Co-activation of VTA DA and GABA neurons mediates nicotine reinforcement.

Smoking is the most important preventable cause of mortality and morbidity worldwide. This nicotine addiction is mediated through the nicotinic acetylcholine receptor (nAChR), expressed on most neurons, and also many other organs in the body. Even within the ventral tegmental area (VTA), the key brain area responsible for the reinforcing properties of all drugs of abuse, nicotine acts on several different cell types and afferents. Identifying the precise action of nicotine on this microcircuit, in vivo, is important to understand reinforcement, and finally to develop efficient smoking cessation treatments. We used a novel lentiviral system to re-express exclusively high-affinity nAChRs on either dopaminergic (DAergic) or γ-aminobutyric acid-releasing (GABAergic) neurons, or both, in the VTA. Using in vivo electrophysiology, we show that, contrary to widely accepted models, the activation of GABA neurons in the VTA plays a crucial role in the control of nicotine-elicited DAergic activity. Our results demonstrate that both positive and negative motivational values are transmitted through the dopamine (DA) neuron, but that the concerted activity of DA and GABA systems is necessary for the reinforcing actions of nicotine through burst firing of DA neurons. This work identifies the GABAergic interneuron as a potential target for smoking cessation drug development.

Computational approaches to the neurobiology of drug addiction.

To increase our understanding of drug addiction--notably its pharmacological and neurobiological determinants--researchers have begun to formulate computational models of drug self-administration. Currently, one can roughly distinguish between three classes of models which all have in common to attribute to brain dopamine signaling a key role in addiction. The first class of models contains quantitative pharmacological models that describe the influence of pharmacokinetic and pharmacodynamic factors on drug self-administration. These models fail, however, to explain how the drug self-administration behavior is acquired and how it eventually becomes rigid and compulsive with extended drug use. Models belonging to the second class circumvent some of these limitations by modeling how drug use usurps the function of dopamine in reinforcement learning and action selection. However, despite their behavioral plausibility, these latter models lack neurobiological plausibility and ignore the potential role of opponent processes in addiction. The third class of models attempts to surmount these pitfalls by providing a more realistic picture of the midbrain dopamine circuitry and of the complex action of drugs of abuse in the output of this circuitry. Here we provide a brief overview of these different models to illustrate the potential contribution of mathematical modeling to our understanding of the neurobiology of drug addiction.

The first examples of (S)-2-hydroxyacid dehydrogenases catalyzing the transfer of the pro-4S hydrogen of NADH are found in the archaea.

Reduction of 2-oxoacids to the corresponding (S)-2-hydroxyacids is an important transformation in biochemistry. To date all (S)-2-hydroxyacid dehydrogenases belonging to the L-lactate/L-malate dehydrogenase family have been found to transfer the pro-4R hydrogen of either NADH or NADPH to C-2 of the 2-oxoacid substrates during their reduction. Here, we report that recombinantly generated (S)-2-hydroxyacid dehydrogenases present in the methanoarchaea Methanococcus jannaschii and Methanothermus fervidus use the pro-4S hydrogen of NADH to reduce a series of 2-oxoacids to the corresponding (S)-2-hydroxyacids. This information as well as the low sequence identity between these archaeal enzymes and the L-lactate/L-malate family of enzymes indicate that these enzymes are not evolutionary related and therefore constitute a new class of (S)-2-hydroxyacid dehydrogenases.

Methanococcus jannaschii generates L-proline by cyclization of L-ornithine.

Cell extracts of Methanococcus jannaschii have been shown to readily convert L-ornithine to L-proline. This cyclization reaction proceeds with the loss of only the C-2 nitrogen, as has been documented for ornithine cyclodeaminase (EC 4.3.1.12). Since no gene homologous to that coding for ornithine cyclodeaminase is present in the genome of M. jannaschii, these results indicate that proline biosynthesis in M. jannaschii is accomplished by a previously unrecognized enzyme.

Biosynthesis of the phosphodiester bond in coenzyme F(420) in the methanoarchaea.

