Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: A potential new treatment for hepatocellular carcinoma

Although shikonin, a naphthoquinone derivative, has showed anti-cancer activity, its precise molecular anti-tumor mechanism remains to be elucidated. In this study, we investigated the effects of shikonin on human hepatocellular carcinoma (HCC) in vitro and in vivo. Our results showed that shikonin induced apoptosis of Huh7 and BEL7402 but not nontumorigenic cells. ROS generation was detected, and ROS scavengers completely inhibited shikonin-induced apoptosis, indicating that ROS play an essential role. Although the JNK activity was significantly elevated after shikonin treatment, JNK was not linked to apoptosis. However, downregulation of Akt and RIP1/NF-κB activity was found to be involved in shikonin-induced apoptosis. Ectopic expression of Akt or RIP1 partly abrogated the effects of shikonin, and Akt inhibitor and RIP1 inhibitor synergistically induced apoptosis in conjunction with shikonin treatment. ROS scavengers blocked shikonin-induced inactivation of Akt and RIP1/NF-κB, but Akt or RIP1/NF-κB did not regulate ROS generation, suggesting that Akt and RIP1/NF-κB signals are downstream of ROS generation. In addition, the results of xenograft experiments in mice were consistent with in vitro studies. Taken together, our data show that shikonin, which may be a promising agent in the treatment of liver cancer, induced apoptosis in HCC cells through the ROS/Akt and RIP1/NF-κB pathways.     

Analysis of CaM-kinase signaling in cells

A change in intracellular free calcium is a common signaling mechanism that modulates a wide array of physiological processes in most cells. Responses to increased intracellular Ca²⁺ are often mediated by the ubiquitous protein calmodulin (CaM) that upon binding Ca²⁺ can interact with and alter the functionality of numerous proteins including a family of protein kinases referred to as CaM-kinases (CaMKs). Of particular interest are multifunctional CaMKs, such as CaMKI, CaMKII, CaMKIV and CaMKK, that can phosphorylate multiple downstream targets. This review will outline several protocols we have used to identify which members and/or isoforms of this CaMK family mediate specific cellular responses with a focus on studies in neurons. Many previous studies have relied on a single approach such as pharmacological inhibitors or transfected dominant-negative kinase constructs. Since each of these protocols has its limitations, that will be discussed, we emphasize the necessity to use multiple, independent approaches in mapping out cellular signaling pathways.     

CIN85 regulates ubiquitination and degradative endosomal sorting of the EGF receptor

CIN85 has been demonstrated to interact with a number of proteins involved in endocytosis and intracellular sorting. However, the exact functional role of CIN85 in endocytosis remains unclear. We have investigated whether CIN85 plays a role in EGF-induced EGF receptor (EGFR) internalization, as previously suggested, or whether CIN85 is rather involved in endosomal sorting of the EGFR. When over-expressing a dominant negative interfering CIN85 mutant consisting of three SH3 domains only, we found that internalization of EGF was inhibited. However, when knocking down CIN85 by RNAi, the EGF–EGFR uptake appeared similar to in control cells. Furthermore, in CIN85 depleted cells, EGF-induced ubiquitination of the EGFR was decreased, and degradation of EGF–EGFR complexes was delayed. Our data further demonstrated that depletion of CIN85 increased the recycling of EGF, suggesting that CIN85 plays a role in endosomal sorting of the ubiquitinated EGFR. Our data also demonstrated that CIN85 was constitutively associated with Hrs, and this strengthens the hypothesis of a functional role of CIN85 in endosomal EGFR sorting.     

Using Knowledge of Protein Structural Constraints to Predict the Evolution of HIV-1

The high levels of sequence diversity and rapid rates of evolution of HIV-1 represent the main challenges for developing effective therapies. However, there are constraints imposed by the three-dimensional protein structure that affect the sequence space accessible to the evolution of HIV-1. Here, we present a strategy for predicting the set of possible amino acid replacements in HIV. Our approach is based on the identification of likely amino acid changes in the context of these structural constraints using environment-specific substitution matrices as well as considering the physical constraints imposed by local structure. Assessment of the power of various published algorithms in predicting the evolution of HIV-1 Gag P17 shows that it is possible to use these methods to make accurate predictions of the sequence diversity. Our own method, SubFit, uses knowledge of local structural constraints; it achieves similar prediction success with the best-performing methods. We also show that erroneous predictions are largely due to infrequently occurring amino acids that will probably have severe fitness costs for the protein. Future improvements; for example, incorporating covariation and immunological constraints will permit more reliable prediction of viral evolution.     

Does small-perimeter fencing inhibit mule deer or pronghorn use of water developments?

Wildlife water development can be an important habitat management strategy in western North America for many species, including both pronghorn (Antilocapra americana) and mule deer (Odocoileus hemionus). In many areas, water developments are fenced (often with small-perimeter fencing) to exclude domestic livestock and feral horses. Small-perimeter exclosures could limit wild ungulate use of fenced water sources, as exclosures present a barrier pronghorn and mule deer must negotiate to gain access to fenced drinking water. To evaluate the hypothesis that exclosures limit wild ungulate access to water sources, we compared use (photo counts) of fenced versus unfenced water sources for both pronghorn and mule deer between June and October 2002–2008 in western Utah. We used model selection to identify an adequate distribution and best approximating model. We selected a zero-inflated negative binomial distribution for both pronghorn and mule deer photo counts. Both pronghorn and mule deer photo counts were positively associated with sampling time and average daily maximum temperature in top models. A fence effect was present in top models for both pronghorn and mule deer, but mule deer response to small-perimeter fencing was much more pronounced than pronghorn response. For mule deer, we estimated that presence of a fence around water developments reduced photo counts by a factor of 0.25. We suggest eliminating fencing of water developments whenever possible or fencing a big enough area around water sources to avoid inhibiting mule deer. More generally, our results provide additional evidence that water development design and placement influence wildlife use. Failure to account for species-specific preferences will limit effectiveness of management actions and could compromise research results. © 2011 The Wildlife Society.     

Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC₅₀ of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.     

Dispersion,contagion, and population stability of red scale,Aonidiella aurantii,in citrus orchards with low or zero insecticide use

Red scale, Aonidiella aurantii Maskell (Hemiptera: Diaspididae), has a contagious distribution among fruit and among trees largely due to the fact that most young settle on the same fruit as their mothers. The pattern of distribution and degree of dispersion of red scale within various sets of contiguous citrus trees in several orchards were recorded each autumn for 4 years. Changes in density and site occupancy within trees and within contiguous groups of trees were made to assess the strength of any link between populations and their dynamics on different trees. Aonidiella aurantii was found to have a very clumped distribution whereby trees with a level of scale infestation well above average for their block could be adjacent to trees with few or no detectable scale. However, patches of high density on a given tree usually did not recur for more than 2 years but were replaced with above average infestations on other trees. The stability of the red scale populations varied from block to block. Some blocks had few or no infested trees over the study period whereas others had widely fluctuating levels of infestation. Dispersion coefficients at ‘within tree’ and ‘within block’ levels were similar. Theoretical and statistical models of the relation of average scale levels to the percentage of fruit or trees infested were less precise than they are for other pests because of high variation of clumping intensity of scale within any block.