Translational regulation in the anoxic turtle, <Emphasis Type="Italic">Trachemys scripta elegans</Emphasis>

Liver cancer is the sixth most common cancer worldwide and 3rd most common cause of cancer-related death. Hepatocellular carcinoma (HCC) represents more than 90% of primary liver cancer and is a major public health problem. Due to the advanced stages of HCC at the time of diagnosis, utilizing the conventional treatment for solid tumors frequently ends with treatment failure, recurrence, or poor survival. HCC is highly refractory to chemotherapy and other systemic treatments, and locoregional therapies or selective internal radiation therapies are largely palliative. Considering how the pathogenesis of HCC often induces an immunosuppressed state which is further amplified by post-treatment recurrence and reactivation, immunostimulation provides a potential novel approach for the treatment of HCC. Immune response(s) of the body may be potentiated by immunomodulation of various effector cells such as B-cells, T-cells, Treg cells, natural killer cells, dendritic cells, cytotoxic T-lymphocytes, and other antigen-presenting cells; cellular components such as genes and microRNA; and molecules such as proteins, proteoglycans, surface receptors, chemokines, and cytokines. Targeting these effectors individually has helped in the development of newer therapeutic approaches; however, combinational therapies targeting multi-faceted biomarkers have yielded better results. Still, there is a need for further research to develop novel therapeutic strategies which may act as either complementary or an alternative treatment to the standard therapy protocols of HCC. This review focuses on potential cellular and molecular targets, as well as the role of virotherapy and combinational therapy in the treatment of HCC.     

Stress-induced antioxidant defense and protein chaperone response in the freeze-tolerant wood frog <Emphasis Type="Italic">Rana sylvatica</Emphasis>

Freeze tolerance is an adaptive response utilized by the wood frog Rana sylvatica to endure the sub-zero temperatures of winter. Survival of whole body freezing requires wood frogs to trigger cryoprotective mechanisms to deal with potential injuries associated with conversion of 65–70% of total body water into ice, including multiple consequences of ice formation such as cessation of blood flow and cell dehydration caused by water loss into ice masses. To understand how wood frogs defend against these stressors, we measured the expression of proteins known to be involved in the antioxidant defense and protein chaperone stress responses in brain and heart of wood frogs comparing freezing, anoxia, and dehydration stress. Our results showed that most stress proteins were regulated in a tissue- and stress-specific manner. Notably, protein levels of the cytosolic superoxide dismutase (SOD1) were upregulated by 1.37?±?0.11-fold in frozen brain, whereas the mitochondrial SOD2 isoform rose by 1.38?±?0.37-fold in the heart during freezing. Catalase protein levels were upregulated by 3.01?±?0.47-fold in the brain under anoxia stress, but remained unchanged in the heart. Similar context-specific regulatory patterns were also observed for the heat shock protein (Hsp) molecular chaperones. Hsp27 protein was down-regulated in the brain across the three stress conditions, whereas the mitochondrial Hsp60 was upregulated in anoxic brain by 1.73?±?0.38-fold and by 2.13?±?0.57-fold in the frozen heart. Overall, our study provides a snapshot of the regulatory expression of stress proteins in wood frogs under harsh environment conditions and shows that they are controlled in a tissue- and stress-specific manner.     

Antioxidant systems and anoxia tolerance in a freshwater turtle Trachemys scripta elegans

In the search for MBP phosphorylating activities in Dictyostelium discoideum, we have found a proteolysis-activated protein kinase. This activity which is distributed between the soluble and the particulate fractions of the cell, uses MBP and histone as substrate and has a molecular mass of 140 kDa as detected in an in situ' assay.This protein kinase has several features shared by the protein kinase C family, such as substrate specificity and sensitivity to proteolysis, but its molecular mass is much larger than that described for the known protein kinase C isoforms.To better characterize this activity we have studied its sensitivity to several protein kinase C inhibitors and activators. This protein kinase is activated neither by phorbol ester nor by phosphatidylserine or Ca²⁺. The activity is inhibited by staurosporine and PKC pseudosubstrate, but is not affected by the specific protein kinase C inhibitor bisindolylmaleimide.These data lead us to propose that proteolytically activated Dictyostelium protein kinase belongs to the recently described protein kinase C-related family.     

The Respiratory Chain of Plant Mitochondria: IV. Oxidation Rates of the Respiratory Carriers of Mung Bean Mitochondria in the Presence of Cyanide

