Erythroid promoter confines FGF2 expression to the marrow after hematopoietic stem cell gene therapy and leads to enhanced endosteal bone formation

Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1(+) hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1(+) cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation.     

Perturbation of pulmonary immune functions by carbon nanotubes and susceptibility to microbial infection

Occupational and environmental pulmonary exposure to carbon nanotubes (CNT) is considered to be a health risk with a very low threshold of tolerance as determined by the United States Center for Disease Control. Immortalized airway epithelial cells exposed to CNTs show a diverse range of effects including reduced viability, impaired proliferation, and elevated reactive oxygen species generation. Additionally, CNTs inhibit internalization of targets in multiple macrophage cell lines. Mice and rats exposed to CNTs often develop pulmonary granulomas and fibrosis. Furthermore, CNTs have immunomodulatory properties in these animal models. CNTs themselves are proinflammatory and can exacerbate the allergic response. However, CNTs may also be immunosuppressive, both locally and systemically. Studies that examined the relationship of CNT exposure prior to pulmonary infection have reached different conclusions. In some cases, pre-exposure either had no effect or enhanced clearance of infections while other studies showed CNTs inhibited clearance. Interestingly, most studies exploring this relationship use pathogens which are not considered primary pulmonary pathogens. Moreover, harmony across studies is difficult as different types of CNTs have dissimilar biological effects. We used Pseudomonas aeruginosa as model pathogen to study how helical multi-walled carbon nanotubes (HCNTs) affected internalization and clearance of the pulmonary pathogen. The results showed that, although HCNTs can inhibit internalization through multiple processes, bacterial clearance was not altered, which was attributed to an enhanced inflammatory response caused by pre-exposure to HCNTs. We compare and contrast our findings in relation to other studies to gauge the modulation of pulmonary immune response by CNTs.     

Interaction of acyl coenzyme A substrates and analogues with pig kidney medium-chain acyl-coA dehydrogenase

Several alkylthio coenzyme A (CoA) derivatives (from ethyl- to hexadecyl-SCoA) have been synthesized to probe the substrate binding site in the flavoprotein medium-chain acyl-CoA dehydrogenase from pig kidney. All bind to apparently equivalent sites with a stoichiometry of four per tetramer. A plot of log Kd vs: hydrocarbon chain length is linear from 2 to 16 carbons with a free energy of binding of 390 cal/methylene group. These data suggest an acyl-binding site of moderate hydrophobicity and imply that the observed substrate specificity of the medium-chain dehydrogenase is not achieved simply by the length of the hydrocarbon binding pocket. Extrapolation of the graph to zero chain length predicts a Kd of 1 mM for the CoA moiety. The difference between this value and the experimentally determined value of 206 microM may be attributed to a contribution from the ionization of the sulfhydryl group in CoASH. The interaction of several eight-carbon intermediates of beta-oxidation (trans-2- and trans-3-octenoyl-CoA and L-3-hydroxy- and 3-ketooctanoyl-CoA) with the dehydrogenase has also been studied. All but the L-3-OH derivative bind tightly to the enzyme (with Kd values in the 50-90 nM range) and are very effective inhibitors of the dehydrogenation of octanoyl-CoA. The trans-3-enoyl analogue produces an immediate, intense, long-wavelength band (lambda max = 820 nm), which probably represents a charge-transfer interaction between the delocalized alpha-carbanion donor and oxidized flavin as the acceptor. The L-3-OH analogue is a reductant of the flavin, yielding 3-ketooctanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunomodulatory compounds from Pestalotiopsis leucothës, an endophytic fungus from Tripterygium wilfordii

The immunomodulatory effects of three compounds designated BS, GS, and YS produced by Pestalotiopsis leucothës, an endophytic fungus isolated from Tripterygium wilfordii, were evaluated. The 50% inhibition concentration (IC₅₀) value of BS in the proliferative assay with various stimulating agents such as phytohemagglutinin-M (PHA-M), phorbol myristate acetate (PMA)/ionomycin, mixed lymphocyte reaction (MLR) and poke weed mitogen (PWM) was 0.35, 1.6, 0.8 and 5.4 μg/ml, respectively. In addition, BS significantly inhibited the production of cytokines such as interleukin (IL)-1β, IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, by peripheral blood mononuclear cells (PBMNC) and soluble IL-2 receptor expression at concentrations greater than 1 μg/ml. Inhibition of PHA stimulated PBMNC proliferation and IL-2 and sIL-2R production by BS indicates that it is a T-cell specific immunosuppressant. However, BS also moderately inhibited immunoglobulin (Ig) G and M at concentrations greater than 1 μg/ml suggesting that it also has B cell immunosuppressive effects. YS was 10% less active than BS in all assay systems. In contrast, GS exhibited both suppression and enhancement of PBMNC proliferation in the presence of various stimulants. However, GS inhibited PWM stimulated PBMNC proliferation and IL-4 and IgG and IgM production at concentrations above 1 μg/ml. All three fungal compounds altered the percentage of T-lymphocyte subpopulations only at high concentrations. Cell viability was not affected at the immunosuppressive concentrations of these compounds. In conclusion, work from our laboratory has identified three potentially potent immunomodulatory compounds from P. leucothës. These compounds have variable effects on T- and B-cells and monocytes. They may partially explain the immunosuppressive activity of T. wilfordii. In addition, they may represent a new source of immunomodulatory compounds for the treatment of human immune mediated diseases.     

