Attenuation of landfill leachate by clay liner materials in laboratory columns: 2. Behaviour of inorganic contaminants.

The chemical attenuation of inorganic contaminants in methanogenic landfill leachate, spiked with heavy metals (Cd, Cd, Ni and Zn), by two UK clay liner materials was compared in laboratory columns over 15 months. Ammonium was attenuated by ion-exchange but this attenuation was finite and when exhausted, NH4 passed through the liners at concentrations found in the leachate. The breakthrough behaviour of NH4 could be described by a simple distribution coefficient. Heavy metals were attenuated by sorption and precipitation of metal sulphide and carbonate compounds near the top of the liner. Adequate SO4 and CaCO3 in the liner is necessary to ensure the long term retention of heavy metals, and pH buffering agents added to stabilise reactive metal fractions should be admixed with the liner. Some metals may not be chemically attenuated by clay liners due to the formation of stable complexes with organic and/or colloidal fractions in leachate. Flushing of the liners with oxygenated water after leachate caused mobilisation of attenuated contaminants. Sorbed NH4 was released by the liners but groundwater loadings were manageable. Re-oxidation of metal sulphides under these conditions resulted in the release of heavy metals from the liners when the pH buffering capacity was poor. Contaminant attenuation by the clay liners was similar and the attenuation of NH4 and heavy metals could be predicted from the geochemical properties of the liner using simple tests. A conceptual model of clay liner performance is presented. Chemical attenuation of inorganic pollutants can be included in containment liner design to produce a dual reactive-passive barrier for landfills.

Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+ T cell recognition.

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) includes a unique glycine-alanine repeat domain that inhibits the endogenous presentation of cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by blocking proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated diseases. Here, we show that cotranslational ubiquitination combined with N-end rule targeting enhances the intracellular degradation of EBNA1, thus resulting in a dramatic reduction in the half-life of the antigen. Using DNA expression vectors encoding different forms of ubiquitinated EBNA1 for in vivo studies revealed that this rapid degradation, remarkably, leads to induction of a very strong CTL response to an EBNA1-specific CTL epitope. Furthermore, this targeting also restored the endogenous processing of HLA class I-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific CTLs. These observations provide, for the first time, evidence that the glycine-alanine repeat-mediated proteasomal block on EBNA1 can be reversed by specifically targeting this antigen for rapid degradation resulting in enhanced CD8+ T cell-mediated recognition in vitro and in vivo.

Immunotherapeutic strategies for EBV-associated malignancies.

Advances in our understanding of the role of cytotoxic T lymphocytes (CTLs) in the control of Epstein-Barr virus (EBV)-associated malignancies and the overall biology of these diseases have led to the development of novel therapeutic strategies designed to specifically target viral antigens expressed in these malignancies. Long-term success of many of these strategies is constrained by the latency phenotypes adopted by different diseases. Adoptive transfer of polyclonal virus-specific CTLs has been used successfully to reverse the outgrowth of malignancies such as post-transplant lymphoproliferative disease (PTLD). On the other hand, limited viral gene expression in other EBV-associated malignancies such as Burkitt's lymphoma, Hodgkin's disease and nasopharyngeal carcinoma limits the efficacy of immunotherapeutic strategies used for PTLD. Preclinical studies based on specific targeting of viral antigens expressed in these malignancies have provided very encouraging results and thus are likely to serve as an important platform for the treatment of human patients.

Characterization of Munc-18c and syntaxin-4 in 3T3-L1 adipocytes. Putative role in insulin-dependent movement of GLUT-4.

