CCR5 internalisation and signalling have different dependence on membrane lipid raft integrity

The chemokine receptor, CCR5, acts as a co-receptor for human immunodeficiency virus entry into cells. CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains termed lipid rafts or caveolae. Cholesterol is essential for CCL4 binding to CCR5 and for keeping the conformational integrity of the receptor. Filipin treatment leads to loss of caveolin-1 from the membrane and therefore to a collapse of the caveolae. We have found here that sequestration of membrane cholesterol with filipin did not affect receptor signalling, however a loss of ligand-induced internalisation of CCR5 was observed. Cholesterol extraction with methyl-beta-cyclodextrin (MCD) reduced signalling through CCR5 as measured by release of intracellular Ca(2+) and completely abolished the inhibition of forskolin-stimulated cAMP accumulation with no effect on internalisation. Pertussis toxin (PTX) treatment inhibited the intracellular release of calcium that is transduced via Galphai G-proteins. Depletion of cholesterol destroyed microdomains in the membrane and switched CCR5/G-protein coupling to a PTX-independent G-protein. We conclude that cholesterol in the membrane is essential for CCR5 signalling via the Galphai G-protein subunit, and that integrity of lipid rafts is not essential for effective CCR5 internalisation however it is crucial for proper CCR5 signal transduction via Galphai G-proteins.     

Expression of matrix metalloproteinases-2 and -9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.     

An autopsy study of maternal mortality in Mozambique: the contribution of infectious diseases

Background

Maternal mortality is a major health problem concentrated in resource-poor regions. Accurate data on its causes using rigorous methods is lacking, but is essential to guide policy-makers and health professionals to reduce this intolerable burden. The aim of this study was to accurately describe the causes of maternal death in order to contribute to its reduction, in one of the regions of the world with the highest maternal mortality ratios.

Methods and Findings

We conducted a prospective study between October 2002 and December 2004 on the causes of maternal death in a tertiary-level referral hospital in Maputo, Mozambique, using complete autopsies with histological examination. HIV detection was done by virologic and serologic tests, and malaria was diagnosed by histological and parasitological examination. During 26 mo there were 179 maternal deaths, of which 139 (77.6%) had a complete autopsy and formed the basis of this analysis. Of those with test results, 65 women (52.8%) were HIV-positive. Obstetric complications accounted for 38.2% of deaths; haemorrhage was the most frequent cause (16.6%). Nonobstetric conditions accounted for 56.1% of deaths; HIV/AIDS, pyogenic bronchopneumonia, severe malaria, and pyogenic meningitis were the most common causes (12.9%, 12.2%, 10.1% and 7.2% respectively). Mycobacterial infection was found in 12 (8.6%) maternal deaths.

Conclusions

In this tertiary hospital in Mozambique, infectious diseases accounted for at least half of all maternal deaths, even though effective treatment is available for the four leading causes, HIV/AIDS, pyogenic bronchopneumonia, severe malaria, and pyogenic meningitis. These observations highlight the need to implement effective and available prevention tools, such as intermittent preventive treatment and insecticide-treated bed-nets for malaria, antiretroviral drugs for HIV/AIDS, or vaccines and effective antibiotics for pneumococcal and meningococcal diseases. Deaths due to obstetric causes represent a failure of health-care systems and require urgent improvement.     

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.     

A DIGE approach for the assessment of rat soleus muscle changes during unloading: effect of acetyl-L-carnitine supplementation

After hind limb suspension, a remodeling of postural muscle phenotype is observed. This remodeling results in a shift of muscle profile from slow-oxidative to fast-glycolytic. These metabolic changes and fiber type shift increase muscle fatigability. Acetyl-L-carnitine (ALCAR) influences the skeletal muscle phenotype of soleus muscle suggesting a positive role of dietary supplementation of ALCAR during unloading. In the present study, we applied a 2-D DIGE, mass spectrometry and biochemical assays, to assess qualitative and quantitative differences in the proteome of rat slow-twitch soleus muscle subjected to disuse. Meanwhile, the effects of ALCAR administration on muscle proteomic profile in both unloading and normal-loading conditions were evaluated. The results indicate a modulation of troponin I and tropomyosin complex to regulate fiber type transition. Associated, or induced, metabolic changes with an increment of glycolytic enzymes and a decreased capacity of fat oxidation are observed. These metabolic changes appear to be counteracted by ALCAR treatment, which restores the mitochondrial mass and decreases the glycolytic enzyme expression, suggesting a normalization of the metabolic shift observed in unloaded animals. This normalization is accompanied by a maintenance of body weight and seems to prevent a switch of fiber type.     