Increased efflux of glutathione conjugate in acutely diabetic cardiomyocytes

In diabetes, cell death and resultant cardiomyopathy have been linked to oxidative stress and depletion of antioxidants like glutathione (GSH). Although the de novo synthesis and recycling of GSH have been extensively studied in the chronically diabetic heart, their contribution in modulating cardiac oxidative stress in acute diabetes has been largely ignored. Additionally, the possible contribution of cellular efflux in regulating GSH levels during diabetes is unknown. We used streptozotocin to make Wistar rats acutely diabetic and after 4 days examined the different processes that regulate cardiac GSH. Reduction in myocyte GSH in diabetic rats was accompanied by increased oxidative stress, excessive reactive oxygen species, and an elevated apoptotic cell death. The effect on GSH was not associated with any change in either synthesis or recycling, as both gamma-glutamylcysteine synthetase gene expression (responsible for bio syn thesis) and glutathione reductase activity (involved with GSH recycling) remained unchanged. However, gene expression of multidrug resistance protein 1, a transporter implicated in effluxing GSH during oxidative stress, was elevated. GSH conjugate efflux mediated by multidrug resistance protein 1 also increased in diabetic cardiomyocytes, an effect that was blocked using MK-571, a specific inhibitor of this transporter. As MK-571 also decreased oxidative stress in diabetic cardiomyocytes, an important role can be proposed for this transporter in GSH and reactive oxygen species homeostasis in the acutely diabetic heart.     

Protein tyrosine kinase-dependent modulation of voltage-dependent potassium channels by genistein in rat cardiac ventricular myocytes

Effects of the isoflavone protein tyrosine kinase (PTK) inhibitor genistein on voltage-dependent K(+) currents, i.e., transient outward K(+) current (I(to)), sustained K(+) current (I(ss)), and inward rectifier K(+) current (I(K1)) were studied in rat cardiac ventricular myocytes. It was found that I(to) was reversibly inhibited by genistein in a concentration-dependent manner (IC(50)=28.1 microM), while I(ss) was suppressed by genistein with IC(50) of 18.5 microM. In addition, I(K1) (at -50 mV) was significantly decreased by 36.3+/-4.4% with 25 microM genistein. The inhibition of I(to), I(ss), and I(K1) by genistein was significantly reversed by the application of the protein tyrosine phosphatase inhibitor sodium orthovanadate (1 mM). However, I(to), I(ss), and I(K1) were not affected by the non-isoflavone PTK inhibitor tyrphostin A23 (100 microM) and PP2 (1 microM). These results indicate that activation of I(to), I(ss), and I(K1) channels is modulated by genistein-sensitive PTKs in rat ventricular myocytes.     

The effect of age at introduction and number of milk-portions on the behaviour of group housed dairy calves fed by a computer controlled milk feeder

This study is an investigation of the effect of age at introduction (6 days versus 14 days) and number of milk-portions (four milk-portions a day versus eight milk-portions a day) on integration into a large dynamic group of calves, fed by a computer controlled milk feeder. Forty calves (Jerseys, Danish Reds and Holstein-Friesians) were allocated equally to the two age conditions (A6 and A14) and the two milk-portion conditions (M4 and M8) in a 2 × 2 factorial design, according to sex and breed, and introduced into the group in pairs (one A6 and one A14). The behaviour of each pair was video-recorded for 8 h from 8 a.m. to 4 p.m. and 1 h from 1 to 2 a.m. on days 1, 8 and 15 after introduction.

The A6 calves performed less licking and sniffing, changed posture more often and tended to spend less time standing than the A14 calves. In the course of time A14 calves lay closer to other calves than the A6 calves.

The M8 calves, which were offered the same daily milk allowance as the M4 calves stood for a longer time in the milk feeding station and the M8 calves also sucked the empty teat more frequently than the M4 calves. Finally, the M8 calves initiated more social play behaviour than the M4 calves. On the first day after introduction the M8 calves lay closer to other calves than the M4 calves.

The results suggest that calves integrate better into a group when introduced at the age of 14 days than at the age of 6 days. Distributing the milk into eight daily milk-portions, rather than four milk-portions in the first period after introduction, increased milk feeder occupancy, which may facilitate learning to use the milk feeder. Surprisingly, more milk-portions also stimulated play behaviour, which is suggested to be due to M8 calves encountering more calves in the milk feeder area.     

Maintaining connexin43 gap junctional communication in v-Src cells does not alter growth properties associated with the transformed phenotype

Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine "normal" cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.