Parathyroid hormone-related protein is a hypertrophy factor for human mesangial cells: Implications for diabetic nephropathy

Hypertrophy of human mesangial cells (HMC) is among the earliest characteristics in patients with diabetic nephropathy (DN). Recently, we observed the upregulation of parathyroid hormone (PTH)-related protein (PTHrP) in experimental DN, associated with renal hypertrophy. Herein, we first examined whether PTHrP was overexpressed in human DN, and next assessed the putative role of this protein on high glucose (HG)-induced HMC hypertrophy. As previously found in mice, kidneys from diabetic patients showed an increased tubular and glomerular immunostaining for PTHrP. In HMC, HG medium increased PTHrP protein expression associated with the development of hypertrophy as assessed by cell protein content. This effect was also induced by PTHrP(1-36). HG and PTHrP(1-36)-induced hypertrophy were associated with an increase in cyclin D1 and p27Kip1 protein expression, a decreased cyclin E expression, and the prevention of cyclin E/cdk2 complex activation. Both PTHrP neutralizing antiserum (α-PTHrP) and the PTH/PTHrP receptor antagonist (JB4250) were able to abolish HG induction of hypertrophy, the aforementioned changes in cell cycle proteins, and also TGF-β1 up-regulation. Moreover, the capability of both HG and PTHrP(1-36) to induce HMC hypertrophy was abolished by α-TGFβ1. These data show for the first time that PTHrP is upregulated in the kidney of patients with DN. Our findings also demonstrate that PTHrP acts as an important mediator of HG-induced HMC hypertrophy by modulating cell cycle regulatory proteins and TGF-β1.     

Homers regulate calcium entry and aggregation in human platelets: a role for Homers in the association between STIM1 and Orai1

Homer is a family of cytoplasmic adaptor proteins that play different roles in cell function, including the regulation of G-protein-coupled receptors. These proteins contain an Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) homology 1 domain that binds to the PPXXF sequence motif, which is present in different Ca2?-handling proteins such as IP3 (inositol 1,4,5-trisphosphate) receptors and TRPC (transient receptor potential canonical) channels. In the present study we show evidence for a role of Homer proteins in the STIM1 (stromal interaction molecule 1)-Orai1 association, as well as in the TRPC1-IP3RII (type II IP3 receptor) interaction, which might be of relevance in platelet function. Treatment of human platelets with thapsigargin or thrombin results in a Ca2?-independent association of Homer1 with TRPC1 and IP3RII. In addition, thapsigargin and thrombin enhanced the association of Homer1 with STIM1 and Orai1 in a Ca2?-dependent manner. Interference with Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, reduced STIM1-Orai1 and TRPC1- IP3RII associations, as compared with the introduction of the inactive PPKKRR peptide. The PPKKFR peptide attenuates thrombin-evoked Ca2? entry and the maintenance of thapsigargin-induced store-operated Ca2? entry. Finally, the PPKKFR peptide attenuated thrombin-induced platelet aggregation. The findings of the present study support an important role for Homer proteins in thrombin-stimulated platelet function, which is likely to be mediated by the support of agonist-induced Ca2? entry.     

Whole-body vibration increases upper and lower body muscle activity in older adults: potential use of vibration accessories

The current study examined the effects of whole-body vibration (WBV) on upper and lower body muscle activity during static muscle contractions (squat and bicep curls). The use of WBV accessories such as hand straps attached to the platform and a soft surface mat were also evaluated. Surface electromyography (sEMG) was measured for the medial gastrocnemius (MG), vastus lateralis (VL), and biceps brachii (BB) muscles in fourteen healthy older adults (74.8±4.5 years; mean±SD) with a WBV stimulus at an acceleration of 40 m s(-2) (30 Hz High, 2.5 mm or 46 Hz Low, 1.1 mm). WBV increased lower body (VL and MG) sEMG vs baseline (no WBV) though this was decreased with the use of the soft mat. The addition of the bicep curl with hand straps had no effect on lower body sEMG. WBV also increased BB sEMG vs baseline which was further increased when using the hand straps. There was no upper body effect of the soft mat. This study demonstrates WBV increases both lower and upper body muscle activity in healthy older adults. Moreover, WBV accessories such as hand straps attached to the platform or a soft surface mat may be used to alter exercise intensity.     

