Positive crosstalk between ERK and p38 in melanoma stimulates migration and in vivo proliferation

Melanoma is one of the most therapy-resistant cancers. Activating mutations in BRAF and NRAS are the source of extracellular signal regulated protein kinase (ERK) pathway activation. We show that melanoma cell lines, originating in different metastatic sites, with BRAF or NRAS mutations, in addition to active mitogen activated protein kinase (MAPK)-ERK, also have highly activated stress activated protein kinase (SAPK)-p38. This is in direct contrast to carcinoma cells in which the activity of the two kinases appears to be mutually exclusive; high level of p38 activity inhibits, through a negative feedback, ERK activity and prevents tumorigenesis. Melanomas are insensitive to ERK inhibition by p38 and utilize p38-signaling pathway for migration and growth in vivo. We found a positive functional loop linking the high ERK activity to surface expression of alphaVbeta3-integrin. This integrin, by interacting with vitronectin, induces p38 activity and increases IL-8 production, enhancing cell migration. Because the negative loop from p38 to ERK is lost, the two kinases can remain simultaneously activated. Inhibition of ERK and p38 activities is more effective in blocking in vivo growth than inhibition of each kinase individually. Future therapies might have to consider targeting of both pathways.     

Endothelium Trans Differentiated from Wharton's Jelly Mesenchymal Cells Promote Tissue Regeneration: Potential Role of Soluble Pro-Angiogenic Factors

Background

Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton''s jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton''s jelly (hWMSCs) can accelerate tissue repair in vivo.

Methods

Mesenchymal stem cells were isolated from human Wharton''s jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO).

Results

hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures.

Conclusion

These results demonstrate that mesenchymal stem cells isolated from Wharton''s jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.     

Abi1 is essential for the formation and activation of a WAVE2 signalling complex

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.     

The interaction between dopamine D2-like and beta-adrenergic receptors in the prefrontal cortex is altered by mood-stabilizing agents

Several studies have suggested the involvement of biogenic monoaminergic neurotransmission in bipolar disorder and in the therapy for this disease. In this study, the effects of the mood-stabilizing drugs lithium, carbamazepine or valproate on the dopaminergic and adrenergic systems, particularly on D2-like and beta-adrenergic receptors, were studied both in cultured rat cortical neurones and in rat prefrontal cortex. In vitro and in vivo data showed that stimulation of beta-adrenergic receptors with isoproterenol increased cyclic adenosine monophosphate (cAMP) levels and this effect was significantly inhibited by lithium, carbamazepine or valproate. The activation of dopamine D2-like receptors with quinpirole decreased the isoproterenol-induced rise in cAMP in control conditions. This inhibition was observed in vivo after chronic treatment of the rats with carbamazepine or valproate, but not after treatment with lithium or in cultured rat cortical neurones after 48 h exposure to the three mood stabilizers. Dopamine D2 and beta1-adrenergic receptors were found to be co-localized in prefrontal cortical cells, as determined by immunohistochemistry, but western blot experiments revealed that receptor levels were differentially affected by treatment with the three mood stabilizers. These data show that mood stabilizers affect D2 receptor-mediated regulation of beta-adrenergic signalling and that each drug acts by a unique mechanism.     

Responsive regions for direct organogenesis in sunflower cotyledons

Regeneration efficiency from three different regions of cotyledonary explants was examined in six sunflower inbred lines. Proximal, middle and distal regions from seedling cotyledons were cultured on regeneration medium supplemented with growth regulators. Plant regeneration by direct organogenesis was observed after four weeks. Significant differences among inbred lines were found for regeneration percentage and average number of shoots per total explants. Also a decreasing regeneration capacity was observed from proximal to distal sections for all inbred lines. Regeneration ability from cotyledonary explants in this species is strongly influenced by the genotype and by the region from which the explant was obtained. The distance to the cotyledonary node plays a preponderant role in the expression of shoot forming capacity. Shoot differentiation via seedling cotyledons depends upon the presence of the proximal region of cotyledon regardless of the genotype.     

Susceptibility of important Gram-negative pathogens to tigecycline and other antibiotics in Latin America between 2004 and 2010

Background

The evolving epidemiology of methicillin resistant Staphylococcus aureus (MRSA) is characterized by the emergence of infections caused by non multiresistant MRSA carrying staphylococcal chromosomal cassette (SCC)mec IV or V in the healthcare settings. A molecular epidemiological analysis of non multiresistant MRSA isolates from four acute general hospitals was performed in Palermo, Italy, during a one year period.

Methods

For the purpose of the study, MRSA isolates were defined as non multiresistant when they were susceptible to at least three classes of non ??-lactam antibiotics. Seventy-five isolates were submitted to antimicrobial susceptibility testing, multilocus sequence typing (MLST) and polymerase chain reaction (PCR) for SCCmec, accessory gene regulator (agr) groups, arginine catabolic mobile element (ACME) and Panton Valentine leukocidin (PVL) toxin genes. For epidemiological typing, Multiple-Locus Variable-Number Tandem Repeat Fingerprinting (MLVF) was performed on all isolates and pulsed field gel electrophoresis (PFGE) on ST8 isolates.

Results

Non multiresistant MRSA isolates were isolated from all hospitals. Resistances to ciprofloxacin, macrolides and tetracycline were the most prevalent. MLST attributed 46 isolates with ST22, 13 with ST8, eight with ST1, three with ST50 and three with ST398. SCCmec type IV was found in all isolates. PVL was detected in one ST22 isolate. All isolates tested negative for the ACME element. MLVF identified 31 different patterns, some subtype clusters ranging in size between two and 22 isolates. The closely related PFGE patterns of the ST8 isolates differed from USA300.

Conclusions

A polyclonal circulation of non multiresistant MRSA along with blurring of boundaries between healthcare associated (HA)-MRSA and community associated (CA)-MRSA appear to be occurring in our epidemiological setting. A better understanding of spread of MRSA with the support of molecular typing can provide invaluable information in the epidemiological, microbiological and clinical fields.     

Suboptimal growth temperatures enhance the biological activity of cultured cyanobacterium Gloeocapsa sp.

The cytotoxic, antibacterial, and antifungal activities of cyanobacterium Gloeocapsa sp. strain Gacheva 2007/R-06/1 were investigated and the possibility for an enhancement of these activities by changing the culture conditions evaluated. Fatty acids of this cyanobacterium were found to be active against Streptococcus pyogenes. Exopolysaccharides inhibited the growth of both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. Both exopolysaccharides and fatty acid mixtures also significantly decreased the viability of human cervical carcinoma cells, HeLa. Greater biological activities were exhibited by Gloeocapsa sp., cultured at suboptimal temperatures (15–26°C) than at optimal and supraoptimal ones. In comparison with higher light intensity, the low-light cultivation stimulated the cytotoxicity of the fatty acids. In general, low temperatures decreased the growth of Gloeocapsa sp., but promoted its biological activity. Prolonged cultivation also had a beneficial impact on the bioactivity. Compared to 4 days, the 17-day cultivation resulted in fourfold higher antibacterial and antifungal activities of exopolysaccharides and more than twice increases in their cytotoxicity. The study revealed that this cyanobacterial isolate is a new source of natural products with potential for pharmacological and medical applications.