Identification of Mycobacterium avium subsp. paratuberculosis in wild cervids ( Cervus elaphus hippelaphus and Capreolus capreolus ) from Northwestern Italy

Seventy-seven red deer (Cervus elaphus hippelaphus), 40 roe deer (Capreolus capreolus) from the Northwestern (NW) Alps (Turin Province, NW Italy) and 29 roe deer from the NW Apennines (Alessandria province, NW Italy) were examined for the presence of Mycobacterium avium subsp. paratuberculosis (MAP) by culture, IS900 nested polymerase chain reaction (PCR) and IS1311 PCR restriction endonuclease analysis for strain characterisation. MAP identification (nested PCR and/or culture) allowed us to detect 32.9% MAP-infected red deer and 22.5% infected roe deer in the NW Alps and 41.4% MAP infected roe deer in the NW Apennines. On the basis of the polymorphism present in the IS1311 sequence, all MAP isolates were characterised as cattle strains. Our results show that MAP circulates widely among populations of wild cervids in NW Italy.     

Influence of topography on the location and density of armadillo burrows (Dasypodidae: Xenarthra) in the central Amazon, Brazil

Armadillos (Dasypodidae: Xenarthra) are the principal group of burrow-excavating mammals in Amazonian forest. Little is known about how the landscape's physical characteristics influence where these organisms choose to construct their burrows. We measured topographic characteristics of armadillo burrow placement and evaluated whether these variables affect overall burrow density. Our study took place on the study sites of the Biological Dynamics of Forest Fragments Project in the central Amazon, Brazil. To determine if armadillos exhibit habitat use preferences in constructing their burrows, we quantified the declination and elevation of available terrain along transect lines on our study sites and compared this with the characteristics of areas containing burrows. We also tested whether topographic variables were related to burrow density. Armadillos preferentially build burrows on low elevation, inclined terrain, and declination was positively related to burrow density. Low, inclined areas correspond to slopes next to stream basins. It may require less effort for armadillos to excavate these areas, while also providing proximity to water. The results of this study indicate that topographic information is important to consider when using burrows to estimate habitat use and density of armadillos in terra-firme forests of the central Amazon.     

Pathophysiological changes of gram-negative bacterial infection can be reproduced by a synthetic peptide mimicking loop L7 sequence of Haemophilus influenzae porin

Several in vivo models have been used to dissect the molecular mechanisms that contribute to activate the coagulation and fibrinolytic systems by bacteria and bacterial products but many aspects remain poorly understood. In this study we examined the in vivo effect of the synthetic peptide corresponding to loop L7 from Haemophilus influenzae type b (Hib) porin to evaluate its role on the coagulative/fibrinolytic cascade and the circulating markers of endothelial injury. Plasma was obtained from rats injected intravenously with loop L7, Hib porin or a scrambled peptide and tested for fragment 1+2 (F1+2), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type I (PAI-1) antigen, von Willebrand factor (vWF) and soluble E-selectin (sE-selectin). The coagulative/fibrinolytic cascade was impaired as shown by PAI-1 level increased. Concomitantly, E-selectin, a marker of endothelial injury, was also significantly elevated. In addition either loop L7 or Hib porin injection induced hyperglycaemia and inflammatory cytokine production. The data were correlated with hemodynamic functions. The results indicate that loop L7 plays an essential role in the pathophysiologic events observed during gram-negative infection. These findings may have implications for the development of alternative therapies to counteract excessive inflammatory responses during septic shock.     

Brucella lipoproteins mimic dendritic cell maturation induced by Brucella abortus

Infection with Brucella abortus induces a pro-inflammatory response that drives T cell responses toward a Th1 profile. The mechanism by which this bacterium triggers this response is unknown. Dendritic cells (DC) are crucial mediators at the host-pathogen interface and are potent Th1-inducing antigen-presenting cells. Thus, we examined the mechanism whereby B. abortus stimulate human DC maturation. B. abortus-infected DC increased the expression of CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40 and induced the production of TNF-alpha, IL-6, IL-10 and IL-12. Both phenomena were not dependent on bacterial viability since they were also induced by heat-killed B. abortus (HKBA). B. abortus LPS was unable to induce markers up-regulation or cytokine production. We next investigated the capacity of the outer membrane protein 19 (Omp19) as a B. abortus lipoprotein model to induce DC maturation. Lipidated Omp19 (L-Omp19), but not its unlipidated form, increased the expression of cell surface markers and the secretion of cytokines. L-Omp19-matured DC also have decreased endocytic activity and displayed enhanced T cell stimulatory activity in a MLR. Pre-incubation of DC with anti-TLR2 mAb blocked L-Omp19-mediated cytokine production. These results demonstrate that B. abortus lipoproteins can stimulate DC maturation providing a mechanism by which these bacteria generate a Th1-type immune response.     

High data output and automated 3D correlative light-electron microscopy method

Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections. We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of our method are (i) the possibility to correlate several hundreds of events at the same time, (ii) the possibility to perform three-dimensional (3D) correlation, (iii) the possibility to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the possibility to combine the high data analysis capability of FLM with the high precision-accuracy of transmission electron microscopy in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach.