Malgorzata Slocinska

Insects Antiviral and Anticancer Peptides: New Leads for the Future?

Insect produce wide range of protein and peptides as a first fast defense line against pathogen infection. These agents act in different ways including insect immune system activation or by direct impact on the target tumor cells or viruses. It has been shown that some of the insect peptides suppress viral gene and protein expression, rybosilate DNA, whereas others cause membrane lysis, induce apoptosis or arrest cell cycle. Several of the purified and characterized peptides of insect origin are very promising in treating of serious human diseases like human immunodeficiency virus (HIV), herpex simplex virus (HSV) or leukaemia. However, some obstacles need to be overcome. Cytotoxic activity of peptides, susceptibility to proteases or high cost of production remain still unsolved problems. Reports on the peptides antiviral and antitumour mechanisms are scanty. Thus, in this review we present characteristic, mode of action and potential medical applications of insects origin peptides with the antiviral and antitumour activity.

Reduced cardiac output is associated with decreased mitochondrial efficiency in the non-ischemic ventricular wall of the acute myocardial-infarcted dog.

Cardiogenic shock is the leading cause of death among patients hospitalized with acute myocardial infarction (MI). Understanding the mechanisms for acute pump failure is therefore important. The aim of this study is to examine in an acute MI dog model whether mitochondrial bio-energetic function within non-ischemic wall regions are associated with pump failure. Anterior MI was produced in dogs via ligation of left anterior descending (LAD) coronary artery, that resulted in an infract size of about 30% of the left ventricular wall. Measurements of hemodynamic status, mitochondrial function, free radical production and mitochondrial uncoupling protein 3 (UCP3) expression were determined over 24 h period. Hemodynamic measurements revealed a > 50% reduction in cardiac output at 24 h post infarction when compared to baseline. Biopsy samples were obtained from the posterior non-ischemic wall during acute infarction. ADP/O ratios for isolated mitochondria from non-ischemic myocardium at 6 h and 24 h were decreased when compared to the ADP/O ratios within the same samples with and without palmitic acid (PA). GTP inhibition of (PA)-stimulated state 4 respiration in isolated mitochondria from the non-ischemic wall increased by 7% and 33% at 6 h and 24 h post-infarction respectively when compared to sham and pre-infarction samples. This would suggest that the mitochondria are uncoupled and this is supported by an associated increase in UCP3 expression observed on western blots from these same biopsy samples. Blood samples from the coronary sinus measured by electron paramagnetic resonance (EPR) methods showed an increase in reactive oxygen species (ROS) over baseline at 6 h and 24 h post-infarction. In conclusion, mitochondrial bio-energetic ADP/O ratios as a result of acute infarction are abnormal within the non-ischemic wall. Mitochondria appear to be energetically uncoupled and this is associated with declining pump function. Free radical production may be associated with the induction of uncoupling proteins in the mitochondria.

UCP4 expression changes in larval and pupal fat bodies of the beetle Zophobas atratus under adipokinetic hormone treatment

We investigated the influence of adipokinetic hormone (AKH), an insect neurohormone, on uncoupling protein 4 (ZaUCP4) expression and activity in larval and pupal fat body mitochondria of the beetle Zophobas atratus in relation to intermediary metabolism. Homologous Tenmo-AKH was administered to the beetle larvae and pupae as either a single dose or as two doses of 20pmol during a 24h interval. In the larval and pupal fat bodies, downregulation of ZaUCP4 expression at the mRNA and protein levels was observed 24h and 48h after AKH treatment, respectively. In both developmental stages, ZaUCP4 activity was lowered in fat body mitochondria 48h after AKH treatment. In the AKH-injected larvae, changes in ZaUCP4 expression were accompanied by the mobilization of carbohydrate reserves, no change in the concentration of total lipids and an increase in the free fatty acid level. In contrast, AKH had no effect on carbohydrate metabolism in the pupal fat body but induced lipid mobilization. It seems that AKH influences ZaUCP4 expression by triggering multiple events and that it has different physiological roles in controlling intermediary metabolism in the fat body of the beetle larvae and pupae.

Molecular identification and functional characterisation of uncoupling protein 4 in larva and pupa fat body mitochondria from the beetle Zophobas atratus

Uncoupling protein 4 (UCP4) is a member of the UCP subfamily that mediates mitochondrial uncoupling, and sequence alignment predicts the existence of UCP4 in several insects. The present study demonstrates the first molecular identification of a partial Zophobas atratus UCP4-coding sequence and the functional characterisation of ZaUCP4 in the mitochondria of larval and pupal fat bodies of the beetle. ZaUCP4 shows a high similarity to predicted insect UCP4 isoforms and known mammalian UCP4s, both at the nucleotide and amino acid sequence levels. Bioenergetic studies clearly demonstrate UCP function in mitochondria from larval and pupal fat bodies. In non-phosphorylating mitochondria, ZaUCP activity was stimulated by palmitic acid and inhibited by the purine nucleotide GTP. In phosphorylating mitochondria, ZaUCP4 activity decreased the yield of oxidative phosphorylation. ZaUCP4 was immunodetected with antibodies raised against human UCP4 as a single 36-kDa band. A lower expression of ZaUCP4 at the level of mRNA and protein and a decreased ZaUCP4 activity were observed in the Z. atratus pupal fat body compared with the larval fat body. The different expression patterns and activity of ZaUCP4 during the larval-pupal transformation indicates an important physiological role for UCP4 in insect fat body development and function during insect metamorphosis.

