A phase 2, double-blind, placebo-controlled trial of a valproate/lithium combination in ALS patients.

BACKGROUND: Few treatments are currently available for amyotrophic lateral sclerosis (ALS). A combination of lithium carbonate and valproic acid (VPA-Li) was shown to inhibit motor neuron death and delay disease progression. METHODS: Outpatients with a typical ALS presentation were enrolled in a randomized, placebo-controlled trial to assess the efficacy of orally administered VPA-Li. Changes in a functional scale score (ALSFRS-R) and survival rate were chosen as primary outcome variables. Secondary outcome variables included BMI, respiratory monitoring, quality of life, and a global impression of the treatment. RESULTS: Out of 42 patients enrolled, 20 individuals receiving VPA-Li and 18 on placebo treatment were included in the final analysis. Forty-five percent of patients receiving VPA-Li completed the trial, whereas only 22.22% of patients in the placebo group attended the final visit 18 months later (P = 0.09). Major changes in the ALSFRS-R score were observed, including a decrease of 1.195 points/month in the placebo group (95% CI: 0.7869-1.6031) and of 0.5085 under VPA-Li treatment (95% CI: 0.2288-0.7882) between months 6 and 14. Adverse events included bad mouth taste, constipation, and anorexia. Survival rate, body weight, and quality of life were positive outcomes by the end of the trial despite a high sample reduction, especially in the placebo group. The inclusion of 212 subjects in each group would confirm these differences. CONCLUSIONS: Combined VPA-Li treatment associated with slower ALS progression and better secondary outcomes. This dual treatment overcame the futility threshold and merits further investigation in ALS.

Degradation of dibutyl phthalate by Paenarthrobacter sp. Shss isolated from Saravan landfill, Hyrcanian Forests, Iran.

Phthalic acid esters are predominantly used as plasticizers and are industrially produced on the million ton scale per year. They exhibit endocrine-disrupting, carcinogenic, teratogenic, and mutagenic effects on wildlife and humans. For this reason, biodegradation, the major process of phthalic acid ester elimination from the environment, is of global importance. Here, we studied bacterial phthalic acid ester degradation at Saravan landfill in Hyrcanian Forests, Iran, an active disposal site with 800 tons of solid waste input per day. A di-n-butyl phthalate degrading enrichment culture was established from which Paenarthrobacter sp. strain Shss was isolated. This strain efficiently degraded 1 g L-1 di-n-butyl phthalate within 15 h with a doubling time of 5 h. In addition, dimethyl phthalate, diethyl phthalate, mono butyl phthalate, and phthalic acid where degraded to CO2, whereas diethyl hexyl phthalate did not serve as a substrate. During the biodegradation of di-n-butyl phthalate, mono-n-butyl phthalate was identified in culture supernatants by ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry. In vitro assays identified two cellular esterase activities that converted di-n-butyl phthalate to mono-n-butyl phthalate, and the latter to phthalic acid, respectively. Our findings identified Paenarthrobacter sp. Shss amongst the most efficient phthalic acid esters degrading bacteria known, that possibly plays an important role in di-n-butyl phthalate elimination at a highly phthalic acid esters contaminated landfill.

Ataxia Severity Correlates with White Matter Degeneration in Spinocerebellar Ataxia Type 7.

BACKGROUND AND PURPOSE: There is a scarcity of information on the effect of white matter degeneration in patients with spinocerebellar ataxia type 7. Therefore, we investigated the WM integrity in a large group of patients with spinocerebellar ataxia type 7 by using Tract-Based Spatial Statistics. MATERIALS AND METHODS: Thirty-three patients with a molecular diagnosis of spinocerebellar ataxia type 7 and their age- and sex-matched healthy controls participated in this study. The patients' ataxia severity was evaluated with the Scale for the Assessment and Rating of Ataxia. Voxelwise analyses of diffusion metrics, including fractional anisotropy and mean diffusivity, were performed with Tract-Based Spatial Statistics. The correlation between WM abnormalities and ataxia severity was then calculated. RESULTS: Tract-Based Spatial Statistics analysis revealed WM abnormalities in the cerebellum and the cerebellar peduncles, as well as in other major cortical and subcortical pathways. Further analysis between the Scale for the Assessment and Rating of Ataxia score and WM mean diffusivity showed significant associations only in key areas related to motor control and visuospatial processing, including the cerebellar WM, the middle occipital WM, the superior cerebellar peduncle, and bilateral anterior thalamic radiation. No significant associations between fractional anisotropy and the Scale for the Assessment and Rating of Ataxia were found. CONCLUSIONS: These results suggest a significant contribution of local cerebellar and cerebellar-midbrain connections to ataxic impairment in spinocerebellar ataxia type 7. The results also suggest an involvement of cortical WM abnormalities including tracts within the occipital and frontal cortices. These findings contribute to a more comprehensive view of the clinical impact of the white matter degeneration in spinocerebellar ataxia type 7.