We have previously identified three mammalian Sec1/Munc-18 homologues in adipocytes (Tellam, J. T., McIntosh, S., and James, D. E. (1995) J. Biol. Chem. 270, 5857-5863). These proteins are thought to modulate the interaction between vesicle membrane and target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and thus regulate intracellular vesicular transport. This study aimed to further characterize these Munc-18 isoforms and to define their potential role in the trafficking of GLUT-4 in adipocytes, a process reported to involve the vesicle membrane SNARE, VAMP-2. Using an in vitro binding assay with recombinant fusion proteins, we show that Munc-18a and Munc-18b bind to syntaxin-1A, -2, and -3, while Munc-18c binds only to syntaxin-2 and -4. The specific interaction between Munc-18c and syntaxin-4 is of interest because aside from syntaxin-1A, which is not expressed in adipocytes, syntaxin-4 is the only syntaxin that binds to VAMP-2. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-2. The subcellular distribution of syntaxin-4 and Munc-18c was almost identical, both being enriched in the plasma membrane, and both exhibiting an insulin-dependent movement out of an intracellular membrane fraction similar to that observed for GLUT-4. Munc-18b had a similar distribution to Munc-18c and so may also be involved in vesicle transport to the cell surface, whereas Munc-18a was undetectable by immunoblotting in adipocytes. Microinjection of a syntaxin-4 antibody into 3T3-L1 adipocytes blocked the insulin-dependent recruitment of GLUT-4 to the cell surface. These data suggest that syntaxin-4/Munc-18c/VAMP-2 may play a role in the docking/fusion of intracellular GLUT-4-containing vesicles with the cell surface in adipocytes.

Identification of a mammalian Golgi Sec1p-like protein, mVps45.

Our understanding of lysosomal biogenesis and general vesicular transport in animal cells has been greatly enhanced by studies of vacuolar biogenesis in yeast. Genetic screens have identified a number of proteins that play direct roles in the proper sorting of vacuolar hydrolases. These include t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and Sec1p-like proteins, which have recently been implicated as key regulators of vesicle fusion. In this study we have extended these observations in yeast and have isolated and characterized a novel member of the Sec1p-like family of proteins from mammalian cells, mVps45. mVps45 shares a high level of identity with the Saccharomyces cerevisiae Sec1p-like protein Vps45p that is believed to function with the t-SNARE Pep12p in the fusion of Golgi-derived transport vesicles with a prevacuolar compartment. We found that mVps45 is a ubiquitously expressed peripheral membrane protein that localized to perinuclear Golgi-like and trans-Golgi network compartments in Chinese hamster ovary cells. We found that mVps45 could bind specifically to yeast Pep12p and to the mammalian Pep12p-like protein, syntaxin 6, in vitro.

Novel isoform of syntaxin 1 is expressed in mammalian cells.

Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor protein required for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Syntaxin 1A consists of 288 amino acid residues including a 265-residue N-terminal region exposed to the cytoplasm and a C-terminal hydrophobic stretch of 23 residues believed to anchor syntaxin to the plasma membrane. Using a human fat-cell library we have isolated a novel cDNA clone of syntaxin 1A containing an insert of 91 bp in codon 226. This insert and subsequent frame shift generated a cDNA that codes for a truncated protein of 260 residues without the C-terminal transmembrane domain characteristic of the syntaxin family. Analysis of the deduced amino acid sequence of the new cDNA clone, termed syntaxin 1C, showed that it was identical for the first 226 residues with the previously described neural syntaxin 1A, and diverged thereafter. The truncated protein lacked the botulinum neurotoxin C cleavage site (Lys253-Ala254), a feature of the syntaxin 1A protein, because of the novel C-terminal domain of 34 residues. The new C-terminal region contained a single cysteine residue and was moderately rich in proline, with three repeats of a PXP motif. The insert occurred within the region encoding the coiled-coil motifs required for interactions with synaptobrevin, alpha-SNAP (SNAP being soluble N-ethylmaleimide-sensitive factor attachment protein) and n-Sec1/Munc-18 (n-Sec1 being the rat brain homologue of yeast Sec1p and Munc-18 the mammalian homologue of Caenorhabditis elegans unc-18, but five residues outside the domain previously mapped as being required for binding SNAP-25. Interaction studies in vitro suggested that unlike syntaxin 1A, which binds to both Munc-18a and- 18b, syntaxin 1C binds only to Munc-18b. The new isoform syntaxin 1C, which might be generated by alternative splicing of the syntaxin 1 gene, was expressed in several human tissues, including brain. Immuno-precipitation and immunoblotting with the monoclonal antibody HPC-1 and a polyclonal antibody raised against a peptide corresponding to the unique C-terminal 35 residues of syntaxin 1C failed to detect syntaxin 1C at the protein level in extracts of muscle, fat or brain.

