Presence Of Histidine Research Articles

FCγRIIa Polymorphism Is Not Associated with Overall Response in Diffuse Large B-Cell Lymphoma Treated with Rituximab.

Abstract 4994 BackgroundLow-affinity receptor for the Fc region of immunoglobulin G (IgG) (FcγR) is constitutively expressed on resting human neutrophils. These receptor, termed FcγRIIa display biallelic polymorphism which have functional consequences with respect to binding and/or ingestion of targets opsonized by human IgG subclass antibodies. Rituximab is a chimeric monoclonal antibody directed against CD20, an antigen found in most B-cell malignancies. Multiple mechanisms have been proposed for the activity of Rituximab, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and a direct proapoptotic effect. F(ab')2 Rituximab homodimers were shown to be effective in inducing apoptosis of B-cell lymphoma cell lines in vitro. Recently, it have been established that ADCC is important as predominant mechanism of lymphoma cell clearance and that Fcγ receptors (FcγRs) are critical for the in vivo actions of Rituximab in non-Hodgkin lymphoma (NHL). A genomic polymorphism at amino acid 131 of FcγRIIA has been described whereby the presence of Histidine (H) rather than Arginine is associated with responses to the CD20-directed immunoglobulin G1 (IgG1) Rituximab among patients with indolent lymphoma. FcγRIIA genotype have been associated with a better clinical and molecular response in follicular lymphoma patients treated as first line therapy with Rituximab alone and in patients with diffuse large B-cell lymphoma (DLBCL) treated with the concomitant administration of Rituximab and CHOP (R-CHOP). MethodsHere we analyzed the role of specific polymorphism of activating FcγRIIA in 42 patients with DLBCL treated with R-CHOP concerning prediction complete response (CR) using a polymerase chain reaction-restriction fragment length polymorphism method. ResultsThe median age of the patients was 48 years (15 to 82). Out of the 42 patients 18 (42.8%) were stage III-IV and 17 (40.4%) had more than 2 factors of the International Prognostic Index. Thirty-seven (87.6%) had CR, 1 (2.3%) had partial remission (PR) and 4 (10.1%) had refractory disease (RD). Four ( 10.8%) of the patients that acquired CR relapsed. Deaths occurred in 3 (7.1%) patients with follow up of 2 years. Eight (19%) patients showed polymorphism HH, 21 (50%) HR and 13 (31%) RR. The univariate analysis did not show correlation between FcγRIIa H/H or R allele polymorphism and CR (p > 0.05). ConclusionContrary to recent report we showed that FcγRIIa polymorphism is not associated to overall response in patients with DLBCL treated with Rituximab. DisclosuresNo relevant conflicts of interest to declare.

Caenorhabditis elegans metallothionein isoform specificity – metal binding abilities and the role of histidine in CeMT1 and CeMT2

Two metallothionein (MT) isoforms have been identified in the model nematode Caenorhabditis elegans: CeMT1 and CeMT2, comprising two polypeptides that are 75 and 63 residues in length, respectively. Both isoforms encompass a conserved cysteine pattern (19 in CeMT1 and 18 in CeMT2) and, most significantly, as a result of their coordinative potential, CeMT1 includes four histidines, whereas CeMT2 has only one. In the present study, we present a comprehensive and comparative analysis of the metal [Zn(II), Cd(II) and Cu(I)] binding abilities of CeMT1 and CeMT2, performed through spectroscopic and spectrometric characterization of the recombinant metal-MT complexes synthesized for wild-type isoforms (CeMT1 and CeMT2), their separate N- and C-terminal moieties (NtCeMT1, CtCeMT1, NtCeMT2 and CtCeMT2) and a DeltaHisCeMT2 mutant. The corresponding in vitro Zn/Cd- and Zn/Cu-replacement and acidification/renaturalization processes have also been studied, as well as protein modification strategies that make it possible to identify and quantify the contribution of the histidine residues to metal coordination. Overall, the data obtained in the present study are consistent with a scenario where both isoforms exhibit a clear preference for divalent metal ion binding, rather than for Cu coordination, although this preference is more pronounced towards cadmium for CeMT2, whereas it is markedly clearer towards Zn for CeMT1. The presence of histidines in these MTs is revealed to be decisive for their coordination performance. In CeMT1, they contribute to the binding of a seventh Zn(II) ion in relation to the M(II)(6)-CeMT2 complexes, both when synthesized in the presence of supplemented Zn(II) or Cd(II). In CeMT2, the unique C-terminal histidine abolishes the Cu-thionein character that this isoform would otherwise exhibit.

