D-amino acid levels in human physiological fluids

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied, considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always <1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often >90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study. © 1993 Wiley-Liss, Inc.     

Lithium-Induced Hypothyroidism: Oxidative Stress and Osmotic Fragility Status in Rats

The present study was conducted to explore the possible effects of different doses of lithium carbonate on thyroid functions, erythrocyte oxidant–antioxidant status, and osmotic fragility. Twenty-four Wistar-type male rats were equally divided into three groups: groups I and II received 0.1 and0.2 % lithium carbonate in their drinking water, respectively, for 30 days. The rats in group III served as controls, drinking tap water without added lithium. At the end of the experimental period, the erythrocyte osmotic fragility and the levels of triiodothyronine (T₃), thyroxine (T₄), thyroid-stimulating hormone (TSH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were measured in blood samples. Compared to controls, there was a statistically significant increase of TSH but decreases of the T₃ and T₄ levels in group II. Both experimental groups showed a statistically significant increase of the maximum osmotic fragility limit. The minimum osmotic fragility values of the animals in group II were statistically higher than those of controls. The standard hemolytic increment curve of both experimental groups was shifted to the right when compared to the curve obtained from the controls. Also, relative to controls, the activities of MDA and SOD were significantly higher and the GSH level lower in group II, but not so in group I. The results of the present study show that treatment with lithium carbonate may result in thyroid function abnormalities, increased oxidative damage, and possible compromise of the erythrocyte membrane integrity resulting from increased osmotic fragility.     

Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.     

In vitro study of the metabolic effects of D-amino acids

While the L -configuration of amino acids predominates in all known living systems, D -enantiomers of amino acids have been detected with highly sensitive chromatographic techniques in human physiological fluids. In the present study, the survival of Chinese hamster ovary cells (CHO) and HeLa cells was inhibited by exposure to high concentrations of some D - or L -amino acids. Inhibition of colony formation, though, was not necessarily observed to be chiral-dependent. Some L -amino acids (L AAS) were found to be toxic while other D -amino acids (D AAS) were innocuous in both cultures. This is contradictory to the previous observations that D AAS were generally considered to be harmful. Frequently it was implied, although not experimentally proven, that the L AAS were not toxic. One of the metabolites produced by oxidative deamination of D - or L AAS is hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS) that is decomposed by catalase. Increased intracellular H₂O₂ can result in peroxidation of lipids. We measured catalase activity and the lipid peroxide levels (LPO) after incubating cells in either D - or L AAS. The amino acids (AAS) that were found to inhibit colony formation were found to be associated with higher levels of catalase activity and LPO. Therefore, we hypothesize that enhanced ROS generation may be, in part, responsible for the observed toxicity of some amino acids. © 1996 Wiley-Liss, Inc.     

Detection of Entamoeba histolytica/Entamoeba dispar in stool specimens by using enzyme-linked immunosorbent assay

Entamoeba histolytica actually comprises two genetically distinct but morphologically indistinguishable species. E. histolytica can cause invasive intestinal and extra intestinal disease, while E. dispar cannot. Identification and differentiation of E. dispar and E. histolytica in stool sample by microscopy is imprecise. Several weeks of culture and isoenzyme analysis are required to differentiate E. histolytica from E. dispar. In this study, we have used an enzyme-linked immunosorbent assay (ELISA) for detection of E. histolytica/E.dispar and compared it with microscopy. Eighty-eight samples were evaluated, trichrome staining was positive in 20.4% (18) and ELISA was positive in 29.5% (26). Both tests were positive in 14 (15.9%) samples, 4 (4.5%) only with direct microscopy, and 12 (13.6%) only with ELISA. Both tests were negative in 58 (65.9%) samples. Microscopy has low sensitivity and high specificity, low negative predictive value and high positive predictive value in comparison with ELISA. E. histolytica/E. dispar antigen detection ELISA tests are inexpensive compared to the specific tests, yield objective results and do not require experienced microscopists and can therefore be recommended for screening of stools worldwide and the results can be taken for treatment that are fitting with its clinic.     

Tracing Myelin Protein Zero (P0) <Emphasis Type="Italic">in vivo</Emphasis> by construction of P0-GFP fusion proteins

Background  

Mutations in P0, the major protein of the myelin sheath in peripheral nerves, cause the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH). We reported earlier a de novo insertional mutation c.662_663GC (Ala221fs) in a DSS patient. The c.662_663GC insertion results in a frame shift mutation Ala221fs altering the C-terminal amino acid sequence. The adhesion-relevant intracellular RSTK domain is replaced by a sequence similar to Na+/K+ ATPase. To further clarify the molecular disease mechanisms in this sporadic patient we constructed wild type P0 and the c.662_663GC mutant expression cassettes by site-specific mutagenesis and transfected the constructs into insect cells (S2, High5). To trace the effects in live cells, green fluorescent protein (GFP) has been added to the carboxyterminus of the wild type and mutated P0 protein.     

