Synergistic activation of plasma membrane H+– ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin

The regulation of the H⁺-ATPase of plasma membrane is a crucial point in the integration of transport processes at this membrane. In this work the regulation of H⁺-ATPase activity induced by changes in turgor pressure was investigated and compared with the stimulating effect of fusicoccin (FC). The exposure of cultured cells of Arabidopsis thaliana L. (ecotype Landsberg 310–14-2) to media containing mannitol (0. 15 or 0. 3 M ) or polyethylene glycol 6000 (PEG) (15. 6% or 22% w/v) resulted in a decrease in the turgor pressure of the cells and in a strong stimulation of H⁺ extrusion in the incubation medium. The osmotica-induced H⁺ extrusion was (1) inhibited by the inhibitor of plasma membrane H⁺-ATPase, erythrosin B (EB), (2) dependent on the external K⁺ concentration, (3) associated with a net K⁺ influx, and (4) lead to an increase of cellular malate content. These results show that the reduction of external osmotic potential stimulates the activity of plasma membrane H⁺-ATPase
The effect of mannitol was only partially inhibited by treatments with cycloheximide (CH) and cordycepin, which block protein and mRNA synthesis, respectively. All the effects of osmotica were qualitatively and quantitatively similar to those induced by 5 μ M FC. However, when FC and mannitol (or PEG) were fed together, their effects on H⁺ extrusion appeared synergistic, irrespective of whether FC was present at suboptimal or optimal concentrations. This behaviour suggests that the modes of action of FC and of the osmotica on H⁺-ATPase activity differ at least in some step(s)     

Thermosensitive hybrid hyaluronan/p(HPMAm-lac)-PEG hydrogels enhance cartilage regeneration in a mouse model of osteoarthritis

Osteoarthritis (OA), due to cartilage degeneration, is one of the leading causes of disability worldwide. Currently, there are not efficacious therapies to reverse cartilage degeneration. In this study we evaluated the potential of hybrid hydrogels, composed of a biodegradable and thermosensitive triblock copolymer cross-linked via Michael addition to thiolated hyaluronic acid, in contrasting inflammatory processes underlying OA. Hydrogels composed of different w/w % concentrations of hyaluronan were investigated for their degradation behavior and capacity to release the polysaccharide in a sustained fashion. It was found that hyaluronic acid was controllably released during network degradation with a zero-order release kinetics, and the release rate depended on cross-link density and degradation kinetics of the hydrogels. When locally administered in vivo in an OA mouse model, the hydrogels demonstrated the ability to restore, to some extent, bone remineralization, proteoglycan production, levels of Sox-9 and Runx-2. Furthermore, the downregulation of proinflammatory mediators, such as TNF-α, NFkB, and RANKL and proinflammatory cytokines was observed. In summary, the investigated hydrogel technology represents an ideal candidate for the potential encapsulation and release of drugs relevant in the field of OA. In this context, the hydrogel matrix could act in synergy with the drug, in reversing phenomena of inflammation, cartilage disruption, and bone demineralization associated with OA.     

P62 deficiency shifts mesenchymal/stromal stem cell commitment toward adipogenesis and disrupts bone marrow homeostasis in aged mice

With advancing age have been observed bone and bone marrow phenotypic alterations due to the impaired bone tissue homeostatic features, involving bone remodeling, and bone marrow niche ontogeny. The complex “inflamm-aging” pathological scenario that culminates with osteopenia and mesenchymal/stromal and hematopoietic stem cell commitment breakdown, is controlled by cellular and molecular intramural components comprising adapter proteins such as the sequestosome 1 (p62/SQSTM1). p62, a “multiway function” protein, has been reported as an effective anti-inflammatory, bone-building factor. In this view, we considered for the first time the involvement of p62 in aging bone and bone marrow of 1 year and 2 years p62^−/− mice. Interestingly, p62 deficiency provoked accelerated osteopenia and impaired niche operational activities within the bone marrow. The above findings unearthed the importance of p62 in mesenchymal stem cell maintenance/differentiation schedule in old animals and provide, at least in part, a mechanistic scenario of p62 action.     

