Zinc stable isotope fractionation upon accelerated oxidative weathering of sulfidic mine waste.

Accelerated oxidative weathering in a reaction cell (ASTM D 5744 standard protocol) was performed over a 33 week period on well characterized, sulfidic mine waste from the Kidd Creek Cu-Zn volcanogenic massive sulfide deposit, Canada. The cell leachate was monitored for physicochemical parameters, ion concentrations and stable isotope ratios of zinc. Filtered zinc concentrations (<0.45 µm) in the leachate ranged between 4.5 mg L(-1) and 1.9 g L(-1)-potentially controlled by pH, mineral solubility kinetics and (de)sorption processes. The zinc stable isotope ratios varied mass-dependently within +0.1 and +0.52‰ relative to IRMM 3702, and were strongly dependent on the pH (rpH-d66Zn=0.65, p<0.005, n=31). At a pH below 5, zinc mobilization was governed by sphalerite oxidation and hydroxide dissolution-pointing to the isotope signature of sphalerite (+0.1 to +0.16‰). Desorption processes resulted in enrichment of (66)Zn in the leachate reaching a maximum offset of +0.32‰ compared to the proposed sphalerite isotope signature. Over a period characterized by pH=6.1 ± 0.6, isotope ratios were significantly more enriched in (66)Zn with an offset of ≈ 0.23‰ compared to sphalerite, suggesting that zinc release may have been derived from a second zinc source, such as carbonate minerals, which compose 8 wt.% of the tailings. This preliminary study confirms the benefit of applying zinc isotopes alongside standard monitoring parameters to track principal zinc sources and weathering processes in complex multi-phase matrices.

Occurrence and behaviour of dissolved, nano-particulate and micro-particulate iron in waste waters and treatment systems: new insights from electrochemical analysis.

Cyclic-, Differential Pulse- and Steady-state Microdisc Voltammetry (CV, DPV, SMV) techniques have been used to quantify the occurrence and fate of dissolved Fe(ii)/Fe(iii), nano-particulate and micro-particulate iron over a 12 month period in a series of net-acidic and net-alkaline coal mine drainages and passive treatment systems. Total iron in the mine waters is typically 10-100 mg L(-1), with values up to 2100 mg L(-1). Between 30 and 80% of the total iron occurs as solid phase, of which 20 to 80% is nano-particulate. Nano-particulate iron comprises 20 to 70% of the nominally "dissolved" (i.e. <0.45 µm) iron. Since coagulation and sedimentation are the only processes required to remove solid phase iron, these data have important implications for the generation or consumption of acidity during water treatment. In most waters, the majority of truly dissolved iron occurs as Fe(ii) (average 64 ± 22%). Activities of Fe(ii) do not correlate with pH and geochemical modelling shows that no Fe(ii) mineral is supersaturated. Removal of Fe(ii) must proceed via oxidation and hydrolysis. Except in waters with pH < 4.4, activities of Fe(iii) are strongly and negatively correlated with pH. Geochemical modelling suggests that the activity of Fe(iii) is controlled by the solubility of hydrous ferric oxides and oxyhydroxysulfates, supported by scanning and transmission electron microscopic analysis of solids. Nevertheless, the waters are generally supersaturated with respect to ferrihydrite and schwertmannite, and are not at redox equilibrium, indicating the key role of oxidation and hydrolysis kinetics on water treatment. Typically 70-100% of iron is retained in the treatment systems. Oxidation, hydrolysis, precipitation, coagulation and sedimentation occur in all treatment systems and - independent of water chemistry and the type of treatment system - hydroxides and oxyhydroxysulfates are the main iron sinks. The electrochemical data thus reveal the rationale for incomplete iron retention in individual systems and can thus inform future design criteria. The successful application of this low cost and rapid electrochemical method demonstrates its significant potential for real-time, on-site monitoring of iron-enriched waters and may in future substitute traditional analytical methods.

Activation of the dentate gyrus by stimulation of the contralateral perforant pathway: evoked potentials and long-term potentiation after ipsi- and contralateral induction.

