The ATP/metallothionein interaction: NMR and STM

We have previously established that ATP binds to mammalian metallothionein-2 (MT). The interaction between ATP and MT and the associated conformational change of the protein affect the sulfhydryl reactivity and zinc transfer potential of MT [Jiang, L.-J., Maret, W., and Vallee, B. L. (1998) The ATP-metallothionein complex. Proc. Natl. Acad. Sci. U.S.A. 95, 9146-9149]. NMR spectroscopic investigations have now provided further evidence for the interaction. (35)Cl NMR spectroscopy has further identified chloride as an additional biological MT ligand, which can interfere with the interaction of ATP with MT. (1)H NMR/TOCSY spectra demonstrate that ATP binding affects the N- and C-terminal amino acids of the MT molecule. Scanning tunneling microscopy recorded images of single MT molecules in buffered solutions. Moreover, this technique demonstrates that the otherwise nearly linear MT molecule bends by about 20 degrees at its central hinge region between the domains in the presence of ATP. These results may bear on the development of mild obesity in MT null mice and the role of MT in the regulation of energy balance. The interaction suggests a mechanism for the cellular translocation, retention, and reactivity of the ATP*MT complex in the mitochondrial intermembrane space. Both MT and ATP are localized there, and MT and thionein alternately bind and release zinc, thereby affecting mitochondrial respiration.     

Beta-alanine protection against hypoxic liver injury in the rat

Liver hypoxia still represents an important cause of liver injury during shock and liver transplantation. We have investigated the protective effects of beta-alanine against hypoxic injury using isolated perfused rat livers and isolated rat hepatocyte suspensions. Perfusion with hypoxic Krebs-Henseleit buffer increased liver weight and caused a progressive release of lactate dehydrogenase (LDH) in the effluent perfusate. The addition of 5 mmol/l beta-alanine to the perfusion buffer completely prevented both weight increase and LDH leakage. These findings were confirmed by histological examinations showing that beta-alanine blocked the staining by trypan blue of either liver parenchymal and sinusoidal cells. Studies performed in isolated hepatocytes revealed that beta-alanine exerted its protective effects by interfering with Na+ accumulation induced by hypoxia. The addition of gamma-amino-butyric acid, which interfered with beta-alanine uptake by the hepatocytes or of Na+/H+ ionophore monensin, reverted beta-alanine protection in either hepatocyte suspensions or isolated perfused livers. We also observed that liver receiving beta-alanine were also protected against LDH leakage and weight increase caused by the perfusion with an hyposmotic (205 mosm) hypoxic buffer obtained by decreasing NaCl content from 118 to 60 mmol/l. This latter effect was not reverted by blocking K+ efflux from hepatocyte with BaCl(2) (1mmol/l). Altogether these results indicated that beta-alanine protected against hypoxic liver injury by preventing Na+ overload and by increasing liver resistance to osmotic stress consequent to the impairment of ion homeostasis during hypoxia.     

Pharmacophore-based molecular docking to account for ligand flexibility

Rapid computational mining of large 3D molecular databases is central to generating new drug leads. Accurate virtual screening of large 3D molecular databases requires consideration of the conformational flexibility of the ligand molecules. Ligand flexibility can be included without prohibitively increasing the search time by docking ensembles of precomputed conformers from a conformationally expanded database. A pharmacophore-based docking method whereby conformers of the same or different molecules are overlaid by their largest 3D pharmacophore and simultaneously docked by partial matches to that pharmacophore is presented. The method is implemented in DOCK 4.0.     

CNTNAP2 is disrupted in a family with Gilles de la Tourette syndrome and obsessive compulsive disorder

Gilles de la Tourette syndrome (GTS) is a sporadic or inherited complex neuropsychiatric disorder characterized by involuntary motor and vocal tics. There is comorbidity with disorders like obsessive compulsive disorder and attention deficit hyperactivity disorder. Until now linkage analysis has pointed to a number of chromosomal locations, but has failed to identify a clear candidate gene(s). We have investigated a GTS family with a complex chromosomal insertion/translocation involving chromosomes 2 and 7. The affected father [46,XY,inv(2) (p23q22),ins(7;2) (q35-q36;p21p23)] and two affected children [46,XX,der(7)ins(7;2)(q35-q36;p21p23) and 46,XY,der(7)ins(7;2)(q35-q36;p213p23)] share a chromosome 2p21-p23 insertion on chromosome 7q35-q36, thereby interrupting the contactin-associated protein 2 gene (CNTNAP2). This gene encodes a membrane protein located in a specific compartment at the nodes of Ranvier of axons. We hypothesize that disruption or decreased expression of CNTNAP2 could lead to a disturbed distribution of the K(+) channels in the nervous system, thereby influencing conduction and/or repolarization of action potentials, causing unwanted actions or movements in GTS.     

In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1

The role of fibroblast growth factors and their receptors (FGFRs) in the regulation of normal hematopoietic stem cells is unknown. Here we show that, in mouse bone marrow, long-term repopulating stem cells are found exclusively in the FGFR(+) cell fraction. During differentiation toward committed progenitors, stem cells show loss of FGFR expression. Prolonged culture of bone marrow cells in serum-free medium supplemented with only FGF-1 resulted in robust expansion of multilineage, serially transplantable, long-term repopulating hematopoietic stem cells. Thus, we have identified a simple method of generating large numbers of rapidly engrafting stem cells that have not been genetically manipulated. Our results show that the multipotential properties of stem cells are dependent on signaling through FGF receptors and that FGF-1 plays an important role in hematopoietic stem cell homeostasis.     