Secretome compartment is a valuable source of biomarkers for cancer-relevant pathways

In principle, targeted therapies have optimal activity against a specific subset of tumors that depend upon the targeted molecule or pathway for growth, survival, or metastasis. Consequently, it is important in drug development and clinical practice to have predictive biomarkers that can reliably identify patients who will benefit from a given therapy. We analyzed tumor cell-line secretomes (conditioned cell media) to look for predictive biomarkers; secretomes represent a potential source for potential biomarkers that are expressed in intracellular signaling and therefore may reflect changes induced by targeted therapy. Using Gene Ontology, we classified by function the secretome proteins of 12 tumor cell lines of different histotypes. Representations and hierarchical relationships among the functional groups differed among the cell lines. Using bioinformatics tools, we identified proteins involved in intracellular signaling pathways. For example, we found that secretome proteins related to TGF-beta signaling in thyroid cancer cells, such as vasorin, CD109, and βIG-H3 (TGFBI), were sensitive to RPI-1 and dasatinib treatments, which have been previously demonstrated to be effective in blocking cell proliferation. The secretome may be a valuable source of potential biomarkers for detecting cancer and measuring the effectiveness of cancer therapies.     

Transfer of chirality by dithiophosphate ligands and chiral discrimination in the stereoselective formation of square-planar Ni(II) complexes

In the formation reaction of Ni(2+) with the chiral racemic ligand, (R)(R)bdtp(-)/(S)(S)bdtp(-), bdtp(-) = [SSPOCH)CH(3))CH(CH(3))O](-), cyclo- O,O'-[1,2-dimethylethylene] dithiophosphato ion, the meso-complex Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)-bdtp] is stereoselectively produced. The meso-complex was compared with the enantiopure crystals of (+)(589)Ni[(R)(R)(lambda)bdtp](2) or (-)(589)Ni[(S)(S)(delta)bdtp](2), as well as racemic crystals, rac-(+/-)Ni[bdtp](2), which were prepared from the solution containing the two enantiomers in a 1:1 ratio. Dissociation constants in solutions indicate different stability of the meso and enantiopure complexes depending on the solvent, whereas a more efficient crystal packing, weak H-bonding, and nonbonding interactions contribute to stabilization of the meso-species over the racemic one. Molecular structures show that the outer five-membered ligand ring adopts the half-chair conformation C(2) with either the lambda or the delta chirality and the methyl groups are in equatorial (e) positions. Enantiopure ligands of (+)(589)Ni[(R)(R)(lambda)bdtp](2) and (-)(589)Ni[(S)(S)(delta)bdtp](2) induce chirality into the symmetric SSNiSS chromophore with slightly helical distortion. Thus, their CD spectra exhibit weak negative or positive Cotton effects at 662 nm. CD spectra in L(+)- and D(-)diethyltartrate of the meso-complex and racemic crystal, rac-(+/-)Ni[bdtp](2), exhibit different weak Cotton effects of opposite sign. Complexes dissociate in methanol; rac-(+/-)Ni[bdtp](2) in methanol undergoes a crystallization-induced second-order asymmetric transformation which finally yields crystals of the meso-Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)bdtp] complex.     

Plasmodium vivax Population Structure and Transmission Dynamics in Sabah Malaysia

Despite significant progress in the control of malaria in Malaysia, the complex transmission dynamics of P. vivax continue to challenge national efforts to achieve elimination. To assess the impact of ongoing interventions on P. vivax transmission dynamics in Sabah, we genotyped 9 short tandem repeat markers in a total of 97 isolates (8 recurrences) from across Sabah, with a focus on two districts, Kota Marudu (KM, n = 24) and Kota Kinabalu (KK, n = 21), over a 2 year period. STRUCTURE analysis on the Sabah-wide dataset demonstrated multiple sub-populations. Significant differentiation (F _ST  = 0.243) was observed between KM and KK, located just 130 Km apart. Consistent with low endemic transmission, infection complexity was modest in both KM (mean MOI  = 1.38) and KK (mean MOI  = 1.19). However, population diversity remained moderate (H _E  = 0.583 in KM and H _E  = 0.667 in KK). Temporal trends revealed clonal expansions reflecting epidemic transmission dynamics. The haplotypes of these isolates declined in frequency over time, but persisted at low frequency throughout the study duration. A diverse array of low frequency isolates were detected in both KM and KK, some likely reflecting remnants of previous expansions. In accordance with clonal expansions, high levels of Linkage Disequilibrium (I _A ^S >0.5 [P<0.0001] in KK and KM) declined sharply when identical haplotypes were represented once (I _A ^S  = 0.07 [P = 0.0076] in KM, and I _A ^S = -0.003 [P = 0.606] in KK). All 8 recurrences, likely to be relapses, were homologous to the prior infection. These recurrences may promote the persistence of parasite lineages, sustaining local diversity. In summary, Sabah''s shrinking P. vivax population appears to have rendered this low endemic setting vulnerable to epidemic expansions. Migration may play an important role in the introduction of new parasite strains leading to epidemic expansions, with important implications for malaria elimination.     

Host genetic background effect on the frequency of mouse mammary tumor virus-induced rearrangements of the int-1 and int-2 loci in mouse mammary tumors.

The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.     

A robust cell cycle control mechanism limits E2F-induced proliferation of terminally differentiated cells in vivo

Terminally differentiated cells in Drosophila melanogaster wings and eyes are largely resistant to proliferation upon deregulation of either E2F or cyclin E (CycE), but exogenous expression of both factors together can bypass cell cycle exit. In this study, we show this is the result of cooperation of cell cycle control mechanisms that limit E2F-CycE positive feedback and prevent cycling after terminal differentiation. Aberrant CycE activity after differentiation leads to the degradation of E2F activator complexes, which increases the proportion of CycE-resistant E2F repressor complexes, resulting in stable E2F target gene repression. If E2F-dependent repression is lost after differentiation, high anaphase-promoting complex/cyclosome (APC/C) activity degrades key E2F targets to limit cell cycle reentry. Providing both CycE and E2F activities bypasses exit by simultaneously inhibiting the APC/C and inducing a group of E2F target genes essential for cell cycle reentry after differentiation. These mechanisms are essential for proper development, as evading them leads to tissue outgrowths composed of dividing but terminally differentiated cells.     

Effects of endothelin-1 and flunarizine on human trabecular meshwork cell contraction

Trabecular meshwork (TM) cells are now considered to play an active role in the aqueous outflow mechanism because they exhibit smooth muscle-like contractile properties. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been proposed to play a role in the local regulation of aqueous outflow and intraocular pressure (IOP) control. We propose an in vitro culture model as a method for the study of ET-1-induced human TM (HTM) cell contractility and for the study of whether pre-incubation with flunarizine, a calcium-channel blocker, can inhibit the action of ET-1. Experiments were performed on semiconfluent HTM cells (primary cultures established from normotensive human donor eyes) at the second passage, with phosphate-buffered saline (PBS) as a control. The contractile status of the cells was evaluated by a morphometric analysis of cell area, assuming that HTM cells in culture are able to reduce their area as a consequence of cytoskeletal contraction, rather than regulatory volume decrease. After incubation with 10 microM ET-1 for 5 mins, we observed a reduction of HTM cell area with respect to PBS-treated cells: 2425 +/- 876 microm2 versus 3125 +/- 987 microm2 (P < 0.001); and cells exhibited a retraction in shape and a reduction in number of indented profiles. Administration of ET-1 at progressively lower doses produced a corresponding lower reduction of HTM cell area, suggesting a dose-response effect of ET-1. Pre-incubation with 10 microM flunarizine strongly inhibited the ET-1 effect on HTM cell contraction: 2806 +/- 865 microm2 versus 2910 +/- 846 microm2 (P = not significant). Our data indicate that ET-1 induced a statistically significant reduction in the area of HTM cells versus controls, and that ET-1 can directly influence the aqueous outflow. Moreover, we observed that flunarizine inhibited the effect of ET-1 on the HTM cells.     

Oral administration of lactoferrin increases hemoglobin and total serum iron in pregnant women.

Iron deficiency anemia (IDA) during pregnancy continues to be of world-wide concern. IDA is a risk factor for preterm delivery and subsequent low birth weight, and possibly for poor neonatal health. Iron supplementation in pregnancy is a widely recommended practice, yet intervention programs have met with many controversies. In our study, 300 women at different trimesters of pregnancy were enrolled in a trial of oral administration of ferrous sulfate (520 mg once a day) or 30% iron-saturated bovine lactoferrin (bLf) (100 mg twice a day). Pregnant women refusing treatment represented the control group. In this group hemoglobin and total serum iron values measured after 30 d without treatment decreased significantly, especially in women at 18-31 weeks of pregnancy. In contrast, after 30 d of oral administration of bLf, hemoglobin and total serum iron values increased and to a greater extent than those observed in women treated orally for 30 d with ferrous sulfate, independently of the trimester of pregnancy. Unlike ferrous sulfate, bLf did not result in any side effects. These findings lead us to hypothesize that lactoferrin could influence iron homeostasis directly or through other proteins involved in iron transport out of the intestinal cells into the blood.