The biochemical route for the formation of the phosphodiester bond in coenzyme F(420), one of the methanogenic coenzymes, has been established in the methanoarchaea Methanosarcina thermophila and Methanococcus jannaschii. The first step in the formation of this portion of the F(420) structure is the GTP-dependent phosphorylation of L-lactate to 2-phospho-L-lactate and GDP. The 2-phospho-L-lactate represents a new natural product that was chemically identified in Methanobacterium thermoautotrophicum, M. thermophila, and Mc. jannaschii. Incubation of cell extracts of both M. thermophila and Mc. jannaschii with [hydroxy-(18)O, carboxyl-(18)O(2)]lactate and GTP produced 2-phospho-L-lactate with the same (18)O distribution as found in both the starting lactate and the lactate recovered from the incubation. These results indicate that the carboxyl oxygens are not involved in the phosphorylation reaction. Incubation of Sephadex G-25 purified cell extracts of M. thermophila or Mc. jannaschii with 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo), 2-phospho-L-lactate, and GTP or ATP lead to the formation of F(420)-0 (F(420) with no glutamic acids). This transformation was shown to involve two steps: (i) the GTP- or ATP-dependent activation of 2-phospho-L-lactate to either lactyl(2)diphospho-(5')guanosine (LPPG) or lactyl(2)diphospho-(5')adenosine (LPPA) and (ii) the reaction of the resulting LPPG or LPPA with Fo to form F(420)-0 with release of GMP or AMP. Attempts to identify LPPG or LPPA intermediates by incubation of cell extracts with L-[U-(14)C]lactate, [U-(14)C]2-phospho-L-lactate, or [8-(3)H]GTP were not successful owing to the instability of these compounds toward hydrolysis. Synthetically prepared LPPG and LPPA had half-lives of 10 min at 50 degrees C (at pH 7.0) and decomposed into GMP or AMP and 2-phospho-L-lactate via cyclic 2-phospho-L-lactate. No evidence for the functioning of the cyclic 2-phospho-L-lactate in the in vitro biosynthesis could be demonstrated. Incubation of cell extracts of M. thermophila or Mc. jannaschii with either LPPG or LPPA and Fo generated F(420)-0. In summary, this study demonstrates that the formation of the phosphodiester bond in coenzyme F(420) follows a reaction scheme like that found in one of the steps of the DNA ligase reaction and in the biosynthesis of coenzyme B(12) and phospholipids.

Identification of an archaeal 2-hydroxy acid dehydrogenase catalyzing reactions involved in coenzyme biosynthesis in methanoarchaea.

Two putative malate dehydrogenase genes, MJ1425 and MJ0490, from Methanococcus jannaschii and one from Methanothermus fervidus were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze pyridine nucleotide-dependent oxidation and reduction reactions of the following alpha-hydroxy-alpha-keto acid pairs: (S)-sulfolactic acid and sulfopyruvic acid; (S)-alpha-hydroxyglutaric acid and alpha-ketoglutaric acid; (S)-lactic acid and pyruvic acid; and 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid and 1-oxo-1,3,4, 6-hexanetetracarboxylic acid. Each of these reactions is involved in the formation of coenzyme M, methanopterin, coenzyme F(420), and methanofuran, respectively. Both the MJ1425-encoded enzyme and the MJ0490-encoded enzyme were found to function to different degrees as malate dehydrogenases, reducing oxalacetate to (S)-malate using either NADH or NADPH as a reductant. Both enzymes were found to use either NADH or NADPH to reduce sulfopyruvate to (S)-sulfolactate, but the V(max)/K(m) value for the reduction of sulfopyruvate by NADH using the MJ1425-encoded enzyme was 20 times greater than any other combination of enzymes and pyridine nucleotides. Both the M. fervidus and the MJ1425-encoded enzyme catalyzed the NAD(+)-dependent oxidation of (S)-sulfolactate to sulfopyruvate. The MJ1425-encoded enzyme also catalyzed the NADH-dependent reduction of alpha-ketoglutaric acid to (S)-hydroxyglutaric acid, a component of methanopterin. Neither of the enzymes reduced pyruvate to (S)-lactate, a component of coenzyme F(420). Only the MJ1425-encoded enzyme was found to reduce 1-oxo-1,3,4,6-hexanetetracarboxylic acid, and this reduction occurred only to a small extent and produced an isomer of 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid that is not involved in the biosynthesis of methanofuran c. We conclude that the MJ1425-encoded enzyme is likely to be involved in the biosynthesis of both coenzyme M and methanopterin.

Identification of the gene encoding sulfopyruvate decarboxylase, an enzyme involved in biosynthesis of coenzyme M.

The products of two adjacent genes in the chromosome of Methanococcus jannaschii are similar to the amino and carboxyl halves of phosphonopyruvate decarboxylase, the enzyme that catalyzes the second step of fosfomycin biosynthesis in Streptomyces wedmorensis. These two M. jannaschii genes were recombinantly expressed in Escherichia coli, and their gene products were tested for the ability to catalyze the decarboxylation of a series of alpha-ketoacids. Both subunits are required to form an alpha(6)beta(6) dodecamer that specifically catalyzes the decarboxylation of sulfopyruvic acid to sulfoacetaldehyde. This transformation is the fourth step in the biosynthesis of coenzyme M, a crucial cofactor in methanogenesis and aliphatic alkene metabolism. The M. jannaschii sulfopyruvate decarboxylase was found to be inactivated by oxygen and reactivated by reduction with dithionite. The two subunits, designated ComD and ComE, comprise the first enzyme for the biosynthesis of coenzyme M to be described.