HES 130/0.42 shows less alteration of pharmacokinetics than HES 200/0.5 when dosed repeatedly.

BACKGROUND: Hydroxyethyl starches (HES) accumulate in the circulation when administered repeatedly. Accumulation is thought to be partly responsible for undesirable effects (tissue storage, blood coagulation impairment, and itching). HES 130/0.42 with low molecular weight and a low level of substitution has recently been developed in order to reduce those risks. METHODS: In healthy volunteers, the pharmacokinetics of HES 130/0.42/6:1 were investigated using a crossover design with HES 200/0.5 serving as control. Fifty grams of either HES were administered in 4 h day-1 for a period of five consecutive days. HES serum concentrations were used for computation of pharmacokinetic coefficients. Change between the first and fifth infusion in the area under the concentration curve (AUC) served as the primary measurement. RESULTS: Although the circulation was freed from the load with HES 130/0.42 within 20 h after end of the previous infusion, the amount of HES 200/0.5 increased continuously from one administration to the other. AUC and elimination half-life (t1/2) were significantly lower with HES 130/0.42. AUC and t1/2 of HES 200/0.5 showed an increase between the first and the fifth administration whereas only a minimal shift was present with HES 130/0.42. Haemodilution via HES 200/0.5 did not change over time. CONCLUSIONS: Repeated administration of HES 130/0.42 shows no accumulation and fewer tendencies to time-dependent changes in pharmacokinetic parameters than HES 200/0.5. The improved reproducibility may improve drug safety, particularly as the accumulation of residual starch with HES 200/0.5 does not contribute to the colloid's volume effect, but may rather increase the risk of undesired reactions.

Molecular weight of hydroxyethyl starch: is there an effect on blood coagulation and pharmacokinetics?

BACKGROUND: The development of hydroxyethyl starches (HES) with low impact on blood coagulation but higher volume effect compared with the currently used HES solutions is of clinical interest. We hypothesized that high molecular weight, low-substituted HES might possess these properties. METHODS: Thirty pigs were infused with three different HES solutions (20 ml kg(-1)) with the same degree of molar substitution (0.42) but different molecular weights (130, 500 and 900 kDa). Serial blood samples were taken over 24 h and blood coagulation was assessed by Thromboelastograph analysis and analysis of plasma coagulation. In addition, plasma concentration and in vivo molecular weight were determined and pharmacokinetic data were computed based on a two-compartment model. RESULTS: Thromboelastograph analysis and plasma coagulation tests did not reveal a more pronounced alteration of blood coagulation with HES 500 and HES 900 compared with HES 130. In contrast, HES 500 and HES 900 had a greater area under the plasma concentration-time curve [1542 (142) g min litre(-1), P<0.001, 1701 (321) g min litre(-1), P<0.001] than HES 130 [1156 (223) g min litre(-1)] and alpha half life (t(alpha)(1/2)) was longer for HES 500 [53.8 (8.6) min, P<0.01] and HES 900 [57.1 (12.3) min, P<0.01] than for HES 130 [39.9 (10.7) min]. Beta half life (t(beta)(1/2)), however, was similar for all three types of HES [from 332 (100) to 381 (63) min]. CONCLUSIONS: In low-substituted HES, molecular weight is not a key factor in compromising blood coagulation. The longer initial intravascular persistence of high molecular weight low-substituted HES might result in a longer lasting volume effect.

Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl-CoA reductase.