The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function

Brain-derived neurotrophic factor (BDNF) modulates hippocampal plasticity and hippocampal-dependent memory in cell models and in animals. We examined the effects of a valine (val) to methionine (met) substitution in the 5' pro-region of the human BDNF protein. In human subjects, the met allele was associated with poorer episodic memory, abnormal hippocampal activation assayed with fMRI, and lower hippocampal n-acetyl aspartate (NAA), assayed with MRI spectroscopy. Neurons transfected with met-BDNF-GFP showed lower depolarization-induced secretion, while constitutive secretion was unchanged. Furthermore, met-BDNF-GFP failed to localize to secretory granules or synapses. These results demonstrate a role for BDNF and its val/met polymorphism in human memory and hippocampal function and suggest val/met exerts these effects by impacting intracellular trafficking and activity-dependent secretion of BDNF.     

Analysis of beta-catenin, Ki-ras, and microsatellite stability in azoxymethane-induced colon tumors of BDIX/Orl Ico rats

The aim of the study reported here was to investigate whether the azoxymethane (AOM)-induced colon cancer rat model mimics the human situation with regard to microsatellite stability, changes in expression of beta-catenin, and/or changes in the sequence of the proto-oncogene Ki-ras. Colon cancer was induced by administration of four weekly doses of AOM (15 mg/kg of body weight per week) separated by a one-week break between the second and third injections. As the histopathologic characteristics of this model resemble those of the human counterpart, further characterization of the genetic changes was undertaken. The animals were euthanized 28 to 29 weeks after the first AOM injection, and tumor specimens were taken for histologic and DNA analyses. Since microsatellite variation was found in only a few (< 2%) specimens, the model can be considered as having stable microsatellites. This result is in accordance with those of similar studies in other rat models and with most human colorectal cancers. Immunohistochemical analyses of beta-catenin did not reveal loss of gene activity, nor did the sequencing of Ki-ras reveal mutations. These results are in contrast to most findings in comparable rat studies. The deviations may be due to differences in exposure to the carcinogen or difference in strain and/or age. The lack of beta-catenin and Ki-ras alterations in this colon cancer model is unlike human sporadic colorectal cancers where these genetic changes are common findings.     

Creatine supplementation in health and disease: What is the evidence for long-term efficacy?

Creatine supplementation is an established ergogenic aid in sports and is now claimed to have therapeutical applications in a variety of diseases. The available literature mainly covers the short-term (one to several weeks) effects of creatine supplementation on skeletal muscle function in health and disease, which is of little help to evaluate the long-term (two or more months) potential of creatine as a drug in chronic disorders, such as neurodegenerative diseases or muscular dystrophies. Recent findings in healthy humans indicate that the beneficial effect on muscle function and muscle total creatine content may disappear when creatine is continuously ingested for more than two or three months. The mechanism for this habituation to chronic creatine exposure is poorly understood. The primary purpose of the present review article is to critically evaluate the available evidence for long-term efficacy of creatine administration and to hypothesize about ways to optimize creatine administration regimens.     

Cholesterol and copper in the liver of rabbit inbred strains with differences in dietary cholesterol response

In order to investigate whether cholesterol intake influences the hepatic copper content of rabbits, we compared the hepatic copper content of two rabbit inbred strains after feeding the animals a control or a cholesterol-rich diet. One strain was not reactive to dietary cholesterol (IIIVO/JU), whereas the other strain was reactive to dietary cholesterol (AX/JU). The coefficient of inbreeding (F) >0.95 for both strains. Dietary cholesterol-reactive rabbits when compared with their non-reactive counterparts had a higher hepatic copper content. The consumption of a hypercholesterolemic diet decreased liver copper concentration (expressed in micro g/g dry weight) in both strains of rabbits, which was (in part) due to dietary-induced hepatomegaly. A decrease in the absolute hepatic copper content was found only in the dietary cholesterol-reactive inbred strain. It is discussed that differences in glucocorticoid levels may be responsible for the strain difference in liver copper content. The cholesterol effect on the hepatic copper content in the reactive strain might be caused by an increased bilirubin secretion.     

Mechanism of Osmotic Activation of the Quaternary Ammonium Compound Transporter (QacT) of Lactobacillus plantarum

The accumulation of quaternary ammonium compounds in Lactobacillus plantarum is mediated via a single transport system with a high affinity for glycine betaine (apparent K_m of 18 μM) and carnitine and a low affinity for proline (apparent K_m of 950 μM) and other analogues. Mutants defective in the uptake of glycine betaine were generated by UV irradiation and selected on the basis of resistance to dehydroproline (DHP), a toxic proline analogue. Three independent DHP-resistant mutants showed reduced glycine betaine uptake rates and accumulation levels but behaved similarly to the wild type in terms of direct activation of uptake by high-osmolality conditions. Kinetic analysis of glycine betaine uptake and efflux in the wild-type and mutant cells is consistent with one uptake system for quaternary ammonium compounds in L. plantarum and a separate system(s) for their excretion. The mechanism of osmotic activation of the quaternary ammonium compound transport system (QacT) was studied. It was observed that the uptake rates were inhibited by the presence of internal substrate. Upon raising of the medium osmolality, the QacT system was rapidly activated (increase in maximal velocity) through a diminished inhibition by trans substrate as well as an effect that is independent of intracellular substrate. We also studied the effects of the cationic amphipath chlorpromazine, which inserts into the cytoplasmic membrane and thereby influences the uptake and efflux of glycine betaine. The results provide further evidence for the notion that the rapid efflux of glycine betaine upon osmotic downshock is mediated by a channel protein that is responding to membrane stretch or tension. The activation of QacT upon osmotic upshock seems to be brought about by a turgor-related parameter other than membrane stretch or tension.