Role of Prolactin Receptors in Lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.     

Bone Cell-autonomous Contribution of Type 2 Cannabinoid Receptor to Breast Cancer-induced Osteolysis

The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.     

Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozymes IX and XII with polyfluorinated aromatic/heterocyclic sulfonamides

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX (CA IX) and XII (CA XII) are involved in acidification of hypoxic tumors, a process correlated with poor prognosis and clinical outcome of patients harboring such tumors. This process may be reversed by inhibiting these enzymes with potent sulfonamide/sulfamate inhibitors. A series of such aromatic/heterocyclic sulfonamides incorporating 2,3,5,6-tetrafluorobenzoyl-, 2,3,5,6-tetrafluoro- phenylsulfonyl- and pentafluorophenylureido moieties has been investigated for its interaction with the catalytic domain of the human isozymes hCA IX and hCA XII. Some of these compounds showed excellent inhibitory properties against both isozymes IX and XII, with several subnanomolar inhibitors detected for the first time. These sulfonamides may constitute valuable candidates for the development of novel antitumor therapies based on the inhibition of such tumor-associated CA isozymes.     

Personality,Relationship Conflict,and Teamwork-Related Mental Models

This study seeks to explore whether neuroticism, agreeableness, and conscientiousness moderate the influence of relationship conflict experienced in groups on changes in group members'' evaluative cognitions related to teamwork quality (teamwork-related mental models). Data from 216 students, nested in 48 groups were analyzed using a multilevel modeling approach. Our results show that the experience of relationship conflict leads to a negative shift from the pre-task to the post-task teamwork-related mental models. Moreover, the results indicate that conscientiousness buffered the negative association between relationship conflict and the change in teamwork-related mental models. Our results did not support the hypothesized moderating effect of agreeableness and show that the detrimental effect of relationship conflict on the shift in teamwork-related mental models is accentuated for group members scoring low rather than high on neuroticism. These findings open new research venues for exploring the association between personality, coping styles and change in teamwork-related mental models.     

Selective Vulnerability of Spinal and Cortical Motor Neuron Subpopulations in delta7 SMA Mice

Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients.     

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.     

Use of locally weighted scatterplot smoothing (LOWESS) regression to study selection signatures in Piedmontese and Italian Brown cattle breeds

Selection is the major force affecting local levels of genetic variation in species. The availability of dense marker maps offers new opportunities for a detailed understanding of genetic diversity distribution across the animal genome. Over the last 50 years, cattle breeds have been subjected to intense artificial selection. Consequently, regions controlling traits of economic importance are expected to exhibit selection signatures. The fixation index (F_st) is an estimate of population differentiation, based on genetic polymorphism data, and it is calculated using the relationship between inbreeding and heterozygosity. In the present study, locally weighted scatterplot smoothing (LOWESS) regression and a control chart approach were used to investigate selection signatures in two cattle breeds with different production aptitudes (dairy and beef). F_st was calculated for 42 514 SNP marker loci distributed across the genome in 749 Italian Brown and 364 Piedmontese bulls. The statistical significance of F_st values was assessed using a control chart. The LOWESS technique was efficient in removing noise from the raw data and was able to highlight selection signatures in chromosomes known to harbour genes affecting dairy and beef traits. Examples include the peaks detected for BTA2 in the region where the myostatin gene is located and for BTA6 in the region harbouring the ABCG2 locus. Moreover, several loci not previously reported in cattle studies were detected.     

Diverse post-translational modifications of the pannexin family of channel-forming proteins

The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.