Benzoyl-CoA reductase (BCR) from the bacterium Thauera aromatica catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene. In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP/2e(-). Reduced but not oxidized BCR hydrolyzes ATP to ADP. In this work, purified BCR was shown to catalyze an isotope exchange from [(14)C]ADP to [(14)C]ATP, which was approximately 15% of the ATPase activity in the presence of equimolar amounts of ADP and ATP. In accordance, BCR (alpha beta gamma delta-composition) autophosphorylated its gamma-subunit when incubated with [gamma-(32)P]ATP. Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionite-reduced BCR catalyzed a dephosphorylation associated with the ATPase activity. This finding suggests that the ATPase- and autophosphatase-partial activities of BCR exhibit identical redox dependencies. BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold. In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR. The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage. The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme.

Redox centers of 4-hydroxybenzoyl-CoA reductase, a member of the xanthine oxidase family of molybdenum-containing enzymes.

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and Mössbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds.

Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites.

CCl4-induced liver damage was modeled in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CCl4 metabolites to cell components and/or peroxidative damage as the cause of injury. (1) Covalent binding of 14C-labeled metabolites was detected in hepatocytes immediately after exposure to CCl4. (2) Low oxygen partial pressure increased the reductive metabolism of CCl4 and thus covalent binding. (3) [14C]-CCl4 was bound to lipids and to proteins throughout subcellular fractions. Binding occurred preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of label. (4) The lipid peroxidation potency of CCl4 revealed subtle differences compared to other peroxidative substances, viz., ADP-Fe3+ and cumol hydroperoxide, respectively. (5) CCl4, but not the other peroxidative substances, decreased the rate of triacylglycerol secretion as very low density lipoproteins. (6) The anti-oxidant vitamin E (alpha-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited. (7) The radical scavenger piperonyl butoxide prevented CCl4-induced lipid peroxidation as well as covalent binding of CCl4 metabolites to cell components, and also restored lipoprotein metabolism. The results confirm that covalent binding of the CCl3* radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl3-OO*, initiates lipid peroxidation. The two processes are independent of each other, and the extent to which either process occurs depends on partial oxygen pressure. The former process may result in adduct formation and, ultimately, cancer initiation, whereas the latter results in loss of calcium homeostasis and, ultimately, apoptosis and cell death.

Single turnover EPR studies of benzoyl-CoA reductase.

Benzoyl-CoA reductase (BCR) catalyzes the ATP-driven transport of two electrons from a reduced 2[4Fe-4S] ferredoxin to the aromatic ring of benzoyl-CoA. A mechanism involving radical species and very low potential electrons similar to the Birch reduction of aromatics has been suggested for this reaction. The redox centers of BCR have previously been identified, by EPR- and Mössbauer spectroscopy, to be three cysteine-ligated [4Fe-4S] clusters [Boll et al. (2000) J. Biol. Chem. 275, 31857-31868] with redox potentials more negative than -500 mV. In this work, the catalytic cycle of BCR was studied by freeze-quench experiments; the dithionite reduced enzyme was rapidly mixed with equimolar amounts of benzoyl-CoA and excess MgATP plus dithionite, and subjected to EPR spectroscopic analysis. The turnover period of the enzyme under the conditions used was 3 s. The total S = (1)/(2) spin concentration increased 3-fold very rapidly (within approximately 25 ms). In the course of a single turnover the extent of enzyme reduction decreased again, finally reaching the starting value. An increased magnetic interaction of [4Fe-4S] clusters and the rise of an S = (7)/(2) high-spin EPR signal occurred as second simultaneous and transient events (at approximately 200 ms). Previous work showed that binding of the nucleotide affects the magnetic interaction of [4Fe-4S] clusters, whereas hydrolysis of MgATP is required for the switch to high-spin EPR signals. Finally, two novel transient EPR signals with an isotropic line-shape developed maximally in the late phase of the catalytic cycle ( approximately 1-2 s). These signals differed from those of typical free radicals by shifted g values at g = 2.015 and g = 2.033 and by an unusually fast relaxation rate, suggesting an interaction of these paramagnetic species with [4Fe-4S](+1) clusters. On the basis of these results, we present a proposal for a catalytic cycle involving radical species.

Hepatocyte damage induced by carbon tetrachloride: inhibited lipoprotein secretion and changed lipoprotein composition.