Rats were chronically implanted with stimulation electrodes in the perforant pathway (pp) bilaterally and a recording electrode in the dentate gyrus (DG) unilaterally. Evoked field potentials (EPs) were recorded upon alternating stimulation of the pp on both sides, and long-term potentiation (LTP) was induced. Besides the EP after ipsilateral stimulation, an EP with a latency of approximately 5.5-6.5 ms was also seen upon stimulation of the contralateral pp. This potential was reversibly abolished during pentobarbital anesthesia and irreversibly after lesioning of the ipsilateral angular bundle. Paired-pulse facilitation and paired-pulse depression, depending on interstimulus interval and intensity, were also observed. Therefore, this long-latency potential could be characterized as polysynaptic and induced perhaps by transsynaptic activation via the ipsilateral entorhinal cortex. Ipsilateral tetanization induced strong E/S potentiation of both the ipsilaterally and contralaterally evoked EP, but with different time courses. Tetanization of the contralateral pp did not induce LTP of the ipsilaterally induced EP in the first 4 h. But afterwards a late and slowly developing potentiation occurred. The contralaterally induced EP also showed potentiation of the population spike, which was not immediately detectable but developed slowly over time. The results can be interpreted such that, after stimulation of the pp, the DG on the opposite side cannot only be activated via the weak crossed entorhinal projection but also transsynaptically via an entorhino/entorhinal connection.

Effects of anticonvulsive drugs on pentylenetetrazol kindling and long-term potentiation in freely moving rats.

Drugs with anticonvulsive properties and different mechanisms of action were compared for their influence on long-term potentiation and pentylenetetrazol kindling in freely moving animals. Rats were chronically implanted with a stimulation electrode in the angular bundle and a recording electrode in the dentate gyrus. Field potentials in the dentate gyrus were elicited and long-term potentiation was induced by stimulation of the perforant pathway. The clinically used drugs or the potentially anticonvulsive drugs, diphenylhydantoin (50 mg/kg), diazepam (0.5 mg/kg), pentobarbital (10 mg/kg), dizocilpine (MK 801, 0.2 mg/kg) and CGP 43487 (2-amino-4-methyl-5-phosphono-3-pentenoic acid-carboxyethylester, 10 mg/kg), were injected before tetanization. In behavioural experiments pentylenetetrazol kindling was performed with pretreatment with the substances in dosages indicated above (except MK 801, 0.3 mg/kg). Field potentials recorded in the interval between drug administration and tetanization were influenced only by diphenylhydantoin which enhanced the population spike amplitude to 128% of control values. However, the substances showed different effects on long-term potentiation. MK 801, CGP 43487 and pentobarbital depressed potentiation; diazepam was without effect. Diphenylhydantoin had a minor influence on induction but significantly impaired maintenance of long-term potentiation. Furthermore, MK 801, CGP 43487, diazepam and pentobarbital differentially depressed kindling whereas phenytoin only slightly influenced it. The consequences as to hypothetical common cellular mechanisms for kindling development and long-term potentiation are discussed.

A novel 52 kDa protein induces apoptosis and concurrently activates c-Jun N-terminal kinase 1 (JNK1) in mouse C3H10T1/2 fibroblasts.

A 52 kDa protein (p52) was purified from chicken embryos and its corresponding cDNA was cloned. The p52 cDNA is 1768 bp long and has an open reading frame of 465 amino acids. The sequence of the p52 cDNA shows significant homology with mouse and human cDNAs from the EST database, so do the deduced amino acid sequences, indicating the existence of human and mouse homologues of p52. Northern blot hybridization showed that the p52 mRNA was expressed in a wide range of embryonic and adult tissues. There was more p52 mRNA in embryonic heart and liver than in the brain or muscle. The adult testis had the highest level of p52 mRNA, whereas adult liver had the lowest. Expression of p52 in mouse C3H10T1/2 fibroblasts caused apoptotic cell death, upregulation of transcription factor c-Jun and activation of c-Jun N-terminal kinase 1 (JNK1). In addition, expression of Bcl-2, but not of the dominant negative mutant JNK1, can block the p52-mediated apoptosis. These results indicate that p52 may represent a new cell-death protein inducing apoptosis and activating JNK1 through different pathways.

How to spot depressed patients in your practice.

This is the third in a series of short articles on management of common problems primary care physicians encounter in the office setting. Written by members of Postgraduate Medicine's Editorial Council, these articles offer a personal perspective gleaned from hands-on experience. Here, Dr Matthies presents his approach to management of depression-how to recognize clues to the disorder, how to confront patients with the diagnosis, and where to go from there.

Demethylation of DNA by purified chick embryo 5-methylcytosine-DNA glycosylase requires both protein and RNA.