New fluorogenic triacylglycerol analogs as substrates for the determination and chiral discrimination of lipase activities.

A new type of fluorogenic and isomerically pure 1(3)-O-alkyl-2,3 (3,2)-diacyl glycerols was synthesized that can be used as substrate for the determination of lipase activities. These compounds contain a fluorescent pyrene acyl chain and, as a potent quencher of pyrene fluorescence, a trinitrophenylamino acyl residue. In their intact form, the fluorogens show only low fluorescence intensity. Upon lipase-induced or chemical hydrolysis of the substrates, however, the fluorophore and quencher separate from each other. This leads to a gradual increase in pyrene fluorescence, reflecting the time-dependent progress of lipolysis and, under substrate saturation conditions, lipase activity. This lipase assay is continuous and does not require separation of substrate and reaction products. Short- and long-chain homologues as well as optical isomers of the fluorogenic alkyldiacyl glycerols were hydrolyzed by pancreatic lipase, hepatic lipase, and lipo-protein lipase at highly different rates depending on the substrate or enzyme preparation and source (e.g., postheparin plasma or cultured cells). It is proposed that a useful set of enantiomeric and/or homologous substrates in combination with appropriate reaction media might be applied to the selective determination of a lipase in a mixture of lipases, e.g., hepatic and lipoprotein lipase in PHP, for medical diagnostics.

Identification of coenzyme M biosynthetic 2-phosphosulfolactate phosphatase. A member of a new class of Mg(2+)-dependent acid phosphatases.

Coenzyme M (CoM; 2-mercaptoethanesulfonic acid) is the terminal methyl carrier in methanogenesis. Methanogenic archaea begin the production of this essential cofactor by sulfonating phosphoenolpyruvate to form 2-phospho-3-sulfolactate. After dephosphorylation, this precursor is oxidized, decarboxylated and then reductively thiolated to form CoM. A thermostable phosphosulfolactate phosphohydrolase (EC 3.1.3.-) catalyzing the second step in CoM biosynthesis, was identified in the hyperthermophilic euryarchaeon Methanococcus jannaschii. The predicted ORF MJ1140 in the genome of M. jannaschii encodes ComB, a Mg2+-dependent acid phosphatase that is specific for 2-hydroxycarboxylic acid phosphate esters. Recombinantly expressed purified ComB efficiently hydrolyzes rac-2-phosphosulfolactate, (S)-2-phospholactate, phosphoglycolate and both enantiomers of 2-phosphomalate. In contrast to previously studied phosphoglycolate phosphatases, ComB has a low pH optimum for activity, a narrow substrate specificity and an amino acid sequence dissimilar to any biochemically characterized protein. Like other phosphatases that function via covalent phosphoenzyme intermediates, ComB can catalyze a transphosphorylation reaction. Homologs of comB are identified in all available cyanobacterial genome sequences and in genomes from phylogenetically diverse bacteria and archaea; most of these organisms lack homologs of other CoM biosynthetic genes. The broad and disparate distribution of comB homologs suggests that the gene has been recruited frequently into new metabolic pathways.

Identification of enzymes homologous to isocitrate dehydrogenase that are involved in coenzyme B and leucine biosynthesis in methanoarchaea.

Two putative Methanococcus jannaschii isocitrate dehydrogenase genes, MJ1596 and MJ0720, were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze the NAD- and NADP-dependent oxidative decarboxylation of DL-threo-3-isopropylmalic acid, threo-isocitrate, erythro-isocitrate, and homologs of threo-isocitrate. Neither enzyme was found to use any of the isomers of isocitrate as a substrate. The protein product of the MJ1596 gene, designated AksF, catalyzed the NAD-dependent decarboxylation of intermediates in the biosynthesis of 7-mercaptoheptanoic acid, a moiety of methanoarchaeal coenzyme B (7-mercaptoheptanylthreonine phosphate). These intermediates included (-)-threo-isohomocitrate [(-)-threo-1-hydroxy-1,2, 4-butanetricarboxylic acid], (-)-threo-iso(homo)(2)citrate [(-)-threo-1-hydroxy-1,2,5-pentanetricarboxylic acid], and (-)-threo-iso(homo)(3)citrate [(-)-threo-1-hydroxy-1,2, 6-hexanetricarboxylic acid]. The protein product of MJ0720 was found to be alpha-isopropylmalate dehydrogenase (LeuB) and was found to catalyze the NAD-dependent decarboxylation of one isomer of DL-threo-isopropylmalate to 2-ketoisocaproate; thus, it is involved in the biosynthesis of leucine. The AksF enzyme proved to be thermostable, losing only 10% of its enzymatic activity after heating at 100 degrees C for 10 min, whereas the LeuB enzyme lost 50% of its enzymatic activity after heating at 80 degrees C for 10 min.

Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence.

We have studied the intrinsic tryptophan fluorescence of the lipases from Chromobacterium viscosum (CVL), Pseudomonas species (PSL), and Rhizopus oryzae (ROL) in aqueous buffer, zwitterionic detergent micelles, and isopropanol-water mixtures. It was the purpose of this study to obtain information about biophysical properties of the respective enzymes under conditions that modulate enzyme activities and stereoselectivities to a significant extent. According to their decay-associated emission spectra, CVL tryptophans are located in the hydrophobic interior of the protein. In contrast, the PSL and ROL tryptophans are probably confined to the core and the surface of the lipase. From the tryptophan lifetime distributions it can be concluded that the conformation of CVL is not much affected by detergent or organic solvent (isopropanol). Accordingly, CVL is enzymatically active in these systems and most active in the presence of isopropanol. In contrast, ROL and PSL show high conformational mobility, depending on the solvent, because their lifetime distributions are very different in the presence and absence of detergent or isopropanol. Time-resolved anisotropy studies provided evidence that the lipases exhibit very high internal molecular flexibility. This peculiar feature of lipases is perhaps the key to the great differences in activity and stereoselectivity observed in different reaction media. Furthermore, information about self-association of the lipases in different solvents could be obtained. PSL, but not CVL and ROL, forms aggregates in water. Lipase aggregation can be reversed by the addition of detergent or isopropanol, which competes for the hydrophobic surface domains of this protein. This dissociation could efficiently contribute to the increase in lipase activity in the presence of a detergent or isopropanol.

Blood-aqueous barrier breakdown after penetrating keratoplasty with simultaneous extracapsular cataract extraction and posterior chamber lens implantation.

BACKGROUND: The purpose of this study was to quantify breakdown of the blood-aqueous barrier (BAB) following penetrating keratoplasty (PK) with simultaneous extracapsular cataract extraction and posterior chamber lens implantation (triple procedure) and compare it with the alterations following PK only. METHODS: This study included 72 eyes after triple procedure and 227 eyes after PK only. The diagnosis for PK was Fuchs dystrophy in 39%, keratokonus in 44%, stromal corneal dystrophy in 3% and avascular corneal scars in 6% of cases. The postoperative topical steroid treatment was standardized in both groups. Aqueous flare was quantified using the laser flare-cell meter (FC-1000, Kowa) at defined postoperative intervals (10 days, 6 weeks, then every 3 months until 1 year postoperatively). Patients with conditions associated with impairment of the BAB were excluded from the study. RESULTS: In the early postoperative course, aqueous flare values (photon counts/ms) were significantly higher in patients with triple procedure (21.9 +/- 11.0) than in patients with PK only (9.8 +/- 3.2; P = 0.001). At 6 weeks postoperatively, aqueous flare returned to normal levels in patients after PK only (5.2 +/- 2.3), whereas patients with triple procedure still showed significantly increased flare values (10.8 +/- 5.6; P = 0.01). At 6 months postoperatively, aqueous flare values of patients with triple had returned to normal levels (6.8 +/- 3.8) and did not differ significantly from those after PK only (5.2 +/- 1.9; P = 0.09). CONCLUSION: Our results indicate that triple procedure causes a more extensive and longer-lasting breakdown of the blood-aqueous barrier than PK only. Quantification of aqueous flare with the laser flare-cell meter is useful in the postoperative follow-up after triple procedure. Further studies are required to investigate the clinical relevance of BAB breakdown on endothelial cell count and the incidence of subsequent immunological graft rejection.

[Interim results from the prospective "Erlanger Non-high-risk Penetrating Keratoplasty Study" in 207 patients]. / Zwischenergebnisse der prospektiven "Erlanger Nicht-Hochrisiko-Keratoplastik-Studie" bei 207 Patienten.

BACKGROUND: Immunologic graft rejection targeted against corneal endothelium is the most frequent cause for graft failure after corneal transplantation. The purpose of this prospective study was to assess the frequency, early symptoms, prophylaxis and therapy monitoring of corneal graft rejection following non-high-risk penetrating keratoplasty (PK). PATIENTS AND METHODS: From February 1997 to May 1999, 237 patients undergoing non-high-risk PK have been enrolled in this prospective study. We evaluated 207 patients (103 female, 113 right eyes, recipient age 54 +/- 20 years, donor age 59 +/- 17 years). In 2 randomized treatment studies we compared the efficacy of postoperative short-term (ST = 6 months) versus long-term (LT = 12 months) topical steroid therapy on the incidence of graft rejection and the effect of high- versus low-dose systemic steroid therapy on the prognosis after a graft rejection. Follow-up examinations included, laser-tyndallometry, corneal topography analysis, endothelial cell count and pachymetry. RESULTS: The main indications for PK were keratoconus (n = 93), endothelial dystrophy Fuchs (n = 52) and bullous keratopathy (n = 35). In 151 (73%) patients, non mechanical trephination with the 193 nm Excimer laser was performed. Up to now, 78 patients were randomized into two groups comparing the postoperative therapy with topical steroids. During follow-up (median: ST: 13.5 months; LT: 12.5 months, maximum 25.3 months) episodes of endothelial graft rejection (3 chronic focal, 8 acute diffuse) showed 11 eyes of 11 patients. Five patients each had short-term and long-term topical steroid treatment. In 1 patient the graft rejection occurred before randomization at 6 months. Six patients with graft rejection episodes underwent a PK only (54% of graft rejections, 4.4% of all patients). In the remaining 5 patients, PK was combined with a lens surgery (46% of graft rejections, 6.9% of all patients). Ten of 11 corneal grafts regained their full function under treatment with systemic and local steroids. CONCLUSION: The frequency of episodes of graft rejection in our study was lower than usually reported in the literature. A good compliance of patients appears to be a major factor for improved prognosis of the graft after PK and in case of graft rejection. Until now no significant differences between short-term or long-term postoperative topical steroid therapy could be detected regarding the incidence of corneal graft rejection.

[Transplant endothelium and measuring corneal thickness after non-high-risk keratoplasty with briefly or long-term preserved corneal donor tissue]. / Transplantatendothel und Hornhaut-Pachymetrie nach Nicht-Hochrisiko-Keratoplastik mit kurzzeit- oder langzeitkonserviertem Hornhautspendergewebe.

PURPOSE: The corneal endothelial cell density is essential for the pump function and the transparency of grafts after penetrating keratoplasty (PK). The purpose of this study was to assess corneal endothelial cell density after non-high-risk PK and to check for possible correlations with storage parameters of the donor corneas using two different storage methods. PATIENTS AND METHODS: Endothelial cell density (specular microscope EM 1100, TOMEY, Erlangen) and central corneal thickness (ultrasonic pachymetry SP-2000, TOMEY, Erlangen) were assessed 6 weeks, 3, 6, 9 months and one year postoperatively in 168 non-high-risk PKs. Short-term-preserved donor corneas were used in 89 patients, whereas in 79 patients organ-cultured corneas were used. The donor trephination was performed from the epithelial side using an artificial anterior chamber. The postoperative treatment with topical steroids was standardized. The mean donor post-mortem time was 9.6 +/- 8.0 hours for short-term-preserved and 17.6 +/- 10.5 hours for organ-cultured corneas (p < 0.0001). The storage time was 71 +/- 49 and 380 +/- 167 hours (p < 0.0001), respectively. RESULTS: Endothelial cell density did not differ significantly between the two storage methods (p > 0.05). At 6 weeks postoperatively, the mean endothelial cell density was 2042 +/- 675 cells/mm2 for short-term-preserved corneas and 1972 +/- 522 cells/mm2 for organ-cultured corneas (p = 0.7). Endothelial cell density did not decrease significantly (p > 0.05) within the observation period of 12 months in both groups (after 12 months: 1868 +/- 957 cells/mm2 and 1638 +/- 643 cells/mm2, respectively). The mean corneal thickness was 542 +/- 50 microns for short-term-preserved and 541 +/- 55 microns for organ-cultured corneas and remainded unchanged during the follow-up of 12 months (542 +/- 42 microns and 521 +/- 43 microns, respectively). Neither the group of short-term-preserved corneas nor organ-cultured corneas showed a significant correlation between endothelial cell density or central cornea thickness with post-mortem time or with storage time of the donor corneas at any postoperative stage (p > 0.1). CONCLUSION: During the first year after PK, only a small decrease in endothelial cell density was observed in comparison with the 6-weeks finding. The storage method does not seem to affect the short-term changes of endothelial cell density. Further long-term studies are necessary to assess the clinical relevance of these observations.