Lysine-Functionalized Silver Nanoparticles for Visual Detection and Separation of Histidine and Histidine-Tagged Proteins

A new chromogenic chemosensor based on lysine-functionalized silver nanoparticles 1 was prepared and characterized by transmission electron microscopy (TEM), Fourier transform Raman, and ultraviolet-visible (UV-vis) spectroscopy. The color changes of nanoparticles 1 in the absence and the presence of metal ion were observed upon addition of various amino acids and proteins in aqueous solution. Among the various amino acids, the sensor 1 in the absence of metal ion shows a novel colorimetric sensor with capability to probe histidine and histidine-tagged proteins. On the other hand, the color changes of 1 in the presence of metal ions such as KCl or NiCl(2) did not occur with any amino acids. Therefore, the sensor 1 in the absence of metal ion responds selectively to histidine, a response which can be attributed to its aggregation induced by histidine with high numbers of electrostatic interactions. This highly selective sensor 1 allows a rapid quantitative assay of histidine to concentrations as low as 5.0 microM, providing a new tool for the direct measurement of histidine and histidine-tagged proteins in vitro system. Furthermore, we examined the effect of pH on absorbance (A(520)) of 1 in the presence of histidine (pH 4-12). The absorbance under basic conditions was higher than that under acidic or neutral conditions, in accord with the stronger aggregation of 1 with histidine by electrostatic interaction between the carboxylate anion of 1 and ammonium protons of histidine under basic conditions.

Elevated cleavage of human immunoglobulin gamma molecules containing a lambda light chain mediated by iron and histidine

Monoclonal antibodies in liquid formulation undergo nonenzymatic hydrolysis when stored at 5 °C for extended periods. This hydrolysis is enhanced at extreme pH and high temperature. In this study we discover that iron in the presence of histidine also enhanced cleavage of human immunoglobulin gamma (IgG) molecules containing a lambda light chain when incubated at 40 °C. The level of cleavage was concentration dependent on both iron and histidine levels. Enhanced cleavage with iron and histidine was not observed on IgG molecules containing a kappa light chain. Using CE-SDS to quantify levels of Fab + Fc, the Fab arm, and free light chain (LC) and heavy chain (HC) fragments, we show that cleavage resulted in elevated levels of free light and heavy chain fragments. Using MS we find elevated scission between residues E/C on the LC resulting in LC fragment 1–215. We also observed enhanced cleavage between S/C residues of the HC resulting in HC fragment 1–217. The corresponding Fab + Fc fragment beginning with cys-218 was not found. Instead, we find elevation of a Fab + Fc fragment that began with aspartic acid (cleavage between C/D). Further studies to understand how iron and histidine enhance cleavage of lambda light chain IgG molecules are ongoing.

A modified suspension test for estimating the mutagenicity of samples containing free and (or) protein-bound histidine

The Ames test has not been very effective in estimating the mutagenicity of histidine-containing samples because external free and (or) protein-bound histidine in these samples would allow the histidine auxotrophs in such test samples to grow more compared with the negative controls that were used as the reference. This could give rise to a false positive.n this study, a modified suspension mutagenicity assay (MS assay) was developed. The tester strains were incubated in Luria-Bertani (LB) broth containing different concentrations of traditional Chinese medicines (TCMs) until the declining phase, and the test samples were assayed to be mutagenic or not by observing whether statistically significant differences were demonstrated in the relative reversion frequencies (RRFs) between the negative control groups and the test groups. Collectively, using LB broth as the test medium and comparing the RRFs in the declining phase made this assay less influenced by the presence of histidine in the test samples.The mutagenicity of some TCMs was measured with the MS assay. The results in MS assay were consistent with those in the mammalian bone marrow chromosomal aberration test, which indicated that the MS assay was appropriate to estimate the mutagenicity of samples containing free and (or) protein-bound histidine.