New metabolic activity of the nonsulfated sulfakinin Zopat-SK-1 in the insect fat body.

Insect sulfakinins are multifunctional neuropeptides homologous to vertebrate gastrin/cholecystokin (CCK) neuropeptide hormones. We investigated the action of the nonsulfated sulfakinin Zopat-SK-1 (pETSDDYGHLRFa) on the levels of chosen metabolites in the Zophobas atratus beetle fat body. Samples of fat body were collected 2h and 24h after hormone injection. The administration of 20pmol of Zopat-SK-1 to feeding larvae significantly increased concentrations of lipids and proteins and decreased the content of glycogen in fat body tissue in the 24h experimental group. In contrast, the only increase in total lipid concentration in prepupal fat bodies was observed 24h after Zopat-SK-1 treatment. Simultaneously, changes in the quality and quantity of free sugars in the hemolymph were measured. In larval hemolymph, a marked increase in free sugar concentration and a decrease in glucose content were observed 24h and 2h after Zopat-SK-1 application, respectively. No changes in the prepupal stage were observed. For the first time we show potent metabolic activity of sulfakinin in the fat body tissue of an insect. Our findings imply a physiological function of the nonsulfated form of sulfakinin in energy storage and release processes in fat body tissue of larvae and prepupae was indicated. We suggest a role for sulfakinin signaling in the regulation of energy metabolism in insect tissues.

Uncoupling proteins of invertebrates: A review

Uncoupling proteins (UCPs) mediate inducible proton conductance in the mitochondrial inner membrane. Herein, we summarize our knowledge regarding UCPs in invertebrates. Since 2001, the presence of UCPs has been demonstrated in nematodes, mollusks, amphioxi, and insects. We discuss the following important issues concerning invertebrate UCPs: their evolutionary relationships, molecular and functional properties, and physiological impact. Evolutionary analysis indicates that the branch of vertebrate and invertebrate UCP4‐5 diverged early in the evolutionary process prior to the divergence of the animal groups. Several proposed physiological roles of invertebrate UCPs are energy control, metabolic balance, and preventive action against oxidative stress.

Cold induced changes in lipid, protein and carbohydrate levels in the tropical insect Gromphadorhina coquereliana

Insects cope with thermal stressors using mechanisms such as rapid cold hardening and acclimation. These mechanisms have been studied in temperate insects, but little is known about their use by tropical insects in response to cold stress. Here, we investigated whether cold stress (1×8 h and 3×8 h at 4°C) triggers a metabolic response in the Madagascar cockroach Gromphadorhina coquereliana. We examined the effects of cold on the levels of selected metabolites in the fat body tissue of G. coquereliana. After cold exposure, we found that the quantity of total protein increased significantly in the insect fat body, whereas glycogen decreased slightly. Using antibodies, we observed upregulation of AQP-like proteins and changes in the HSP70 levels in the fat body of G. coquereliana when exposed to cold. We also examined the content and nature of the free sugars in the G. coquereliana hemolymph and discovered an increase in the levels of polyols and glucose in response to cold stress. These results suggest an important role of the fat body tissue of tropical insects upon cold exposure.

Adipokinetic hormone induces changes in the fat body lipid composition of the beetle Zophobas atratus

In insects, neuropeptide adipokinetic hormone (AKH) released from the corpora cardiaca mobilizes lipids and carbohydrates in the fat body. We examined the developmental differences in the action of Tenmo-AKH, a bioanalogue belonging to the adipokinetic/hypertrahelosemic family (AKH/HrTH), on the lipid composition of larval and pupal fat bodies in the beetle Zophobas atratus. Tenmo-AKH was administered to the beetle larvae and pupae either as a single dose or as two doses of 20 pmol during a 24h interval. Extracts of fat bodies were used to analyse the lipid composition by gas chromatography (GC) combined with mass spectrometry (GC-MS). Control extracts were analyzed using the same method. Fatty acids (FA) and fatty acid methyl esters (FAME) were the most abundant compounds in the fat bodies from both developmental stages. We observed significant differences in their concentrations following hormonal treatment. Tenmo-AKH also induced a distinct increase in larval sterols, fatty alcohols and benzoic acid.

Evidences for an ATP-sensitive potassium channel (KATP) in muscle and fat body mitochondria of insect

In the present study, we describe the existence of mitochondrial ATP-dependent K(+) channel (mitoKATP) in two different insect tissues, fat body and muscle of cockroach Gromphadorhina coquereliana. We found that pharmacological substances known to modulate potassium channel activity influenced mitochondrial resting respiration. In isolated mitochondria oxygen consumption increased by about 13% in the presence of potassium channel openers (KCOs) such as diazoxide and pinacidil. The opening of mitoKATP was reversed by glibenclamide (potassium channel blocker) and 1 mM ATP. Immunological studies with antibodies raised against the Kir6.1 and SUR1 subunits of the mammalian ATP-sensitive potassium channel, indicated the existence of mitoKATP in insect mitochondria. MitoKATP activation by KCOs resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of mitochondrial ATP-sensitive potassium channel in insects.

The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum

Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle.