Preconditioning of cortical neurons by oxygen-glucose deprivation: tolerance induction through abbreviated neurotoxic signaling

Transient exposure of rat cortical cultures to nonlethal oxygen-glucose deprivation (OGD preconditioning) induces tolerance to otherwise lethal oxygen-glucose deprivation (OGD) or N-methyl-D-aspartate 24 h later. This study evaluates the role of cytosolic and mitochondrial Ca²⁺-dependent cellular signaling. Mechanistic findings are placed in context with other models of ischemic preconditioning or known neurotoxic pathways within cortical neurons. Tolerance to otherwise lethal OGD is suppressed by performing OGD preconditioning in the presence of the broad-scope catalytic antioxidants Mn(III)tetra(4-carboxyphenyl)porphyrin (MnTBAP) or Zn(II)tetra(4-carboxyphenyl)porphyrin [Zn(II)TBAP], but not by a less active analog, Mn(III)tetra(4-sulfonatophenyl)porphyrin, or a potent superoxide scavenger, Mn(III)tetra(N-ethyl-2-pyridyl)porphyrin chloride. Inhibitors of adenosine A₁ receptors, nitric oxide synthase, mitogen-activated protein kinase, and poly(ADP-ribose) polymerase fail to suppress OGD preconditioning despite possible links with reactive oxygen species in other models of ischemic preconditioning. Preconditioning is suppressed by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which has been ascribed elsewhere to inhibition of superoxide transport to the cytosol through mitochondrial anion channels. However, although it induces mitochondrial Ca²⁺ uptake, neuronal preconditioning is largely insensitive to mitochondrial uncoupling with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone or 2,4-dinitrophenol. Un-couplers will prevent production of mitochondrial reactive oxygen species, implying nonmitochondrial targets by MnTBAP, Zn(II)TBAP, and DIDS. Emphasizing the importance of an increase in cytosolic Ca²⁺ during preconditioning, a Ca²⁺/calmodulin-dependent protein kinase II inhibitor, KN-62, suppresses development of subsequent tolerance. Summarizing, only those cellular transduction pathways that have the potential to be neurotoxic may be activated by preconditioning in cortical neurons. Finally, a marked decrease in extracellular glutamate is observed during otherwise lethal OGD in preconditioned cultures, suggesting that this end effector may represent a point of convergence across different preconditioning models. N-methyl-D-aspartate; Ca²⁺; antioxidants; mitochondria     

Identification of a novel series of selective 5-HT7 receptor antagonists

Novel 5-HT(7) receptor antagonists containing the benzocycloheptanone core were identified from high throughput screening. Molecular modelling and SAR studies have converted these intractable hits into a more potent, selective and tractable series, exemplified by compound (25), SB-691673.     

Suppression of fructokinase encoded by LeFRK2 in tomato stem inhibits growth and causes wilting of young leaves

Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.     

Determining subcellular localization of novel drug targets by transient transfection in COS cells

Genomics-based approaches are increasingly being used to identify disease-associated genes that represent potential new drug targets. As a first step in the validation of genes of unknown function, we describe a method for rapidly determining the subcellular localization of the gene product. If an immunotherapeutic approach is being considered, it is of particular interest to identify targets that are either on the cell-surface or secreted. Transient expression in COS cells combined with immunofluorescent staining provides a semi-high throughput method for determining the subcellular localization of multiple targets in parallel. COS cells are ideal for this purpose since: (i) they transfect easily; (ii) the high levels of expression that can be achieved transiently allow detection after 24 h; and (iii) the relatively large size and spread morphology of these cells allows the subcellular organelles to be easily visualized. To evaluate the system, we show prototype staining patterns for known cytoplasmic,secreted, Golgi-associated, endoplasmic reticulum-associated, and plasma membrane proteins, as well as data for novel targets. The localization of novel secretory and cell-surface proteins as determined by immunofluorescent staining, was confirmed by independent methods. This revised version was published online in July 2006 with corrections to the Cover Date.     

beta-hydroxy-beta-methylbutyrate (HMB) kinetics and the influence of glucose ingestion in humans

The dietary supplement, beta-hydroxy-beta-methylbutyrate (HMB), has been shown to decrease muscle proteolysis during the stress of exercise and disease. The aim of this investigation was to determine the time course kinetics of HMB and to determine whether oral glucose ingestion alters the kinetics. In Study 1, eight males (32 +/- 10 yrs) participated in two randomize trials: 1) oral ingestion of 1g of HMB with water in capsule form (HMB), and 2) placebo. Blood samples were obtained prior to ingestion of treatment and at 30, 60, 90, 120, 150, and 180 min for the measurement of plasma HMB. Additional blood samples were obtained at 6, 9, and 12 hr. Urine was collected prior to ingestion and at 3, 6, 9, and 12 h for the measurement of urinary HMB. In Study 2, eight males (25 +/- 6 yrs) followed the same study design and testing procedure as for Study 1. Treatments were 1) modified glucose tolerance test (75 g glucose) (GLU), 2) oral ingestion of 3 g of HMB with water (HMB), and 3) ingestion of 3 g of HMB with 75 g of glucose (HMB+GLU). Blood samples were analyzed for insulin, glucose, and HMB. Additional blood samples were obtained at 24h and 36h for the measurement of HMB. Additional urine samples were collected at 24h and 36h. In Study 1, plasma HMB peaked at 120 nmol/ml at 2.0 +/- 0.4 hr in HMB trial. Half-life was 2.37 +/- 0.1 hr. Following the consumption of 1g of HMB, approximately 14% of the HMB consumed accumulated in the urine. In Study 2, plasma glucose and insulin levels were significantly greater in GLU and HMB+GLU treated subjects compared to HMB treated subject at minutes 30, 60 and 90. Plasma HMB peaked at 487.9 +/- 19.0 nmol/ml at 1.0 +/- 0.1 hr in the HMB treated subjects and at 352.1 +/- 15.3 nmol/ml at 1.94 +/- 0.2 hr when subjects consumed HMB+GLU. The time to reach peak was different (P <0.001) between HMB and HMB+GLU. The plasma HMB half-life was less (P = 0.08) 2.38 +/- 0.1 hr in HMB trial compared to 2.69 +/- 0.2 hr in HMB+GLU trial. Area under the plasma HMB curve during the first 3 hr was less (P = 0.002) in the HMB+GLU trial compared to the HMB trial. From 3 h through 36 h the area under the HMB curve tended to be less (P = 0.106) for the HMB+GLU compared to the HMB alone. HMB accumulation in the urine as well as the area under the curve were similar with both HMB (94875.8 +/- 15159.5 nmol/36 hrs) and HMB+GLU (80678.2 +/- 3863.1 nmol/36 hrs). The percentage of the HMB dose that accumulates in the urine was 27% for HMB+GLU and 29% for HMB alone. In conclusion, HMB plasma levels peak within 60 to 120 min depending on the amount of HMB consumed and whether glucose is consumed with HMB. The plasma half-life is approximately 2.5 hr. Plasma HMB reaches baseline levels at approximately 9 hr following ingestion. However, 70 to 85% of the ingested oral HMB is retained in the body for further metabolism.     

Structural and biochemical identification of a novel bacterial oxidoreductase

By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum- and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.     

Synergistic antifungal activity of two chitin-binding proteins from spindle tree (<Emphasis Type="Italic">Euonymus europaeus</Emphasis> L.)

Two structurally different chitin-binding proteins were isolated from bark and leaves of the spindle tree (Euonymus europaeus L.). Both the small hevein-like chitin-binding protein (Ee-CBP) and the classical class-I chitinase (Ee-chitinase) possess antifungal properties, Ee-CBP being far more potent than Ee-chitinase. In addition, Ee-CBP and Ee-chitinase display a pronounced synergistic effect when added together in the test medium. Determination of the biological activities indicates that the synergism between Ee-CBP and Ee-chitinase relies on a different mode of action. Cloning and sequencing of the corresponding genes further revealed that Ee-CBP and Ee-chitinase are simultaneously expressed in bark and leaf tissues, and hence can act synergistically in planta. Moreover, analysis of the deduced sequences allowed the exact relationship between the structurally different Ee-CBP and Ee-chitinase to be corroborated. Both proteins are synthesized as similar chimeric precursors consisting of an N-terminal hevein domain linked to a C-terminal chitinase-like domain by a hinge region. However, whereas in the case of Ee-chitinase the C-terminal chitinase domain remains linked to the N-terminal hevein domain, the corresponding domain is cleaved from the Ee-CBP-precursor resulting in the formation of the hevein-type Ee-CBP. Since both precursors are—apart from the hinge region between the hevein and chitinase domains—very similar, the Ee-CBP/Ee-chitinase system offers a unique opportunity to study the importance of sequence and/or structural information comprised in the hinge region for the posttranslational processing of the respective precursor proteins.Abbreviations AMP Antimicrobial protein - CBP Chitin-binding protein - GlcNAc N-Acetylglucosamine - HCA Hydrophobic cluster analysis - MeJA Methyl jasmonate - PDB Potato dextrose broth - RACE Rapid amplification of cDNA ends - SPR Surface plasmon resonance - UDA Urtica dioica agglutinin - WGA Wheat (Triticum aestivum) germ agglutinin     

Hyper IgE in New Zealand black mice due to a dominant-negative CD23 mutation

Immunoglobulin E (IgE) plays a critical role in both resistance to parasitic infection and allergy to environmental antigens. The IgE response is in turn regulated by the B-cell co-receptor CD23, and CD23-deficient mice show exaggerated IgE responses and airway hyper-responsiveness. In this report, we show that New Zealand black (NZB) mice express a variant CD23 allele, with mutations in both the C-lectin-binding domain and stalk region, which fails to bind IgE at high affinity and has reduced expression on the cell surface. Expression of the variant CD23 chain interferes with trimerisation of the receptor and has a dominant-negative effect leading to reduced IgE binding in crosses between NZB and other strains. Genetic mapping shows that the variant CD23 leads to an exaggerated primary IgE response, which is independent of other strain-specific effects. These results suggest that NZB mice or mice carrying the variant allele will be useful models for studying both allergy and quantitative traits associated with atopy. The exaggerated IgE response provides an explanation for the natural resistance of NZB mice to parasitic infection by Leishmania.     

Gestational ethanol exposure disrupts the expression of FGF8 and Sonic hedgehog during limb patterning

BACKGROUND: Ethanol is known to induce a wide variety of gestational anomalies, including skeletal malformations. Gestational ethanol exposure in mice has been shown to induce postaxial digit loss (ectrodactyly). How ethanol induces limb malformations is not understood. To better understand how ethanol effects limb development, we have utilized a transgenic line of mice that expresses beta-galactosidase in the apical ectodermal ridge (AER) of the limbs throughout gestation. METHODS: Pregnant female mice were injected with 2.9, 3.4, or 3.9 gm/kg ethanol at E9.3 and E9.5; embryos were isolated at E11.25, stained for beta-galactosidase activity, and evaluated for AER defects. Based upon the pattern of defects seen, expression of FGF8 in the AER and Sonic hedgehog in the postaxial mesoderm was evaluated by in situ hybridization. RESULTS: Two distinct phenotypes were seen in response to ethanol that were dose dependent. At 2.9 gm/kg ethanol, the most prevalent phenotype was a mislocalization of the AER to regions both dorsal and ventral to the midline. A higher dosage of 3.4 gm/kg ethanol did not increase the mislocalization phenotype, but resulted in a higher frequency of postaxial loss of the AER and associated mesenchymal tissue. The highest dosage utilized (3.9 gm/kg) resulted in a high frequency of both preaxial and postaxial loss of the AER. Through in situ hybridization, we found that ethanol exposure resulted in a concomitant reduction in FGF8 expression in the AER and Sonic hedgehog expression from the zone of polarizing activity (ZPA). CONCLUSIONS: We propose a model where ethanol disrupts the AER/ZPA positive feedback loop to induce postaxial malformations. Preaxial malformations seen at higher ethanol dosage suggest FGF8 as a critical target of ethanol in producing limb defects.     

Mre11 assembles linear DNA fragments into DNA damage signaling complexes

Mre11/Rad50/Nbs1 complex (MRN) is essential to suppress the generation of double-strand breaks (DSBs) during DNA replication. MRN also plays a role in the response to DSBs created by DNA damage. Hypomorphic mutations in Mre11 (which causes an ataxia-telangiectasia-like disease [ATLD]) and mutations in the ataxia-telangiectasia-mutated (ATM) gene lead to defects in handling damaged DNA and to similar clinical and cellular phenotypes. Using Xenopus egg extracts, we have designed a simple assay to define the biochemistry of Mre11. MRN is required for efficient activation of the DNA damage response induced by DSBs. We isolated a high molecular weight DNA damage signaling complex that includes MRN, damaged DNA molecules, and activated ATM. Complex formation is partially dependent upon Zn²⁺ and requires an intact Mre11 C-terminal domain that is deleted in some ATLD patients. The ATLD truncation can still perform the role of Mre11 during replication. Our work demonstrates the role of Mre11 in assembling DNA damage signaling centers that are reminiscent of irradiation-induced foci. It also provides a molecular explanation for the similarities between ataxia-telangiectasia (A-T) and ATLD.