Anthraquinone profile and chemical fingerprint of Rhamnus saxatilis L. from Italy

Several species belonging to the genus Rhamnus (Rhamnaceae), comprising ones among which are found the most typical plants of the Italian flora, are known to contain biologically active anthraquinone secondary metabolites. Although several Rhamnus species were so far investigated, no information is available concerning the content and relative abundances of anthraquinones in R. saxatilis. In this study we used a simple, reliable, and accurate analytical method to determine the anthraquinones in bark of R. saxatilis. This allowed us also to trace a comparative study on the efficacy of different extraction solvents in ultrasonication time dependent assays. Separation and quantification of anthraquinones were accomplished using a C18 column with the mobile phase of H₂O:methanol (40:60, v/v, 1% formic acid) at a flow rate of 0.7 mL/min and a detection wavelength of 254 nm, while the qualitative analyses were also achieved at a wavelength of 435 nm.Finally, the described HPLC method, was used to obtain a specific chemical fingerprint for this species in comparison with other species from the same family.     

The Shc family protein adaptor, Rai, negatively regulates T cell antigen receptor signaling by inhibiting ZAP-70 recruitment and activation

Rai/ShcC is a member of the Shc family of protein adaptors expressed with the highest abundance in the central nervous system, where it exerts a protective function by coupling neurotrophic receptors to the PI3K/Akt survival pathway. Rai is also expressed, albeit at lower levels, in other cell types, including T and B lymphocytes. We have previously reported that in these cells Rai attenuates antigen receptor signaling, thereby impairing not only cell proliferation but also, opposite to neurons, cell survival. Here we have addressed the mechanism underlying the inhibitory activity of Rai on TCR signaling. We show that Rai interferes with the TCR signaling cascade one of the earliest steps--recruitment of the initiating kinase ZAP-70 to the phosphorylated subunit of the TCR/CD3 complex, which results in a generalized dampening of the downstream signaling events. The inhibitory activity of Rai is associated to its inducible recruitment to phosphorylated CD3, which occurs in the physiological signaling context of the immune synapse. Rai is moreover found as a pre-assembled complex with ZAP-70 and also constitutively interacts with the regulatory p85 subunit of PI3K, similar to neuronal cells, notwithstanding the opposite biological outcome, i.e. impairment of PI-3K/Akt activation. The data highlight the ability of Rai to establish interactions with the TCR and key signaling mediators which, either directly (e.g. by inhibiting ZAP-70 recruitment to the TCR or sequestering ZAP-70/PI3K in the cytosol) or indirectly (e.g. by promoting the recruitment of effectors responsible for signal extinction) prevent full triggering of the TCR signaling cascade.     

Novel optical system for in vitro quantification of full surface strain fields in small arteries: I. Theory and design

Recent advances in vascular biology and pathophysiology have revealed the need to understand better the genetic basis of arterial stiffness, disease progression and responses to clinical intervention. Towards this end, in vitro experiments on arteries from genetically modified mice promise to provide significantly increased insight into both health and disease. The need to test small arteries, often of complex shape, necessitates new methods for experimental arterial mechanics, however, ones that can provide information on local changes in geometry and strain. In this paper, we present a theoretical framework for a new panoramic digital image correlation-based method sufficient to collect such information and we demonstrate the utility of this approach via validation studies on phantoms having dimensions on the order of 500-1000 μm, similar to those of large mouse arteries. In particular, we show that placing the specimen within a conical mirror and imaging the specimen via a gimbal-mounted mirror using a single camera yields stereo information sufficient to quantify the size, shape and deformation along the full length and around the entire circumference of small arteries. In a companion paper, we show further that this approach can be implemented effectively while testing arteries within a physiological solution that maintains native biomechanical properties.     

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.     

Actein inhibits the Na+-K+-ATPase and enhances the growth inhibitory effect of digitoxin on human breast cancer cells

The Na⁺-K⁺-ATPase is a known target of cardiac glycosides such as digitoxin and ouabain. We determined that the enzyme also is a target of the structurally-related triterpene glycoside actein, present in the herb black cohosh. Actein’s inhibition of Na⁺-K⁺-ATPase activity was less potent than that of digitoxin, but actein potentiated digitoxin’s inhibitory effect on Na⁺-K⁺-ATPase activity and MDA-MB-453 breast cancer cell growth. We observed different degrees of signal amplification for the two compounds. Actein’s inhibitory effect on ATPase activity was amplified 2-fold for cell growth inhibition, whereas digitoxin’s signal was amplified 20-fold. Actein induced a biphasic response in proteins downstream of ATPase: low dose and short duration of treatment upregulated NF-κB promoter activity, p-ERK, p-Akt and cyclin D1 protein levels, whereas higher doses and longer exposure inhibited these activities. Actein and digitoxin may be a useful synergistic combination for cancer chemoprevention and/or therapy.     

Gonadal maturation in the blackspot seabream Pagellus bogaraveo: a comparison between a farmed and a wild broodstock

The blackspot seabream Pagellus bogaraveo (Brünnich, 1768) has been regarded as a possible alternative to traditionally cultured Mediterranean species such as seabream and seabass, due to its high market value and good adaptation to captivity. Broodstock establishment and management represent the first step towards reliable production of eggs and fry, which is required to develop aquaculture of this new species. Two different broodstocks were tested for gonadal maturation and spawning, one constituting of wild fish caught as juveniles and reared in tanks until sexual maturity (4 years), and one assembled from wild adult fish caught during or just before the reproductive season. All fish were maintained under the same rearing conditions and fed the same diet. Gonadal stripping and biopsies were performed weekly to monitor maturation in both males and females. Ovarian samples were staged for maturity on the basis of follicular diameter and migration of germinal vesicle. Sperm samples were tested for density (number of spermatozoa ml⁻¹) and motility. The fish reared in captivity reached ovarian maturity during the breeding season of the wild stock. Eggs were obtained by stripping from both farmed and wild specimens, but appeared degenerated as a result of being retained too long in the ovarian cavity due to the absence of spontaneous spawning. Spermiation was prolonged in the farmed fish, but appeared to be blocked in the wild breeders after first sampling. However, the sperm was very viscous and the motile spermatozoa did not exceed 10%.     

