Efficiency of Immunotoxin Cytotoxicity Is Modulated by the Intracellular Itinerary

Pseudomonas exotoxin-based immunotoxins, including LMB-2 (antiTac(F_v)-PE38), are proposed to traffic to the trans-Golgi network (TGN) and move by a retrograde pathway to the endoplasmic reticulum, where they undergo translocation to the cytoplasm, a step that is essential for cytotoxicity. The retrograde transport pathways used by LMB-2 are not completely understood, so it is unclear if transit through specific organelles is critical for maximal cytotoxic activity. In this study, we used Chinese hamster ovary (CHO) cell lines that express chimeric constructs of CD25, the Tac antigen, attached to the cytoplasmic domain of the TGN-targeted transmembrane proteins, TGN38 and furin. These chimeras are both targeted to the TGN, but the itineraries they follow are quite different. LMB-2 was incubated with the two cell lines, and the efficiency of cell killing was determined using cell viability and cytotoxicity assays. LMB-2 that is targeted through the endocytic recycling compartment to the TGN via Tac-TGN38 kills the cells more efficiently than immunotoxins delivered through the late endosomes by Tac-furin. Although the processing to the 37 kDa active fragment was more efficient in Tac-furin cells than in Tac-TGN38 cells, this was not associated with enhanced cytotoxicity – presumably because the toxin was also degraded more rapidly in these cells. These data indicate that trafficking through specific organelles is an important factor modulating toxicity by LMB-2.     

Functional tyrosine residue in the active center of human dipeptidyl peptidase III

Abstract Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased kcat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.     

Divergent effects of leptin on luteinizing hormone and insulin secretion are dose dependent

We have shown recently that fasting permits leptin to modulate both luteinizing hormone (LH) and insulin secretion in cows. In rodents, leptin causes divergent effects on LH and insulin release that are dose dependent. To test the hypothesis that leptin effects on LH and insulin secretion in fasted cows are dose related, we examined the effects of various doses of recombinant ovine leptin (oleptin) in mature cows. Twenty ovariectomized beef cows, each bearing an estradiol implant to maintain basal estradiol concentrations, were used. All cows were fasted for 60 hr with free access to water and were assigned randomly to one of four groups (n = 5/group): 1) saline control; 2) leptin, 0.2 microg/kg; 3) leptin, 2.0 microg/kg; and 4) leptin, 20 microg/kg body wt. Blood samples were collected at 10-min intervals for 6 hr on Days 0 and 2, with saline or oleptin injected intravenously immediately after the first intensive sample on Day 2 (54 hr). Leptin caused a dose-related increase (P < 0.001) in mean concentrations of circulating LH. Stimulation of LH release by leptin was significant at the lowest (141% of control) and middle (122% of control) doses used, but no increase was observed for the highest dose. Increased mean concentrations of LH appeared to result from an augmentation of basal secretion, as pulse characteristics were not affected. After 54 hr of fasting, plasma insulin concentrations were lowered (P < 0.01) in all treatment groups compared to Day 0. After leptin injections, plasma insulin concentrations increased (P < 0.01) and reached highest concentrations during the first hour of sampling. However, this increase was sustained for several hours only in the intermediate (2.0 microg/kg) dose group. Collectively, our results show that leptin has potent positive effects on both LH and insulin secretion in fasted cows, but the anterior pituitary and endocrine pancreas appear to become downregulated in the presence of excess ligand.     

Decoupling of reproductive rates and parental expenditure in a polyandrous butterfly

Current theory postulates that the operational sex ratio (OSR)determines the relative degree of mating competition in thetwo sexes and is in turn influenced by a sexual difference inthe potential reproductive rate (PRR) denned as 1/time out,where time out is the time an individual must spend recoveringfrom a bout of mating activity and/or caring for offspring.In bushcricket mating systems where males provide females witha nuptial gift, relative energy expenditure in offspring influencesthe PRR of males and females and underlies a diet-mediated shiftin the OSR. Here we investigated if there is a similar positiverelationship between relative parental nutrient expenditurein offspring and PRR in the polyandrous butterfly Pieris napi,where female fecundity is strongly dependent on male nuptialgifts at mating. By varying the amount of nutrients femalesreceive at mating and relating this to number of offspring produced,we show that male P. napi have, on average, a nutrient expenditurein offspring equaling that of females. In spite of this, themale reproductive rate is 8–13 times higher than thatof females. Hence the relative degree of parental expenditurein offspring is largely decoupled from the degree of matingcompetition in P. napi. Two alternative explanations are advancedto account for the difference between the butterfly and thebushcricket mating systems.     

Design of Tripeptides as a Competitive Inhibitor for HMG-CoA Reductase

International Journal of Peptide Research and Therapeutics - This study presents a simple approach in design of tripeptides as a competitive inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase...     

Body mass index, waist circumference and waist-to-hip ratio: which anthropometric indicator is better predictor for the hypertension development in women population of the island Cres

The aim of this research was to investigate the prevalence of obesity and high blood pressure and to prove which of three anthropometric indicators of obesity - waist circumference, body mass index (BMI) waist-to-hip ratio - is better predictor for the development of hypertension in women population of the island of Cres. We approached separately groups of women with measured high blood pressure and with previously diagnosed. The research was preformed within the research project "Genetic and biomedical characteristics of the population of the island of Cres". This was the cross sectional study and data were obtained on the sample of 247 females over 18 years old that voluntarily participated in this study. In our study group the prevalence of overweight was 39.0%, obesity 27.5%, increased waist circumference was present in 69.4% while increased blood pressure was found in 53.0% examinees. Our results indicate that age, BMI, impaired glucose concentration and serum cholesterol could be considered as predictors for the development of arterial hypertension, whether measured or previously diagnosed.     

Mitochondria and Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and anti-apoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and –3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.Special issue dedicated to Lawrence F. Eng.     

The first structure of dipeptidyl-peptidase III provides insight into the catalytic mechanism and mode of substrate binding

Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-A crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates.     

Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy

Background  

Bacterial penicillin-binding proteins and β-lactamases (PBP-βLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-βL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-βL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-βL proteins.