Repair of O6-methylguanine to guanine by cysteine in the absence and presence of histidine and by cysteine thiolate anion: a quantum chemical study

O6-methylguanine (O6mG) is known to be a potential mutagenic modification of guanine as it mispairs with thymine in DNA and causes GC to AT transversion mutation. It is experimentally known that O6mG can be repaired to guanine by the protein O6-alkylguanine-DNA alkyltransferase (AGT), a cysteine residue being the main active site. In the present work, the mechanisms of repair of cis-O6-methylguanine (O6mG) to guanine due to its reaction with cysteine in the absence and presence of histidine and with cysteine thiolate anion were investigated theoretically using the B3LYP hybrid functional of density functional theory and the second order Møller-Plesset perturbation (MP2) theory. Reactant, intermediate and product complexes as well as transition states involved in these reactions were fully optimized at the B3LYP/6-31 + G* level of theory in the gas phase. The solvent effect of water was treated using the polarizable continuum model (PCM). Single point energy calculations were performed at the B3LYP/AUG-cc-pVDZ and MP2/6-31 + G* levels of theory in the gas phase and aqueous media. It is found that cysteine alone can repair the cis-O6mG to guanine, but the involvement of histidine along with cysteine lowers down the barrier energy significantly. However, when cysteine thiolate anion is used in place of cysteine, the barrier energy is strongly reduced. These results broadly support the suggestions based on experimental studies.

Involvement of Reactive Oxygen Radicals in Photoinhibition of Primary Photosynthetic Reactions—Effect of Temperature and Oxygen Radical Scavengers

ABSTRACTExposure of leaves or chloroplasts to high light intensity leads to inactivation of photosynthesis. Two processes are observed—inhibition of photochemical activity of both photosystems and photobleaching of pigments. Oxygen evolving complex, located at the oxidizing side of Photosystem II, is the most sensitive component of photosynthetic apparatus to environmental stress factors. In the present work the effect of high light treatment at room and low temperatures on kinetic parameters of flash oxygen yields and oxygen evolution were studied. Isolated thylakoid membranes were subjected to high light illumination for different periods of time at room (22°C) and low (4°C) temperature. Flash oxygen yields were determined using fast oxygen rate electrode. Photochemical activity of photosystem II was measured by Clark oxygen electrode using artificial electron acceptor. Data presented show that the damaging effect of high light treatment on oxygen evolution is lower at 4°C than at 22°C. When high light treatment was carried out in the presence of histidine and DMSO—scavengers of oxygen radicals, the inhibition process was retarded. Data are discussed in terms of different production rate and mobility of oxygen radicals at room and low temperature.

Histidine at codon 154 of the prion protein gene is a risk factor for Nor98 scrapie in goats

Prion protein gene (PRNP) polymorphisms are involved in modulating the appearance of atypical/Nor98 scrapie in sheep, with the alleles AHQ and AF141RQ strongly associated with occurrence of the disease. The presence of histidine at codon 154 has also been detected in Nor98-affected goats, but statistical analysis of the association between Nor98 and goat PRNP polymorphisms has not been reported previously. Here, a case-control study was carried out on eight Nor98-positive goats and 246 negative herdmates belonging to eight Italian Nor98 scrapie outbreaks. The results revealed that histidine at codon 154 is also strongly associated with the disease in goats.

Prey location and prey choice by the freshwater leech Erpobdella octoculata using foraging kairomones

Summary1. The freshwater leech, Erpobdella octoculata, is a generalist predator feeding on prey organisms such as Tubifex spp., Chironomus spp. and Asellus aquaticus. Using different experimental designs, we studied the use of foraging kairomones by the leeches for prey location.2. Leeches were attracted to living as well as to freshly killed larvae of Chironomus sp., to Tubifex sp., and to A. aquaticus offered in Petri dishes covered with gauze. The leeches also reacted to an extract of macerated Tubifex sp. presented in agar.3. Using ion exchange chromatography, the presence of histidine and glutamic acid was demonstrated in water contaminated by living larvae of Chironomus sp. and Tubifex sp. Agar blocks containing a synthetic mixture of these compounds at concentrations above 5 mg mL−1 were attractive to the leeches.4. Choice tests showed that leeches preferred chemical signals from Tubifex sp. over larvae of Chironomus sp. No difference was found between chemicals from Tubifex sp. and A. aquaticus, and A. aquaticus and larvae of Chironomus sp.5. The results demonstrate that E. octoculata uses specific foraging kairomones in searching for prey and indicate that amino acids serve as foraging kairomones.