Changes of lipoprotein secretion and composition in response to CCl4 treatment were studied in monolayer cultures of rat primary hepatocytes. (1) CCl4 decreased secretion of very low density lipoproteins (VLDL) by about 85%, while high density lipoprotein (HDL) secretion was less affected (about 40%). The effect was concentration-dependent. (2) CCl4 significantly inhibited secretion of VLDL- and HDL-associated triglycerides and cholesterol esters. VLDL- and HDL-associated cholesterol was not affected, while secretion of phospholipids was increased. (3) Hepatocytes secreted the apolipoproteins B48, B100, E, C, and A-I. CCl4 reduced secretion of apoproteins associated with VLDL by almost 20%, and by about 75% when associated with HDL. The de novo synthesis of apolipoproteins was attenuated by CCl4. (4) CCl4 caused variations in the apolipoprotein composition in VLDL and HDL. CCl4 intoxication of the liver affected the morphology and/or function of the lipoproteins, which drastically impaired their ability to act as transport vehicles for lipids from the liver to the circulation.

Pathogenesis of carbon tetrachloride-induced hepatocyte injury bioactivation of CCI4 by cytochrome P450 and effects on lipid homeostasis.

The CCl4-induced development of liver damage was studied in monolayer cultures of primary rat hepatocytes: (1) CCl4 caused accumulation of triglycerides in hepatocytes following cytochrome P450 induction with beta-naphthoflavone or metyrapone. Ethanol or a high dose of insulin plus triiodothyronine had the same effect. (2) CCl4 increased the synthesis of fatty acids and triglycerides and the rate of lipid esterification. Cholesterol and phospholipid synthesis from acetate was also increased. (3) CCl4 reduced beta-oxidation of fatty acids as assessed by CO2-release and ketone body formation. Hydrolysis of triglycerides was also reduced. (4) The content of unsaturated fatty acids in microsomal lipids was decreased by almost 50% after incubation with CCl4, while saturated fatty acids increased slightly. (5) CCl4 exerted a pronounced inhibitory effect on the exocytosis of macromolecules (albumin), but did not affect secretion of bile acids from hepatocytes.

Cloning and characterization of the gene encoding the mouse peptide transporter PEPT2.

Here we describe the cDNA structure, genomic organization, chromosomal localization, and promoter analysis of the mouse peptide transporter PEPT2. The PEPT2-cDNA is 3987 bp long and encodes a protein of 729 amino acids. The functional properties, analyzed by expression in Xenopus laevis oocytes, showed a typical PEPT2-phenotype with electrogenic, proton-coupled transport, high substrate affinity, and a broad specificity. Immunoblotting of renal brush-border membranes revealed an apparent molecular mass of PEPT2 of 100 kDa. The murine Pept2 gene was cloned from a 129/SvevTACfBr genomic library. It is 34 kb long and comprises 22 exons and 21 introns. By radiation mapping analysis the Pept2 gene was mapped on central mouse chromosome 16. Two putative transcription start sites lying 35 and 235 bp upstream from the translation start were identified. The Pept2 gene possesses a TATA-less promoter. Functional promoter analysis revealed the core promoter to be located between 432 and 286 bp upstream from the translation start.

EPR and Mössbauer studies of benzoyl-CoA reductase.

Benzoyl-CoA reductase catalyzes the two-electron transfer from a reduced ferredoxin to the aromatic ring of benzoyl-CoA; this reaction is coupled to stoichiometrical ATP hydrolysis. A very low reduction potential (less than -1 V) is required for the first electron transfer to the aromatic ring. In this work the nature of the redox centers of purified benzoyl-CoA reductase from Thauera aromatica was studied by EPR and Mössbauer spectroscopy. The results obtained indicated the presence of three [4Fe-4S] clusters. Redox titration studies revealed that the reduction potentials of all three clusters were below -500 mV. The previously reported S = 7/2 state of the enzyme during benzoyl-CoA-independent ATPase activity (Boll, M., Albracht, S. J. P., and Fuchs, G. (1997) Eur. J. Biochem. 244, 840-851) was confirmed by Mössbauer spectroscopy. Inactivation by oxygen was associated with the irreversible conversion of part of the [4Fe-4S] clusters to [3Fe-4S] clusters. Acetylene stimulated the benzoyl-CoA-independent ATPase activity and induced novel EPR signals with g(av) >2. The presence of simple cubane clusters in benzoyl-CoA reductase as the sole redox-active metal centers demonstrates novel aspects of [4Fe-4S] clusters since they adopt the role of elemental sodium or lithium which are used as electron donors in the analogous chemical Birch reduction of aromatic rings.