The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells.

Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle-associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP-2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.

Molecular identification of two novel Munc-18 isoforms expressed in non-neuronal tissues.

Munc-18, also known as n-Sec1 or rbSec1, is a syntaxin-binding protein thought to play a role in regulating synaptic vesicle exocytosis. Although a gene family of syntaxins has been identified, only a limited subset bind to Munc-18. This implicates the existence of other mammalian Munc-18 homologues that may be involved in a range of vesicle transport reactions. The purpose of the present study was to identify other members of the Munc-18 family by cDNA cloning. Three distinct Munc-18 isoforms, Munc-18a, previously identified in neuronal tissue, and two novel isoforms, Munc-18b and Munc-18c, were isolated from a 3T3-L1 adipocyte cDNA library by screening with a rat brain Munc-18 DNA probe. Munc-18a is identical to Munc-18 and by Northern analysis is expressed predominantly in brain and to a lesser extent in testis and 3T3-L1 cells. Munc-18b is 62% identical to Munc-18 at the amino acid level and is expressed in testis, intestine, kidney, rat adipose tissue, and 3T3-L1 cells. Munc-18c is 51% identical to Munc-18 and is ubiquitously expressed. It is likely, based on these findings, that unique Munc-18/syntaxin interactions may play an important role in generating a combinatorial mechanism for the regulation of vesicle transport in mammalian cells.

Isolation and nucleotide sequence of a cDNA clone encoding rat mitochondrial malate dehydrogenase.

We have determined the complete sequence of the rat mitochondrial malate dehydrogenase (mMDH) precursor derived from nucleotide sequence of the cDNA. A single synthetic oligodeoxynucleotide probe was used to screen a rat atrial cDNA library constructed in lambda gt10. A 1.2 kb full-length cDNA clone provided the first complete amino acid sequence of pre-mMDH. The 1014 nucleotide-long open reading frame encodes the 314 residue long mature mMDH protein and a 24 amino acid NH2-terminal extension which directs mitochondrial import and is cleaved from the precursor after import to generate mature mMDH. The amino acid composition of the transit peptide is polar and basic. The pre-mMDH transit peptide shows marked homology with those of two other enzymes targeted to the rat mitochondrial matrix.

Human renin gene sequence, gene regulation and prorenin processing.

Human DNA coding for renin was identified and sequenced. The gene consisted of 10 exons corresponding to a 1500 nucleotide mRNA was broken up by long stretches of 'nonsense' DNA (introns) and spanned 12,000 base pairs. In addition, the sequence of nucleotides involved in regulation of the gene was determined by sequencing upstream. Prediction of the amino acid sequence of human preprorenin revealed likely sites of processing. This helps explain many past experimental observations. For example, the pro region contained adjacent likely cleavage sites for trypsin and pepsin and so reveals why both trypsin and pepsin can activate prorenin. The structure of human renin had features involved in its highly specific hydrolysis of the Leu10-Val11 bond unique to human angiotensinogen: in particular leucine 224 (instead of valine). Renin gene expression was studied in the mouse by quantification of both renin activity and its mRNA. Sodium depletion, captopril and spironolactone increased expression of Ren-1 in the kidney. The unusual, duplicated, mouse gene, Ren-2, which is expressed in the submandibular gland was, regulated by (dihydro)testosterone in male mice and by thyroid hormone in female mice.

Primary structure of the human renin gene.