The reproductive biology of blackspot sea bream Pagellus bogaraveo in captivity. I. gonadal development, maturation and hermaphroditism

Gonadal development, maturation, spawning and hermaphroditism were investigated in captive blackspot sea bream, Pagellus bogaraveo Brünnich, 1768, during the second, third and fourth years of life. The gonads of 224 fish were examined macroscopically and histologically. Undifferentiated gonads were found in fish smaller than 22.0 cm. Adult fish showed four gonadal differentiations: ovotestes with functional testis and quiescent ovary (Mf), ovotestes with functional ovary and regressed testis (mF), ovotestes with both ovary and testis at a resting stage (mf), and ovaries with no male tissue (F). The overall incidence of gonochoric females F was 41%. Functional males Mf were more frequent in age classes 1+ and 2+, whereas functional females mF predominated in the 3+ age class, above 25.0 cm TL. Histological examination revealed testicular degeneration and atrophy in functional females mF. On the basis of both histological data as well as size and age frequency distribution, it is suggested that the reproductive strategy of P. bogaraveo in captivity is characterized by protandrous hermaphroditism, with a high incidence of female gonochorism. Spawning occurred in March–April, at a size of 28.0 cm and age 3 in males and at 29.5 cm and age 4 in females. The gonosomatic index (GSI) remained constantly low (≤ 0.05) throughout the second and third years of life. A significant increase in GSI was noted in both males and females at the accomplishment of the fourth year of life, coinciding with the spawning season. The results are compared with information available on wild P. bogaraveo and discussed with a view to a possible exploitation of this species in aquaculture, through a reliable control of reproduction.     

Casein kinase II motif-dependent phosphorylation of human papillomavirus E7 protein promotes p130 degradation and S-phase induction in differentiated human keratinocytes

The E7 proteins of human papillomaviruses (HPVs) promote S-phase reentry in differentiated keratinocytes of the squamous epithelia to support viral DNA amplification. In this study, we showed that nuclear p130 was present in the differentiated strata of several native squamous epithelia susceptible to HPV infection. In contrast, p130 was below the level of detection in HPV-infected patient specimens. In submerged and organotypic cultures of primary human keratinocytes, the E7 proteins of the high-risk mucosotrophic HPV-18, the benign cutaneous HPV-1, and, to a lesser extent, the low-risk mucosotropic HPV-11 destabilized p130. This E7 activity depends on an intact pocket protein binding domain and a casein kinase II (CKII) phosphorylation motif. Coimmunoprecipitation experiments showed that both E7 domains were important for binding to p130 in extracts of organotypic cultures. Metabolic labeling in vivo demonstrated that E7 proteins were indeed phosphorylated in a CKII motif-dependent manner. Moreover, the efficiencies of the E7 proteins of various HPV types or mutations to induce S-phase reentry in spinous cells correlated with their relative abilities to bind and to destabilize p130. Collectively, these data support the notion that p130 controls the homeostasis of the differentiated keratinocytes and is therefore targeted by E7 for degradation to establish conditions permissive for viral DNA amplification.     

Immunological Modulation of Human Cardiac Mast Cells

Human mast cells, by elaborating various cytokines, chemokines and proinflammatory mediators play a complex role in several allergic and inflammatory disorders. Mast cells have been identified in human heart tissue in close proximity to the sarcolemma, in perivascular and adventitial locations and in the shoulder region of coronary atheroma. Human heart mast cells (HHMC) can be isolated from patients undergoing heart transplantation and can be immunologically activated in vitro to induce the release of tryptase, chymase, cysteinyl leukotriene C₄ and prostaglandin D₂. Several cytokines (e.g., stem cell factor and TNF-) reside in secretory granules of HHMC. Mast cell density is increased in the hearts of patients with ischemic and idiopathic dilated cardiomyopathy. Cardiac mast cells might contribute to the evolution of atherosclerosis, dilated cardiomyopathy, cardiac and systemic anaphylaxis through the release of cytokines and vasoactive and proinflammatory mediators.     

Modulation of ion uptake across posterior gills of the crab Chasmagnathus granulatus by dopamine and cAMP

Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills of Chasmagnathus granulatus acclimated to 10 per thousand salinity. Addition of cAMP agonists, such as cp-cAMP, forskolin, and IBMX, produced a significant increase in the transepithelial potential difference (Vte), which reflects ion transport activity. Dopamine (DA) also had a stimulatory effect on ion uptake, increasing Vte and Na(+) influx, although this effect was transient, since both variables remained elevated for less than 30 min. In addition, the dose-response curve for DA concentration-Vte was biphasic, and the maximum stimulation was obtained with 10 micromol l(-1). When the effects of forskolin and DA on the Na(+)/K(+)-ATPase activity were tested, they correlated well with the Vte and Na(+) influx experiments; the enzyme activity increased significantly after preincubation of gill fragments for 10 min with forskolin or DA (51 and 64%, respectively), but there was no effect after pre-incubation with DA for 20 min. Finally, KT5720, a specific inhibitor of cAMP-dependent protein kinase (PKA), completely abolished the stimulatory effect of DA on Vte, suggesting the involvement of PKA in this mechanism.