Nonaromatic products from anoxic conversion of benzoyl-CoA with benzoyl-CoA reductase and cyclohexa-1,5-diene-1-carbonyl-CoA hydratase.

The enzymes benzoyl-CoA reductase and cyclohex-1, 5-diene-1-carbonyl-CoA hydratase catalyzing the first steps of benzoyl-CoA conversion under anoxic conditions were purified from the denitrifying bacterium, Thauera aromatica. Reaction products obtained with [ring-(13)C(6)]benzoyl-CoA and [ring-(14)C]benzoyl-CoA as substrates were analyzed by high pressure liquid chromatography and by NMR spectroscopy. The main product obtained with titanium(III) citrate or with reduced [8Fe-8S]-ferredoxin from T. aromatica as electron donors was identified as cyclohexa-1, 5-diene-1-carbonyl-CoA. The cyclic diene was converted into 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the hydratase. Assay mixtures containing reductase, hydratase, and sodium dithionite or a mixture of sulfite and titanium(III) citrate as reducing agent afforded cyclohex-2-ene-1-carbonyl-CoA and 6-hydroxycylohex-2-ene-1-carbonyl-CoA. The potential required for the first electron transfer to the model compound S-ethyl-thiobenzoate yielding a radical anion was determined by cyclic voltammetry as -1.9 V versus a standard hydrogen electrode. The energetics of enzymatic ring reduction of benzoyl-CoA are discussed.

Unusual spectroscopic and electrochemical properties of the 2[4Fe-4S] ferredoxin of Thauera aromatica.

A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica. It contains two [4Fe-4S] clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension. The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both [4Fe-4S] clusters from T. aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR. Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE. X-band EPR spectra recorded at potentials where only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = (3)/(2) and (5)/(2) spin states of one reduced [4Fe-4S] cluster. No typical S = (1)/(2) EPR signals were observed. At lower potentials (less than -500 mV), the more negative [4Fe-4S] cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = (1)/(2) low-spin [4Fe-4S] cluster with a g(av) of 1.94. However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = (1)/(2) and S = (5)/(2) EPR signals. At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >(5)/(2). The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the [4Fe-4S] clusters. The observation of different spin states in T. aromatica ferredoxin is novel among CvFd-type ferredoxins.

Characteristics of dipeptide transport in pig jejunum in vitro.

Characteristics of dipeptide transport in pig jejunum were investigated in vitro by applying the Ussing-chamber technique and mucosal uptake studies. Addition of both glycyl-L-glutamine and glycyl-L-sarcosine (20 mmol.l-1) to the mucosal buffer solution significantly increased the short-circuit current by 2.60 +/- 0.15 and 1.57 +/- 0.20 mu eq.cm-2.h-1, respectively. Concentration-dependent changes in short-circuit current followed Michaelis-Menten kinetics with similar affinity constants for both dipeptides. From unidirectional flux rates for radiolabelled glycyl-L-sarcosine, a net flux rate for glycyl-L-sarcosine of 49.8 +/- 6.7 nmol.cm-2.h-1 was calculated. In mucosal uptake experiments, the apical influx of 14C-labelled glycyl-L-sarcosine into isolated porcine mucosa was pH dependent and significantly inhibited by glycyl-L-glutamine. Moreover, RT-PCR studies with primers derived from rabbit PepT1 identified two PCR fragments of identical size to rabbit PepT1 from pig intestinal mRNA preparations. In conclusion, our studies revealed key features of mammalian intestinal peptide transporters and give evidence for a PepT1-like transporter in the pig jejunum that could significantly contribute to the overall amino acid absorption from the gut.

In vivo and in vitro studies on the regulatory link between 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in rat liver.