Comparative evaluation of N-acetylcysteine and N-acetylcysteineamide in acetaminophen-induced hepatotoxicity in human hepatoma HepaRG cells

Acetaminophen (N-acetyl-p-aminophenol, APAP) is one of the most widely used over-the-counter antipyretic analgesic medications. Despite being safe at therapeutic doses, an accidental or intentional overdose can result in severe hepatotoxicity; a leading cause of drug-induced liver failure in the U.S. Depletion of glutathione (GSH) is implicated as an initiating event in APAP-induced toxicity. N-acetylcysteine (NAC), a GSH precursor, is the only currently approved antidote for an APAP overdose. Unfortunately, fairly high doses and longer treatment times are required due to its poor bioavailability. In addition, oral and intravenous administration of NAC in a hospital setting are laborious and costly. Therefore, we studied the protective effects of N-acetylcysteineamide (NACA), a novel antioxidant, with higher bioavailability and compared it with NAC in APAP-induced hepatotoxicity in a human-relevant in vitro system, HepaRG. Our results indicated that exposure of HepaRG cells to APAP resulted in GSH depletion, reactive oxygen species (ROS) formation, increased lipid peroxidation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase release. Both NAC and NACA protected against APAP-induced hepatotoxicity by restoring GSH levels, scavenging ROS, inhibiting lipid peroxidation, and preserving mitochondrial membrane potential. However, NACA was better than NAC at combating oxidative stress and protecting against APAP-induced damage. The higher efficiency of NACA in protecting cells against APAP-induced toxicity suggests that NACA can be developed into a promising therapeutic option for treatment of an APAP overdose.     

Ultrastructure of endothelium in ovules of Penstemon gentianoides Poir. (Scrophulariaceae) at mature embryo sac phase

In this study ultrastructural differences between endothelial cells of different location in Penstemon gentianoides have been examined with electron microscope at mature embryo sac phase. Embryo sac is of the Polygonum type and surrounded by endothelium except the micropylar region. The cuticle is located primarily around the chalazal three-fourths of the embryo sac. Endothelium cells around the chalaza and toward the micropylar region are rich in cytoplasmic organelles. The cytoplasm of endothelial cells near the central cell has large vacuoles and few organelles. There are also plasmodesmas on the anticlinal walls of endothelial cells. The endothelium and the micropylar integumentary cells play a role in transport of metabolites into the embryo sac.     

Highly active antiretroviral therapy drug combination induces oxidative stress and mitochondrial dysfunction in immortalized human blood-brain barrier endothelial cells

The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, the prevalence of HIV-1-associated neurocognitive disorders has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3'-azido-2',3'-deoxythymidine (AZT) and indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating this condition. The viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72h treatment, in a dose-dependent manner. Reactive oxygen species were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione and malondialdehyde, were altered in the treated groups. Loss of mitochondrial membrane potential, as assessed by fluorescence microscopy and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT+IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with the thiol antioxidant N-acetylcysteine amide reversed some of the pro-oxidant effects of AZT+IDV. Results from our in vitro studies indicate that the AZT+IDV combination may affect the BBB in HIV-infected individuals treated with HAART drugs.     

Spermine and putrescine enhance oxidative stress tolerance in maize leaves

The protective effects of spermine (SPM) and putrescine (PUT) against paraquat (PQ), a herbicide in agriculture and oxidative stress inducer, were investigated in the leaves of maize. Maize leaves were pretreated to SPM and PUT at concentrations of 0.2 and 1 mM and treated with PQ afterwards. Pretreatment with 1 mM of SPM and PUT significantly prevented the losses in chlorophyll and carotenoid levels induced by PQ. Ascorbic acid content in the leaves pretreated with both polyamines was found to be higher than those of the leaves pretreated with water. Also, pretreatment with SPM and PUT was determined to have some effects on the activities of superoxide dismutase (SOD) and peroxidase (POD). 1 mM of SPM increased SOD activity, but PUT has no significant effect on SOD activity. On the other hand, POD activity was recorded to increase slightly in response to both concentrations of SPM and 1 mM of PUT. The results showed that such polyamine pretreated plants may become more tolerant to oxidative stress due to increases in the antioxidative enzymes and antioxidants.