Photoprotection by Porcine Eumelanin Against Singlet Oxygen Production†

Melanin, a major pigment found in retinal pigment epithelium (RPE) cells, is considered to function in dual roles, one protective and one destructive. By quenching free radical species and reactive oxygen species (ROS) melanin counteracts harmful redox stress. However, melanin is also thought to be capable of creating ROS. In this destructive role, melanin increases redox strain in the cell. This study uses readily available eumelanin extracted from porcine RPE cells as a more authentic model than synthetic melanin to determine specific mechanisms of melanin activity with regard to singlet oxygen in the presence and absence of rose bengal, a singlet-oxygen photosensitizer. Optical detection of singlet-oxygen was determined by monitoring the bleaching of p-nitrosodimethylaniline in the presence of histidine. Production of singlet oxygen in aqueous oxygen-saturated solutions of rose bengal without eumelanin was readily accomplished. In contrast, detection of singlet oxygen in oxygen-saturated solutions of eumelanin without rose bengal failed, consistent with results of others. However, a significant decrease in singlet oxygen production by rose bengal was observed in the presence of eumelanin. After correction for light absorption and chemical bleaching of eumelanin, the results show that eumelanin also provides a photoprotective mode arising from chemistry, that is, not just the physical process of light absorption followed by energy dissipation as heat.

Assessment of Macrocyclic Triamine Ligands As Synthons for Organometallic 99mTc Radiopharmaceuticals

In a search of coordination molecules suitable to the fac-{(99m)Tc(CO)3}(+) core as a synthon for (99m)Tc-radiopharmaceuticals, nonradioactive rhenium complexes of two macrocyclic triamine compounds with different chelate ring structures, 1,4,7-triazacyclononane (9N3) and 1,5,9-triazacyclododecane (12N3), were synthesized and characterized. (99m)Tc-labeled 9N3 and 12N3 compounds were also prepared using [(99m)Tc(OH 2)3(CO)3](+) and were characterized by both in vitro and in vivo studies. 9N3 produced a single rhenium complex, whereas 12N3 generated two major complexes. The crystallographic data and infrared absorption wavenumber assigned to the C-O stretch suggested that the coordination geometry of 9N3 would be more suitable to fac-{Re(CO)3}(+) than that of 12N3. In contrast, both 9N3 and 12N3 provided a single (99m)Tc-labeled compound. However (99m)Tc-labeled 9N3 exhibited higher stability than (99m)Tc-labeled 12N3 in rat plasma and in the presence of histidine at an elevated temperature. In biodistribution studies, both (99m)Tc-labeled compounds did not show any specific accumulation of radioactivity in any organs except for the excretory organs such as the liver and kidney. These findings showed that 9N3 would constitute a macrocyclic chelating molecule of choice to prepare (99m)Tc radiopharmaceuticals using a fac-{(99m)Tc(CO)3}(+) core.

The role of ascorbate and histidine in fibrinogen protection against changes following exposure to a sterilizing dose of γ-irradiation

Sodium ascorbate and histidine were employed to protect fibrinogen against modifications followed by a gamma-irradiation process that could potentially inactivate the blood-borne viruses in plasma-derived products. Fibrinogen was irradiated (50 kGy total dose, on dry ice) using a 60Co source. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. Carbonyl groups were measured by the 2,4-dinitrophenylhydrazine-coupled method, and the fibrinogen clotting activity was assessed by different functional assays. In irradiated fibrinogen, the carbonyl group concentration was elevated three-fold versus control; and moderate fragmentation of largely Aalpha and Bbeta chains was revealed. The rate of thrombin-catalyzed fibrinogen polymerization was inhibited (average 50%) with normal fibrinopeptide release and with a minor decrease of total clottable fibrinogen and alpha-polymer formation. Ascorbate reduced the incorporation of carbonyls to the fibrinogen molecule (by > 50% at 50 mmol/l; P < 0.001). Contrary to ascorbate, which alone delayed the fibrinogen polymerization rate, histidine abolished irradiation-induced inhibition of fibrinogen polymerization (by 80% at 50 mmol/l; P < 0.001). In conclusion, even though ascorbate effectively protects fibrinogen from oxidation due to its adverse effects on fibrinogen function, it may not serve as a suitable radioprotective. On the contrary, the first definite evidence is provided that radiation-sterilized fibrinogen in the presence of histidine greatly retains its clotting capability.