The gene encoding human renin has been isolated on two overlapping clones from a bacteriophage lambda library of human DNA. The entire gene spans about 12,000 bp and contains 10 exons separated by 9 intervening sequences. The gene structure is similar to that of human pepsinogen in terms of overall size, homology in the coding regions, position of introns, and sizes of the exons, suggesting that the two genes are evolutionarily related. However, a novel exon coding for only three amino acids was detected that is not present in the pepsinogen gene and whose amino acids are also not found in mouse renin. Although the nucleotide sequence of the 5'-flanking DNA differs from that of the pepsinogen gene, in both cases this region contains a structure of almost perfect dyad symmetry which immediately precedes the TATA box and may have functional importance. Furthermore, sequences resembling the putative consensus sequence for glucocorticoid regulation of gene expression are located approximately 200 and 300 bp upstream from the gene. The overall structural anatomy suggests that the human renin gene evolved by mechanisms that include a duplication of exon segments, particularly those containing the codons for the catalytically important aspartate residues, together with the insertion of other exon and flanking DNA structures. An analysis of human chromosomal DNA demonstrates that there is only one gene with high homology to human renin.

Structure of human renin and expression of the renin gene.

The amino acid sequence of human renin was identified for the first time. This was determined from the nucleotide sequence of exons in the human renin gene identified in a genomic library by recombinant DNA techniques. Examination of amino acid residues involved in the enzymatic hydrolysis by human renin of the unique Leu10-Val11 bond of human angiotensinogen revealed features peculiar to this highly specialized aspartyl protease. The expression of the renin gene was examined with a hybridization probe for renin mRNA in sections and extracts of tissues. In the submandibular gland of mice renin mRNA, like renin, increased during development and in response to testosterone in females; sodium depletion increased renin mRNA in kidney.

Antibody-nucleic acid complexes. Identification of antigenic determinant of a murine monoclonal antibody specific for single-stranded nucleic acids.

Cloned hybrid cells, selected for their ability to secret an IgG 2a immunoglobulin specific for single-stranded (ss) nucleic acids, were obtained by fusion of spleen cells from an unimmunized autoimmune MRL/1 pr male mouse with nonsecreting myeloma cells (MOPC-21, line Sp2/0-Ag 14). Designated MRss-1, this monoclonal antibody was (i) propagated by intraperitoneal injection of hybrid cells to pristane-treated. Balb/c mice, (ii) purified from the bulk of other proteins in ascites extracts by chromatography with DEAE-Sephacel adsorbent, and (iii) radiochemically labeled via reductive methylation using NaB3H4 and formaldehyde. The binding of 3H-labeled antibody to immobilized ssDNA- agarose, calf thymus) or soluble (fd DNA) ssDNA was rapid and dependent upon ssDNA and ionic strength, but not hydrogen ion concentration. Optimal binding occurred in both low and intermediate salt concentration (0.1-0.25 M NaCl), yet was completely abolished above 0.30 M NaCl. The presence of guanine (Gua)-containing mono-, oligo-, and polynucleotides also abolished and/or decreased 3H-labeled antibody binding to ssDNA-agarose. In these competition assays, the amount of Gua-containing mono-and oligonucleotides required to inhibit antibody binding by 50% (0.2-1.0 mg/mL) exceeded those of poly(G), rRNA, and fd DNA (i.e., 0.03-0.1 microgram/mL) by 4 orders of magnitude. In contrast, (deoxy)ribose 5'-phosphate as well as other nucleic acid derivatives devoid of Gua failed to inhibit antibody binding. The above findings were substantiated by the observation that 3H-labeled antibody bound to guanosine (G)- and guanidylate (pG)-conjugated Sepharose, yet not to other nucleoside (A, C, and U)- or nucleotide (pA, pC, and pU)-conjugated adsorbents. Last, the introduction of the methyl group at the N-2, O-6, and N-7 positions in the Gua ring system completely abolished antibody binding. Collectively, these results demonstrate that the MRss-1 antibody recognized single-stranded nucleic acid substrates by virtue of their content of guanidylate residues and, more specificity, by the presence of the Gua base moiety.