Mast Pulses Shape Trophic Interactions between Fluctuating Rodent Populations in a Primeval Forest

How different functional responses of consumers exploiting pulsed resources affect community dynamics is an ongoing question in ecology. Tree masting is a common resource pulse in terrestrial ecosystems that can drive rodent population cycles. Using stable isotope (δ¹³C, δ¹⁵N) analyses, we investigated the dietary response of two fluctuating rodent species, the yellow-necked mouse Apodemus flavicollis and the bank vole Myodes glareolus, to mast events in Białowieża Forest (NE Poland). Rodent hair samples were obtained non-invasively from faeces of their predators for an 11-year period that encompassed two mast events. Spectacular seed crops of deciduous trees, namely oak Quercus robur and hornbeam Carpinus betulus, occur after several intermediate years of moderate seed production, with a post-mast year characterised by a nil crop. While a Bayesian isotopic (SIAR) mixing model showed a variety of potential vegetation inputs to rodent diets, the isotopic niche of the yellow-necked mouse was strongly associated with mast of deciduous trees (>80% of diet), showing no variation among years of different seed crop. However, bank voles showed a strong functional response; in mast years the vole shifted its diet from herbs in deciduous forest (∼66% of diet) to mast (∼74%). Only in mast years did the isotopic niche of both rodent species overlap. Previous research showed that bank voles, subordinate and more generalist than mice, showed higher fluctuations in numbers in response to masting. This study provides unique data on the functional response of key pulse consumers in forest food webs, and contributes to our understanding of rodent population fluctuations and the mechanisms governing pulse–consumer interactions.     

Time of Progression to Osteopenia/Osteoporosis in Chronically HIV-Infected Patients: Screening DXA Scan

Background

Algorithms for bone mineral density (BMD) management in HIV-infected patients are lacking. Our objective was to assess how often a dual-energy x-ray absorptiometry (DXA) scan should be performed by assessing time of progression to osteopenia/osteoporosis.

Methods

All DXA scans performed between 2000 and 2009 from HIV-infected patients with at least two DXA were included. Time to an event (osteopenia and osteoporosis) was assessed using the Kaplan–Meier method. Strata (tertiles) were defined using baseline minimum T scores. Differences between strata in time to an event were compared with the log-rank test.

Results

Of 391 patients (1,639 DXAs), 49.6% had osteopenia and 21.7% osteoporosis at their first DXA scan. Of the 112 (28.6%) with normal BMD, 35.7% progressed to osteopenia; median progression time was 6.7 years. These patients were stratified: “low-risk" (baseline minimum T score >−0.2 SD), “middle-risk" (between −0.2 and −0.6 SD), and “high-risk" (from −0.6 to −1 SD); median progression time to osteopenia was 8.7, >7.2, and 1.7 years, respectively (p<0.0001). Of patients with osteopenia, 23.7% progressed to osteoporosis; median progression time was >8.5 years. Progression time was >8.2 years in “low-risk" tertile (T score between −1.1 and −1.6 SD), >8.5 years in “middle-risk" (between −1.6 and −2), and 3.2 years in “high-risk" (from −2 to −2.4) (p<0.0001).

Conclusions

Our results may help to define the BMD testing interval. The lowest T score tertiles would suggest recommending a subsequent DXA in 1–2 years; in the highest tertiles, ≥6 years. Early intervention in patients with bone demineralization could reduce fracture–related morbidity/mortality.     

Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus

Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded.     

Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G(1) phase. Aberrant expression of CDK4 and CDK6 is a hallmark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM (the calcein acetoxymethyl-ester) is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G(1) phase. The metabolic effects of calcein AM on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phosphate pathway was significantly altered. To elucidate whether these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.     

Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans-Infection Through Recognition of Viral Membrane Gangliosides

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4⁺ T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4⁺ T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.