Synthesis and Structure of Novel Sugar-Substituted Bipyridine Complexes of Rhenium and 99m-Technetium

Novel ligands have been obtained from the reaction of 4,4'-dibromomethyl-2,2'-bipyridine with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylthiol, 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosylthiol or 2,3,4,6-tetra-O-acetyl-alpha-D-thioacetylmannopyranoside in which the sugar residues are thioglycosidically linked to the bipyridine in the 4,4'-position. Cleavage of the acetyl groups affords hydrophilic symmetric ligands with free hydroxyl groups. Reaction of the new glycoconjugated ligands (L) with [Re(CO)(5)Cl] yields fluorescent complexes of general formula [Re(L)(CO)(3)Cl], which were characterised by mass spectrometry, elemental analysis and (1)H and (13)C NMR, IR, UV/Vis and fluorescence spectroscopy. These complexes exhibit excellent solubility and stability in organic solvents or water, depending on the residues of the sugar. One complex, namely tricarbonyl-4,4'-bis[(2,3,4,6-tetra-O-acetyl-beta-D-glycopyranosyl)thiomethyl]-2,2'-bipyridinerheniumtricarbonylo chloride, has been characterised by X-ray crystallography. A non-symmetric structure of the complexes could be assigned. Radiolabelling of the unprotected ligands with [(99m)Tc(H(2)O)(3)(CO)(3)](+) affords the corresponding water-soluble technetium complexes (in quantitative yields), which were characterised by their HPLC radiation traces. The formed complexes are stable for several hours in the presence of histidine but show partial ligand-exchange after one day.

Metabolism of [U-13C]Glutamine and [U-13C]Glutamate in Isolated Rat Brain Mitochondria Suggests Functional Phosphate-Activated Glutaminase Activity in Matrix

One of the forms of phosphate activated glutaminase (PAG) is associated with the inner mitochondrial membrane. It has been debated whether glutamate formed from glutamine in the reaction catalyzed by PAG has direct access to mitochondrial or cytosolic metabolism. In this study, metabolism of [U-(13)C]glutamine (3 mM) or [U-(13)C]glutamate (10 mM) was investigated in isolated rat brain mitochondria. The presence of a functional tricarboxylic (TCA) cycle in the mitochondria was tested using [U-(13)C]succinate as substrate and extensive labeling in aspartate was seen. Accumulation of glutamine into the mitochondrial matrix was inhibited by histidine (15 mM). Extracts of mitochondria were analyzed for labeling in glutamine, glutamate and aspartate using liquid chromatography-mass spectrometry. Formation of [U-(13)C]glutamate from exogenous [U-(13)C]glutamine was decreased about 50% (P<0.001) in the presence of histidine. In addition, the (13)C-labeled skeleton of [U-(13)C]glutamine was metabolized more vividly in the tricarboxylic acid (TCA) cycle than that from [U-(13)C]glutamate, even though glutamate was labeled to a higher extent in the latter condition. Collectively the results show that transport of glutamine into the mitochondrial matrix may be a prerequisite for deamidation by PAG.

Absorption of copper and copper–histidine complexes across the apical surface of freshwater rainbow trout intestine

Bioavailability is integral in mediating the delicate balance between nutritive and potentially toxic levels of copper in fish diets. Brush-border membrane vesicles isolated from freshwater rainbow trout intestine were used to characterise apical copper absorption, and to examine the influence of the amino acid histidine on this process. In the absence of histidine, a low affinity, high capacity copper uptake mechanism was described. However, when expressed as a function of ionic copper (Cu(2+)), absorption was linear, rather than saturable, suggesting that the saturable curve was an artifact of copper speciation. Conversely, in the presence of L: -histidine (780 microM) saturable uptake was characterised. The uptake capacity discerned (J (max) of 354 +/- 81 nmol mg protein(-1) min(-1)) in the presence of histidine indicated a significantly reduced capacity for copper transport than that in the absence of histidine. To determine if copper uptake was achievable through putative histidine uptake pathways, copper and histidine were incubated in the presence of tenfold greater concentrations of amino acids proposed to block histidine transporters. Accounting for changes in copper speciation, significant inhibition of uptake by glycine and lysine were noted at copper levels of 699 and 1,028 microM. These results suggest that copper-histidine complexes may be transportable via specific amino acid-transporters in the brush-border membrane.