The activities of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoA reductase; EC 1.1.1.34), rate-limiting enzyme of cholesterol biosynthesis, and cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), key enzyme of the neutral bile acid synthesis pathway, were measured in the microsomal fraction of rat liver and in rat liver cells to investigate the coordinate regulation of the two pathways. Both enzyme activities exhibited the same diurnal rhythm and responded in a coordinate fashion to fasting or bile acid-feeding (decrease) and to cholestyramine-feeding (increase). Cholesterol-feeding decreased the activity of HMGCoA reductase, increased that of cholesterol 7 alpha-hydroxylase, and concomitantly increased free cholesterol in microsomes. In an ex vivo setting using primary hepatocytes from animals fed a high cholesterol diet the activity of HMGCoA reductase was initially low and that of cholesterol 7 alpha-hydroxylase was elevated. Release of cholesterol into the medium with ongoing incubation caused HMGCoA reductase activity to increase, and that of cholesterol 7 alpha-hydroxylase to decline. Incubation of hepatocytes with a cholesterol-containing lipoprotein fraction stimulated the activity of cholesterol 7 alpha-hydroxylase, but left HMGCoA reductase activity unaffected. The results confirm the idea of a joint regulation of the two key enzymes of cholesterol metabolism in response to the levels of substrate and metabolites, and support the notion that with respect to bile acid and cholesterol levels, respectively, regulation of HMGCoA reductase activity may be secondary to that of cholesterol 7 alpha-hydroxylase. The in vitro studies supply evidence that the effects of cholesterol and bile acid excess or deficiency are direct and do not involve accessory changes of hormone levels or mediators.

Reduced ferroxidase activity in the cerebrospinal fluid from patients with Parkinson's disease.

Recent evidence suggest the implication of transition metals leading to overproduction of free radicals as a possible causal factor in the death of nigral cells associated to Parkinson's disease (PD). Iron depots in the basal ganglia of PD patients have been described; in addition, contents of nigral copper have been found decreased, while its concentration in cerebrospinal fluid (CSF) is raised, particularly the free form of the metal. To search for a possible link between altered copper concentrations and PD, we advanced the hypothesis that ferroxidase activity of ceruloplasmin is decreased in the CSF of PD patients. We studied 35 untreated PD patients, 14 L-3,4-dihydroxyphenylalanine (L-DOPA)-treated PD patients and 26 controls. Both CSF ferroxidase activity and CSF copper content were measured and correlated with the clinical stage of the disease. We found that untreated PD patients had a significant reduction of 40% in CSF ferroxidase while CSF copper was slightly increased as compared with both the values in L-DOPA-treated PD patients and controls. We also found that the fraction of copper linked to ferroxidase in untreated PD is inversely related to the clinical stage of the disease.

The occurrence of 4-amino-4-deoxy-arabinose in LPS of supersusceptible strain Pseudomonas aeruginosa Z61: noncorrelation with polymyxin resistance.

Lipopolysaccharides (LPS) extracted from the supersusceptible strain Pseudomonas aeruginosa Z61 were compared with LPS from other strains with varying antimicrobial susceptibilities. The presence of 4-amino-4-deoxy-arabinose (4-AraN) in P. aeruginosa Z61 LPS was confirmed by gas-liquid chromatography/mass spectrometry (GLC-MS) and quantitated by high-performance liquid chromatography (HPLC). Z61 LPS (compared with wild-type strain PAO1) has reduced amounts of rhamnose and higher concentrations of hydroxy fatty acids, 4-AraN, and phosphates. 31P Nuclear magnetic resonance revealed that Z61 LPS phosphates are configured in monophosphates, phosphodiesters, pyrophosphomonoesters, and glycosidic pyrophosphodiester groups. The presence of 4-AraN in P. aeruginosa LPS did not correlate with antimicrobial resistance.

Investigations on a cell culture medium for storage and transportation of avulsed teeth.

Non-physiologic storage of avulsed teeth leads to a high incidence of root resorption, resulting in poor prognosis. This study investigated the suitability of specially composed cell culture media for storage of extracted teeth for up to 48 hours. Autoradiographic investigations revealed that the proliferative activity of periodontal ligament (PDL) cells of teeth stored in cell culture medium for up to 48 hours increased with storage time. Studies on proliferation of PDL cells after storage of teeth in different media for up to 24 hours demonstrated that the proliferative activity is dependent on the composition of the medium. Immunohistochemical investigations with markers for cell proliferation revealed that pulp cells of extracted immature teeth show numerous proliferations after storage for up to 24 hours in a special cell culture medium but few proliferations after storage in Hanks Balanced Salt Solution (HBSS). The investigations indicate that a special cell culture medium can preserve cell viability of PDL cells adhering to extracted teeth for at least 48 hours. The in vitro results are confirmed by a case presented: After storage of two upper central incisors for 36 hours in the cell culture medium the teeth could be successfully reimplanted after extraoral insertion of titanium posts into the root canal (auto-alloplastic reimplantation). Clinical and radiological follow-up examinations for 12 months revealed normal periodontal healing.