Investigating the Effects of Statins on Cellular Lipid Metabolism Using a Yeast Expression System

In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids.     

Smoothelin-like 1 Protein Regulates Myosin Phosphatase-targeting Subunit 1 Expression during Sexual Development and Pregnancy

Pregnancy coordinately alters the contractile properties of both vascular and uterine smooth muscles reducing systemic blood pressure and maintaining uterine relaxation. The precise molecular mechanisms underlying these pregnancy-induced adaptations have yet to be fully defined but are likely to involve changes in the expression of proteins regulating myosin phosphorylation. Here we show that smoothelin like protein 1 (SMTNL1) is a key factor governing sexual development and pregnancy induced adaptations in smooth and striated muscle. A primary target gene of SMTNL1 in these muscles is myosin phosphatase-targeting subunit 1 (MYPT1). Deletion of SMTNL1 increases expression of MYPT1 30–40-fold in neonates and during development expression of both SMTNL1 and MYPT1 increases over 20-fold. Pregnancy also regulates SMTNL1 and MYPT1 expression, and deletion SMTNL1 greatly exaggerates expression of MYPT1 in vascular smooth muscle, producing a profound reduction in force development in response to phenylephrine as well as sensitizing the muscle to acetylcholine. We also show that MYPT1 is expressed in Type2a muscle fibers in mice and humans and its expression is regulated during pregnancy, suggesting unrecognized roles in mediating skeletal muscle plasticity in both species. Our findings define a new conserved pathway in which sexual development and pregnancy mediate smooth and striated muscle adaptations through SMTNL1 and MYPT1.     

Conjugative transfer frequency of resistance genes from ESBL-producing Enterobacteriaceae strains isolated from patients hospitalized in pediatric wards

The aim of this study was to evaluate the transfer frequency of plasmids encoding extended-spectrum beta-lactamases (ESBLs) from clinical isolates of Enterobacteriaceae to E. coli K12 C600 recipient strain. Additionally, resistance patterns to antimicrobial drugs of the isolates as well as transconjugants were analyzed. Fifty-four clinical strains belonging to the Enterobacteriaceae family were isolated from children hospitalized in Medical University Hospital in Wroc?aw. All the strains studied were identified in automatic ATB system using ID32E tests. Besides, they were ESBL-positive as was confirmed by the double-disc synergy test (DDST). The minimal inhibitory concentration (MIC) was determined for twelve selected antibiotics and chemotherapeutics. The majority of the strains (87%) were able to transfer plasmid-mediated ESBL to E. coli K12 C600 recipient strain with a frequencies ranged from 10(-5) to 10(-1) per donor cell. All the isolates studied as well as their transconjugants were susceptible to imipenem, meropenem and norfloxacin (MIC <1mg/L). On the other hand, these strains displayed high level of resistance (MIC 512 - >1024 mg/L) to cefotaxime, ceftriaxone, gentamycin, amikacin and cotrimoxazole. Genetic markers conferring resistance to aminoglycosides and cotrimoxazole were often co-transferred to recipient strain in conjugation process.     

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF PROTEASES IN LARVAL PREPARATIONS OF THE CEREAL LEAF BEETLE (Oulema melanopus,CHRYSOMELIDAE, COLEOPTERA)

We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole‐larval preparations, permitting studies of the very small whole‐larval preparations. Protein preparations were analyzed using FITC–casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0–4.0). Cysteine protease activity increased at acidic pH and in the presence of β‐mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe²⁺, Ca²⁺, Mg²⁺, Zn²⁺, and K⁺ (10 mM). Fe²⁺ and Zn²⁺ significantly decreased enzyme activity at all pH values, Ca²⁺ and Mg²⁺ at pH 6.2 and Mg²⁺ at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N‐p‐tosyl‐l‐phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, N_α‐tosyl‐l‐lysine chloromethyl ketone hydrochloride, N‐p‐tosyl‐l‐phenylalanine chloromethyl ketone, trans‐epoxysuccinyl‐l‐leucylamido‐(4‐guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin‐like) proteases and metalloproteases act in cereal leaf beetle digestion.     

Amyloid-Precursor-Protein-Lowering Small Molecules for Disease Modifying Therapy of Alzheimer's Disease

Alzheimer''s disease (AD) is the most common form of dementia in the elderly with progressive cognitive decline and memory loss. According to the amyloid-hypothesis, AD is caused by generation and subsequent cerebral deposition of β-amyloid (Aβ). Aβ is generated through sequential cleavage of the transmembrane Amyloid-Precursor-Protein (APP) by two endoproteinases termed beta- and gamma-secretase. Increased APP-expression caused by APP gene dosage effects is a risk factor for the development of AD. Here we carried out a large scale screen for novel compounds aimed at decreasing APP-expression. For this we developed a screening system employing a cell culture model of AD. A total of 10,000 substances selected for their ability of drug-likeness and chemical diversity were tested for their potential to decrease APP-expression resulting in reduced Aβ-levels. Positive compounds were further evaluated for their effect at lower concentrations, absence of cytotoxicity and specificity. The six most promising compounds were characterized and structure function relationships were established. The novel compounds presented here provide valuable information for the development of causal therapies for AD.