Free radical-mediated pre-hemolytic injury in human red blood cells subjected to lead acetate as evaluated by chemiluminescence

The mechanisms by which Pb 2+ induces hemolysis are not completely understood. For this reason, the involvement of oxidative stress in the mechanism of Pb 2+-induced pre-hemolytic lesion was investigated by exposing RBC to Pb 2+ in vitro and then separating the intact non-hemolysed RBC. Oxidative stress was investigated on human RBCs by tert-butyl hydroperoxide-initiated chemiluminescence method (CL). Our results revealed that lead-induced time and concentration-dependent hemolysis and CL time curves showed a very narrow correlation each other. GSH oxidation to GSSG and the stress index also increased significantly. Treatment of lead-exposed RBC with desferrioxamine, an iron-chelating agent or the chain-breaking antioxidant, Trolox, quenched light emission and inhibited hemolysis dramatically. Mannitol and sodium formate, OH scavengers, on the contrary, did not inhibit CL or hemolysis, significantly. These data indicate that lead-induced lipid peroxide formation is mediated by a metal-driven Fenton reaction but do not support the direct involvement of hydroxyl radicals in this process. By contrast, our results revealed a decrease in light emission and decreased hemolysis in the presence of histidine, a singlet oxygen scavenger. Our results suggest that membrane damage and hemolysis of RBC are mediated by Pb 2+ through free radical reactions and that singlet oxygen plays a significant role in this process.

Effect of buffer species on the thermally induced aggregation of interferon-tau

It is now becoming apparent that a common pathway of protein aggregation involves the unimolecular structural rearrangement from the native state to a slightly expanded aggregation-competent species. It is the goal of this study to understand the aggregation and the effects of buffer on the stability of IFN-tau. In this study, the thermally-induced aggregation of interferon-tau (IFN-tau) is described. By monitoring the aggregation rate in the presence of increasing amounts of sucrose, the relative change in surface area (Deltas) for conversion to the aggregation-competent state can be determined. Under conditions of pH 7 and in 20 mM buffer, the protein displays different aggregation rates depending on the nature of the buffer species. The protein aggregates mostly quickly in phosphate buffer, slower in the presence of Tris and slowest in the presence of histidine. The largest value for Deltas occurs for the histidine-containing samples, where aggregation proceeds via a slightly expanded aggregation competent state with a surface area increase of 7.6%. Furthermore, it appears that histidine binds to the native state of IFN-tau, thereby stabilizing the native state and retarding aggregation. Measurement of the second virial coefficient, B(22), for different formulations indicates that inclusion of histidine has only a small effect on repulsion between protein molecules, suggesting that colloidal stabilization is not the dominant mechanism for stabilization of IFN-tau. This study represents the first detailed biophysical study of specific buffer-induced stabilization, resulting in shifting the equilibrium towards the native state and away form the expanded aggregation-competent species.

The effect of alkaline phosphatase inhibitors on intracellular lipid accumulation in preadipocytes isolated from human mammary tissue

A previous study has demonstrated that alkaline phosphatase (AP) may play a role in the control of intracellular lipid accumulation in the rodent preadipocyte cell line, 3T3-L1. The present study investigated whether AP may have a similar function in preadipocytes isolated from human mammary gland tissue. Preadipocyte maturation was induced in the presence or absence of the tissue non-specific AP inhibitors levamisole and histidine, and the tissue-specific AP inhibitor PheGlyGly. Cellular AP activity and adipogenesis were both assessed at 0 and 12 days post-induction of differentiation. After differentiation, AP activity increased 5.1 +/- 1.3-fold in the absence and 8.9 +/- 2.8-fold (P < 0.05) in the presence of levamisole. However, adipogenesis increased 1.95 +/- 0.11-fold in the absence but only 1.36 +/- 0.06-fold (P < 0.001) in the presence of levamisole. There was a 4.2 +/- 2.2-fold increase in AP activity in the absence and a 0.51 +/- 0.46-fold (P < 0.05) decrease in the presence of histidine. Adipogenesis increased 2.09 +/- 0.35-fold in the absence of histidine but only 1.22 +/- 0.30-fold (P < 0.05) in the presence of histidine. PheGlyGly had no effects. Fluorescent microscopy showed AP activity was localized to the triglyceride-containing droplets of the cell. This is the first study to show that tissue non-specific AP inhibitors can block adipogenesis in human preadipocytes.