The half-time for oxidation of cytochrome b(557) in mitochondria from etiolated mung bean (Phaseolus aureus) hypocotyls is 5.8 milliseconds at 24 Celsius in the absence or presence of 0.3 mm KCN, when the oxidation is carried out by injecting a small amount of oxygenated medium into a suspension of mitochondria made anaerobic in the presence of succinate plus malonate. Since oxygen is consumed by the alternate, cyanide-insensitive respiratory pathway of these mitochondria, cycles of oxidation and reduction can be obtained with the oxygen pulses when cyanide is present. Reduced cytochromes (a + a(3)) also become oxidized at nearly the uninhibited rate under these conditions, a(3) completely and a partially. The half-time for oxidation of c(547) is also unaffected by 0.3 mm KCN, but c(549) has a half-time equal to that of c(547) in the presence of KCN, compared to the shorter one observed in the absence of inhibitor. The maximum extent of oxidation of the cytochromes c is about 70% in the presence of 0.3 mm KCN; this oxidation is rapidly followed by an extensive reduction which is synchronous with the reduction of cytochrome a observed under the same conditions. In the presence of cyanide, it appears likely that the cytochromes c and b(557) are oxidized by cytochrome oxidase in oxygen pulse experiments, rather than by the alternate oxidase. The oxidation of cytochrome b(553) is partially inhibited by KCN, but complete oxidation is attained in the aerobic steady state with excess oxygen. If the oxygen pulse experiment is carried out in the presence of sufficient malonate so that entry of reducing equivalents into the respiratory chain occurs at a rate negligible compared to inter-carrier electron transport, the half-time for flavoprotein oxidation is unaffected by 0.3 mm KCN while that for ubiquinone oxidation is but 2-fold larger. The observed net oxidation rate of these two carriers in mung bean mitochondria is more sensitive to the entry rate of reducing equivalents, as set by succinate concentration and malonate to succinate ratio, then it is in skunk cabbage (Symplocarpus foetidus) mitochondria. These observations are interpreted in terms of a respiratory carrier Y, placed between flavoprotein plus ubiquinone and the cytochromes, which is the fork in the split respiratory pathway to the two terminal oxidases and which has lower electron transport capacity in mung bean mitochondria than in skunk cabbage mitochondria.     

The Respiratory Chain of Plant Mitochondria: XVII. Flavoprotein-Cytochrome b(562) Interaction in Antimycin-treated Skunk Cabbage Mitochondria

During the transition from the aerobic steady state with succinate as substrate to anaerobiosis, in suspensions of skunk cabbage (Symplocarpus foetidus) mitochondria treated with antimycin A, cytochrome b(562) becomes reoxidized to the extent of about 20%, synchronously with the reduction of cytochrome c(549). This reoxidation occurs in both the absence and presence of m-chlorobenzhydroxamic acid, a specific inhibitor for the alternate terminal oxidase of plant mitochondria. A flavoprotein component, amounting to 13% to 15% of the total nonfluorescent mitochondrial flavoprotein, undergoes reduction synchronously with the oxidation of cytochrome b(562) during the aerobic to anaerobic transition with succinate as substrate in the presence of both antimycin A and m-chlorobenzhydroxamic acid. This flavoprotein component remains reduced in the presence of cyanide. The half-time for reduction of the flavoprotein component and cytochrome c(549) and for oxidation of cytochrome b(562) during the aerobic to anaerobic transition with succinate as substrate in the presence of both antimycin A and m-chlorobenzhydroxamic acid is 2 seconds. The half-times for oxidation of cytochrome c(549) and the flavoprotein component are 2.1 and 170 milliseconds, respectively, during the anaerobic to aerobic transition induced by addition of 14 mum O(2) to the mitochondrial suspensions. The half-time for reduction of cytochrome b(562) under these conditions is 150 milliseconds, synchronous with the flavoprotein component. The synchrony of the flavoprotein oxidation and of the cytochrome b(562) reduction at a rate much slower than that of cytochrome c(549) oxidation implies that, in antimycin-treated plant mitochondria, the state of the cytochrome b(562)/antimycin complex is regulated by the redox state of this flavoprotein component, rather than by cytochrome c(549). It is tentatively suggested that these two components are not part of the main sequence of the respiratory chain, but may be part of a multienzyme complex active in the hydroxylation reactions required for ubiquinone biosynthesis in the inner mitochondrial membrane.     

A “Love” Dart Allohormone Identified in the Mucous Glands of Hermaphroditic Land Snails

Animals have evolved many ways to enhance their own reproductive success. One bizarre sexual ritual is the “love” dart shooting of helicid snails, which has courted many theories regarding its precise function. Acting as a hypodermic needle, the dart transfers an allohormone that increases paternity success. Its precise physiological mechanism of action within the recipient snail is to close off the entrance to the sperm digestion organ via a contraction of the copulatory canal, thereby delaying the digestion of most donated sperm. In this study, we used the common garden snail Cornu aspersum to identify the allohormone that is responsible for this physiological change in the female system of this simultaneous hermaphrodite. The love dart allohormone (LDA) was isolated from extracts derived from mucous glands that coat the dart before it is stabbed through the partner''s body wall. We isolated LDA from extracts using bioassay-guided contractility measurement of the copulatory canal. LDA is encoded within a 235-amino acid precursor protein containing multiple cleavage sites that, when cleaved, releases multiple bioactive peptides. Synthetic LDA also stimulated copulatory canal contractility. Combined with our finding that the protein amino acid sequence resembles previously described molluscan buccalin precursors, this indicates that LDA is partially conserved in helicid snails and less in other molluscan species. In summary, our study provides the full identification of an allohormone that is hypodermically injected via a love dart. More importantly, our findings have important consequences for understanding reproductive biology and the evolution of alternative reproductive strategies.