization of effector-target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry.

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one-fourth of conjugates between K-562 cells and human PBL that had been cultured with or without IL-2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector-target conjugates are incubated at 37 degrees C in the absence of calcium--thereby precluding lysis--the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.     

Assessment of the Correlation Between Two Defining Criteria for Bidirectional Isthmic Block in the Ablation of Typical Atrial Flutter

Background

A complete, bidirectional conduction block in the cavotricuspid isthmus (CTI) represents the end-point of the typical atrial flutter ablation. We investigated the correlation between two criteria for successful ablation, one based on the atrial bipolar electrogram morphology before and after complete CTI conduction block, compared to the standard criteria of differential pacing and reversal in the right atrial depolarization sequence during coronary sinus (CS) pacing.

Method

We conducted a retrospective study in 111 patients (81 males, average age 62±10 years) who underwent an atrial flutter ablation during September 2007 - July 2009 in the Cardiology - Rehabilitation Hospital, UMF Cluj-Napoca. We assessed the presence of a bidirectional block at the end of the procedure using the standard criteria. We then analyzed the morphology of the bipolar atrial electrograms adjacent to the ablation line, before and after CTI conduction block.

Results

A change from a qRs morphology to a rSr'' morphology when pacing from the coronary sinus and from a rsr'' morphology to a QRS morphology when pacing from the low-lateral right atrium was associated with a CTI conduction block. Sensitivity (Se), specificity(Sp), positive predictive value (PPV), negative predictive value (NPV) were 96%, 89%, 99% and 67% respectively.

Conclusion

Our study suggests that the analysis of the atrial bipolar electrogram next to the ablation line before and after CTI ablation may be used as a reliable criterion to validate CTI conduction block due to its high sensitivity, specificity and positive predictive value.     

Synchronisation of oestrus in dairy cows using prostaglandin F2alpha,gonadotrophin-releasing hormone,and oestradiol cypionate

An oestrous synchronisation protocol was developed for use in lactating dairy cows using PGF(2alpha), GnRH, and oestradiol cypionate (ECP). In experiment 1, lactating dairy cows received two injections of PGF(2alpha) (on days 0 and 11) (PP; n=10) or two injections of PGF(2alpha) (days 0 and 11) and 100 microg of GnRH on day 3 (PGP; n=10). In experiment 2, cows were treated with PGP (n=7), or PGP and 1 mg of ECP at the same time (PGPE(0); n=7) or 1 day after the second PGF(2alpha) injection (PGPE(1); n=7). In experiment 3, 101 lactating dairy cows in a commercial herd were assigned to one of three treatments; PP, PGP, or PGPE(1). Follicular growth was measured by ultrasound in experiments 1 and 2. Every cow (experiments 1, 2, and 3) was blood sampled at selected intervals for progesterone and oestradiol assays and inseminated at oestrus. In experiment 1, a higher percentage of GnRH-treated cows ovulated after the first PGF(2alpha) injection (90% versus 50%; P<0.05). The GnRH-treated cows tended to have a larger dominant follicle present at the time of the second PGF(2alpha) injection (16.5+/-0.5 mm versus 15.0+/-0.7 mm; P<0.10). The percentage of cows that ovulated after the second PGF(2alpha) injection was similar (60%). In experiment 2, cows treated with ECP had higher peak preovulatory concentrations of oestradiol in plasma (6.99+/-0.63 versus 3.63+/-0.63; P<0.01) following the second PGF(2alpha) injection and a higher percentage ovulated (86% versus 43%; P<0.05). A higher percentage of PGPE(1)-treated cows in experiment 3 were observed in standing oestrus and ovulated after the second PGF(2alpha) injection (standing oestrus, 26.4, 34.3, and 62.6%, P<0.01; ovulated, 56, 63, and 78%, P<0.05; PP, PGP, and PGPE(1), respectively). In conclusion, the PGP protocol increased the number of cows that ovulated after the first PGF(2alpha) injection and produced a more mature dominant follicle at the time of the second PGF(2alpha) injection. Adding ECP to PGP (PGPE(1)) enhanced the expression of oestrus and increased ovulation percentage. The combination of PGP and ECP is potentially a new method to routinely synchronise oestrus and ovulation in dairy cows.     

Inverting character of alpha-glucuronidase A from Aspergillus tubingensis

Alpha-glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4-O-methyl-D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue. Reduced aldotetrauronic acid, 4-O-methyl-D-glucuronosyl-alpha-1,2-D-xylopyranosyl-beta-1,4-xylopyranosyl-beta-1,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme. The configuration of the liberated MeGlcA was followed in a D(2)O reaction mixture by (1)H-NMR spectroscopy. It was unambiguously established that MeGlcA was released from the substrate as its beta-anomer from which the alpha-anomer was formed on mutarotation. This result represents the first experimental evidence for the inverting character of a microbial alpha-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139).