Extension of the Single Amino Acid Chelate Concept (SAAC) to Bifunctional Biotin Analogues for Complexation of the M(CO)3+1 Core (M = Tc and Re): Syntheses, Characterization, Biotinidase Stability, and Avidin Binding

Biotin and avidin form one of the most stable complexes known (K(D) = 10(-15) M(-1)) making this pairing attractive for a variety of biomedical applications including targeted radiotherapy. In this application, one of the pair is attached to a targeting molecule, while the other is subsequently used to deliver a radionuclide for imaging and/or therapeutic applications. Recently, we reported a new single amino acid chelate (SAAC) capable of forming stable complexes with Tc(CO)3 or Re(CO)3 cores. We describe here the application of SAAC analogues for the development of a series of novel radiolabeled biotin derivatives capable of forming robust complexes with both Tc and Re. Compounds were prepared through varying modification of the free carboxylic acid group of biotin. Each 99mTc complex of SAAC-biotin was studied for their ability to bind avidin, susceptibility to biotinidase, and specificity for avidin in an in vivo avidin-containing tumor model. The radiochemical stability of the 99mTc(CO)3 complexes was also investigated by challenging each 99mTc-complex with large molar excesses of cysteine and histidine at elevated temperature. All compounds were radiochemically stable for greater than 24 h at elevated temperature in the presence of histidine and cysteine. Both [99mTc(CO)3(L6)]+1 [TcL6; L6 = biotinylamidopropyl-N,N-(dipicolyl)amine] and [99mTc(CO)3(L12a)]+1 (TcL12; L12 = N,N-(dipicolyl)biotinamido-Boc-lysine; TcL12a; L12a = N,N-(dipicolyl)biotinamide-lysine) readily bound to avidin whereas [99mTc(CO)3(L9)]+1 [TcL9; L9 = N,N-(dipicolyl)biotinamine] demonstrated minimal specific binding. TcL6 and TcL9 were resistant to biotinidase cleavage, while TcL12a, which contains a lysine linkage, was rapidly cleaved. The highest uptake in an in vivo avidin tumor model was exhibited by TcL6, followed by TcL9 and TcL12a, respectively. This is likely the result of both intact binding to avidin and resistance to circulating biotinidase. Ligand L6 is the first SAAC analogue of biotin to demonstrate potential as a radiolabeled targeting vector of biotin capable of forming robust radiochemical complexes with both 99mTc and rhenium radionuclides. Computational simulations were performed to assess biotin-derivative accommodation within the binding site of the avidin. These calculations predict that deformation of the surface domain of the binding pocket can occur to accommodate the transition metal-biotin derivatives with negligible changes to the inner-beta-barrel, the region most responsible for binding and retaining biotin and its derivatives. The biological activity and biodistribution of the technetium complexes TcL6, TcL9, and TcL12a were examined in an avidin tumor model. In the avidin bead tumor localization model, TcL6 demonstrated the most favorable localization with a 7:1 ratio of avidin bead implanted muscle versus normal muscle, while TcL9 exhibited a 2:1 ratio. However, TcL9 displayed no specificity for avidin.

New variants in the mitochondrial genomes of schizophrenic patients

The impaired mitochondrial function hypothesis in schizophrenia is based on evidence of altered brain metabolism, morphology, biochemistry and gene expression. Mitochondria have their own genome, which is needed to synthesize some of the subunits of the respiratory chain enzymes. Mitochondrial DNA (mtDNA) is maternally inherited and we observed an excess of maternal transmission of schizophrenia in a set of parent-offspring affected pairs. We therefore hypothesized that mutations in the mtDNA may contribute to the complex genetic basis of schizophrenia. The entire mtDNA of six schizophrenic patients with an apparent maternal transmission of the disease was sequenced and compared to the reference sequence. We have identified 50 variants and among these six have not been previously reported. Three of them were missense variants: MTCO2 7750C>A, MTATP6 8857G>A and MTND4 12096T>A. These were maternally inherited because they were also present in the mtDNA of their respective schizophrenic mothers and none of them were found in 95 control individuals. The MTND4 12096T>A (Leu446His) is a heteroplasmic variant present in five of the six mother-offspring patient pairs that triggers a non-conservative substitution in the ND4 subunit of complex I. Sequence alignment of 110 ND4 peptides from all eukaryotic kingdoms shows that only hydrophobic amino acids are found in this position. Moreover, leucine was conserved or substituted by an isoleucine in all mammalian species. This indicates that the presence of histidine could affect complex I activity in patients with schizophrenia.