We have previously purified and characterized a 5-methylcytosine (5-MeC)-DNA glycosylase from 12 day old chick embryos [Jost,J.P. et al. (1995) J. Biol. Chem. 270, 9734-9739]. The activity of the purified enzyme is abolished upon treatment with proteinase K and ribonuclease A. RNA copurifies with 5-MeC-DNA glycosylase activity throughout all chromatographic steps and preparative gel electrophoresis. RNA with a length of approximately 300-500 nucleotides was isolated from the gel purified enzyme. Upon extensive treatment with proteinase K, the gel eluted and labeled RNA did not show any significant change in molecular mass. The purified RNA incubated alone or in the presence of Mg2+and deoxyribonucleotide phosphates had no 5-MeC-DNA glycosylase or demethylating activities. However, activity of 5-MeC-DNA glycosylase could be restored when the purified RNA was incubated with the inactive protein, free of RNA.

Lumbar radiculopathy and intraspinal gas.

Gas within the spinal canal is uncommon and has been associated with trauma, infection and disc degeneration and vacuum phenomenon. We report a 73-year-old man with a history of intermittent low back pain who developed sharp low back pain with radiation to the left calf and ankle when ambulating or standing. Relief was obtained with sitting or lying down, which led to the clinical impression of spinal stenosis. The neuromuscular exam was unremarkable and no sciatic nerve tension sign was present. Electrodiagnostic studies were consistent with an acute S1 radiculopathy, while a dermatomal somatosensory evoked potential was interpreted as essentially normal. A CT scan showed gas in the left lateral spinal canal at L4-S1. After 2 weeks, spontaneous improvement occurred and the patient resumed normal activities. A repeat CT scan after 0.5 years showed the intraspinal gas was diminished while the L5-S1 disc vacuum phenomenon had worsened and a right disc extrusion occurred. The natural history of intraspinal gas, disc vacuum phenomenon and related complications are discussed.

Purification and cDNA cloning of a transcription factor which functionally cooperates within a cAMP regulatory unit in the porcine uPA gene.

One of cAMP-regulatory sites in the porcine urokinase-type plasminogen activator (uPA) gene resides 3.4 kb upstream of the transcription initiation site and is composed of three protein binding domains, FPA, FPB and FPC. Whereas FPA and FPB contain a CRE-like sequence, the FPC sequence is not related to any known protein recognition sequences, yet all three domains are required to mediate cAMP action on a heterologous promoter. To study the functional cooperation among these three domains we purified and cloned a FPC-binding protein (FPCB) from porcine kidney derived LLC-PK1 cells. Sequence comparisons showed that FPCB is homologous to mouse LFB3 and rat vHNF1. LFB3/vHNF1 is related to a liver specific transcription factor HNF1, it recognizes the same sequence as HNF1 and is highly expressed in kidney cells. FPCB and HNF1 recognition sequences are dissimilar, nevertheless both sequences are recognized by in vitro-translated LFB3 and FPCB, indicating that binding to the two different sequences is an intrinsic character of FPCB/LFB3/vHNF1. In HeLa cells, this cAMP-responsive site was inactive whether FPCB was overexpressed or not, suggesting a requirement for an additional cell-specific factor. These results may suggest a mechanism by which hormonal control is integrated into cell-specific gene regulation.

Non-opioid antitussives and methadone differentially influence hippocampal long-term potentiation in freely moving rats.

Long-term potentiation (LTP) of monosynaptically evoked field potentials (MEFP) in the dentate gyrus of freely moving rats following tetanization of the perforant pathway was investigated after peripheral application of substances which have been shown to influence NMDA receptor-mediated effects (dextromethorphan, methadone) as well as structurally related substances with similar antitussive effects (codeine, normethadone). The noncompetitive NMDA receptor antagonist MK 801 was also tested for comparison. Whereas under control conditions the field e.p.s.p. (excitatory postsynaptic potential) and the population spike of the MEFP were largely uninfluenced by these substances, different effects were seen after the induction of LTP. MK 801 (0.2 mg/kg i.p.) suppressed the induction of LTP of both the field e.p.s.p. and the population spike. Dextromethorphan (40 mg/kg i.p.) also prevented the potentiation of the field e.p.s.p. and the population spike, thus resembling MK 801 in its effect. Codeine (20 mg/kg i.p.), the levorotatory structural analogue of dextromethorphan had no effect. Methadone and normethadone did not influence the potentiation of the field e.p.s.p. or interfere with the induction of potentiation of the population spike but depressed its maintenance. The results obtained with MK 801 confirm those reported by others. Comparison of the effects of dextromethorphan with those of MK 801, suggests that there is a direct interaction with the NMDA receptor-ionophore complex. The effects of methadone and normethadone appear not to be linked to an interaction with opioid receptors, since naloxone did not influence the suppression of LTP caused by methadone. The possibility of interference with the NMDA receptor-ionophore complex is discussed.

Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform.

The trimeric form of protein phosphatase 2A (PP2A1 or polycation-stimulated protein phosphatase H1) was purified to homogeneity from rabbit skeletal muscle. Preparative SDS-polyacrylamide gel electrophoresis was used to purify the individual subunits with relative molecular masses of 36, 55, and 65 kDa. Sequence analysis of five peptides from the 65-kDa regulatory subunit (PR65) suggested that it was identical with the PR65 subunit derived from the dimeric protein phosphatase 2A2. Amino acid sequences derived from the 55-kDa regulatory subunit (PR55) were used to clone human and rabbit cDNAs encoding this protein. The PR55 subunit was found to be encoded by two genes, termed alpha and beta. The open reading frames of the PR55 alpha and beta cDNAs spanned 1341 and 1329 nucleotides, respectively, and predicted proteins with a molecular mass of about 52 kDa that are 86% identical. Comparison of the human PR55 amino acid sequences with the data obtained from the rabbit skeletal muscle protein and a partial rabbit PR55 beta cDNA clone indicated a high degree of conservation. Analysis of the mRNA expression in human cell lines revealed that the PR55 alpha isoform was encoded by two transcripts of about 2.3 and 2.5 kb and a less abundant 4.4-kb mRNA. Whereas a PR55 beta transcript of about 2.3 kb was detected at high levels in the neuroblastoma derived cell line LA-N-1, the level of the mRNA was very low in the other human cell lines analyzed. Interestingly, the PR55 sequence showed limited homology to the catalytic domain (domains VI-IX) of the c-abl protein tyrosine kinase.

Protein chemical and kinetic characterization of recombinant porcine ribonuclease inhibitor expressed in Saccharomyces cerevisiae.

A cDNA encoding porcine ribonuclease inhibitor was used to express this protein in yeast under control of the PHO5 promoter. The recombinant protein was purified to homogeneity with a yield of 0.2 mg/g of yeast cells (wet weight) and was found to be indistinguishable from the inhibitor isolated from porcine liver on the basis of the following criteria: the amino acid composition, the number of free sulfhydryl groups, the molecular weight of the native and the denatured protein, peptide mapping, and amino acid sequence analysis of the N- and C-terminal regions of the protein. A simple method was developed for measuring accurately the slow, tight-biding kinetics of the inhibition of ribonuclease by ribonuclease inhibitor. From the dependence of the observed inhibition constant on the substrate concentration, it could be concluded that RI was competitive with the substrate UpA. The dependence of the observed association rate constant on the substrate concentration was consistent with a two-step mechanism in which the substrate only competed in the second (isomerization) step. The values for the inhibition constant for the inhibition of RNase by the recombinant inhibitor, 67 fM, the association rate constant, 1.5 x 10(8) M-1.s-1, and the dissociation rate constant, 8.3 x 10(-6) s-1, were in good agreement with those obtained for the porcine liver RNase inhibitor.

Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily.

In most tissues, ribonucleases (RNases) are found in a latent form complexed with ribonuclease inhibitor (RI). To examine whether these so-called cytoplasmic RNases belong to the same superfamily as pancreatic RNases, we have purified from porcine liver two such RNases (PL1 and PL3) and examined their primary structures. It was found that RNase PL1 belonged to the same family as human RNase Us [Beintema et al. (1988) Biochemistry 27, 4530-4538] and bovine RNase K2 [Irie et al. (1988) J. Biochem. (Tokyo) 104, 289-296]. RNase PL3 was found to be a hitherto structurally uncharacterized type of RNase. Its polypeptide chain of 119 amino acid residues was N-terminally blocked with pyroglutamic acid, and its sequence differed at 63 positions with that of the pancreatic enzyme. All residues important for catalysis and substrate binding have been conserved. Comparison of the primary structure of RNase PL3 with that of its bovine counterpart (RNase BL4; M. Irie, personal communication) revealed an unusual conservation for this class of enzymes; the 2 enzymes were identical at 112 positions. Moreover, comparison of the amino acid compositions of these RNases with that of a human colon carcinoma-derived RNase, RNase HT-29 [Shapiro et al. (1986) Biochemistry 25, 7255-7264], suggested that these three proteins are orthologous gene products. The structural characteristics of RNases PL1 and PL3 were typical of secreted RNases, and this observation questions the proposed cytoplasmic origin of these RI-associated enzymes.

Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats.

The primary structure of the ribonuclease inhibitor from pig liver has been determined by amino acid sequence analysis. The N alpha-acetylated polypeptide chain of 456 amino acids consists of 15 homologous leucine-rich repeats, characterized by leucyl residues at constant positions. Two types of alternating repeats occur, 29 (A) and 28 (B) residues long. The degree of identity between repeats of a given type ranged from 25 to 60%. Only one deletion in the B-repeat was necessary to perfectly align the leucyl residues between the two repeats. Leucine-rich repeats have previously been found in four membrane-bound proteins and one extracellular protein, and their amphiphilic character suggested that they could be involved in membrane binding. Ribonuclease inhibitor is the first example of a cytoplasmic protein containing this type of repeat. It seems likely, therefore, that leucine-rich repeats can have functions other than forming membrane binding structures.

Low frequency perforant path stimulation as a conditioned stimulus demonstrates correlations between long-term synaptic potentiation and learning.

Stimulation of the perforant path with impulse trains of 15 cps and 670 msec duration was used as a conditioned stimulus in a two-way shuttle box avoidance on rats. Field potentials in the dentate area evoked by test stimuli were measured after the training sessions until the 7th day. Foot-shock and unconditioned escape elicited only a transient slight depression of the population spike amplitude (P) and increased also slightly the slope function (SF) of the population EPSP of the evoked test potentials. The control stimulation of the perforant path without pairing with foot-shock as in conditioning did only slightly increase SF of test potentials, but produced a strong transient inhibition followed by a long lasting moderate depression of P. After conditioning, all animals exhibited the same initial inhibition of P as shown in control stimulation of the perforant path. However during the following 4 hours, good learners with a relearning index greater than 30% developed a significant potentiation of P lasting until the second training session 24 hours later, which resulted in a further enhancement. SF of the evoked test potentials increased in good learners with a similar time course after conditioning but without initial depression. After 7 days P showed still enhanced but non-significant values. Poor learners with a relearning index less than 10% did not develop a potentiation of P after conditioning and initial inhibition, but a long-term depression. Also SF of test potentials decreased in poor learners during 4 hours after conditioning and returned almost to baseline until the following day. After 7 days, P and SF did not differ from baseline. The analysis of the observed synaptic changes by E-S curves demonstrated the post-tetanic LTP seems to differ in some ways from post-conditioning LTP in good learners. The latter exhibits a clear tendency of a right shift contrary to the left shift commonly occurring after tetanization. Furthermore poor learners do not only fail to produce long-term potentiation, but fail to show a change in the opposite direction with a left shift of the E-S curves. The observed correlation of LTP in the conditioning pathway with the learning ability suggests an involvement of LTP at least in the acquisition and early retention of this learned behavior. The results do however not finally clarify the role of LTP in long-term retention.

[The effect of the systemic and intraventricular application of orotic acid on monosynaptic evoked potentials in the dentate gyrus in free-moving rats]. / Einflus von systemisch und intraventrikulär applizierter Orotsäure auf monosynaptisch evozierte Potentiale im Gyrus dentatus der freibeweglichen Ratte.

In 20 freely moving Wistar rats the influence of systemically and intraventricularly applied orotic acid derivatives on monosynaptically evoked field potentials registered in the dentate gyrus on the stimulation of the medial entorhinal cortex was investigated. Both, methylglucamine-orotate and tris-orotate led to an increase of the amplitude of the population spike. The slope function of the field EPSP, however, remained unchanged. The effect has been observed to begin 2 h after application and had a duration of 6-10 h. The data obtained in a monosynaptic pathway targeting on a brain structure which is known to play a crucial role in memory formation support earlier findings of a transient effect of orotic acid on cerebral excitation processes. An influence on mechanisms of spike generation or other postsynaptic membrane processes might be assumed.