Fusion-competent vaccines: broad neutralization of primary isolates of HIV

Current recombinant human immunodeficiency virus (HIV) gp120 protein vaccine candidates are unable to elicit antibodies capable of neutralizing infectivity of primary isolates from patients. Here, "fusion-competent" HIV vaccine immunogens were generated that capture the transient envelope-CD4-coreceptor structures that arise during HIV binding and fusion. In a transgenic mouse immunization model, these formaldehyde-fixed whole-cell vaccines elicited antibodies capable of neutralizing infectivity of 23 of 24 primary HIV isolates from diverse geographic locations and genetic clades A to E. Development of these fusion-dependent immunogens may lead to a broadly effective HIV vaccine.     

Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein

Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.     

Comment on the letter ''imaging in transient regional osteoporosis''

Canada's hospitals are slowly coming to grips with the millennium bug, but Anita Elash reports that no one really knows what impact the move into the year 2000 will have on computers and medical devices, either in the hospital or doctor's office.     

Silicon and aluminium interactions in haemodialysis patients

BACKGROUND: Aluminium toxicity in dialysis patients is well described. Aluminium has a close chemical affinity with silicon. Silicon may have a role in protection against aluminium toxicity. METHODS: We measured serum aluminium and silicon levels from haemodialysis patients from four different centres. RESULTS: Though no relationship was seen across all centres combined, in one centre there was a reciprocal relationship in patients on home haemodialysis (who did not require reverse osmosis). Median (range) aluminium levels were higher, 2.2 (0.4-9.6) micromol/l when serum silicon was less than 150 micromol/l, and lower, 1.1 (0.2-2.8) micromol/l when serum silicon levels were greater than 150 micromol/l (P = 0.03). CONCLUSIONS: In patients treated by haemodialysis without reverse osmosis high serum silicon concentrations were associated with lower serum aluminium concentrations than those with low serum silicon. Further work needs to confirm a preventative role for silicon in the accumulation and subsequent toxicity of aluminium in dialysis patients.     

Purification and characterization of MAR1. A mitochondrial associated ribonuclease from Leishmania tarentolae

A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mitochondrial extract of Leishmania tarentolae and the gene cloned. The purified nuclease has a Km of 100-145 +/- 33 nM and a Vmax of 1.8-2.9 +/- 2 nmol/min, depending on the RNA substrate, and yields a 3'-OH and a 5'-phosphate. Cleavage was limited to several specific sites in the substrate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was affinity-purified. The cleavage specificity of the recombinant enzyme from L. tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820-kilobase pair chromosome and contains an open reading frame of 579 nucleotides. The 18-amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Data base searching revealed two homologues of MAR1 corresponding to unidentified open reading frames in Caenorhabditis elegans (GenBankTM accession number Z69637) and Archaeoglobus fulgidus (GenBankTM accession number AE000943). The function of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be determined.     

Lack of TEL/AML1 fusion in pediatric AML: further evidence for lineage specificity of TEL/AML1

The percutaneous cardiopulmonary support system (PCPS) was used in a 64-year-old woman with cardiogenic shock due to sustained ventricular fibrillation (Vf) caused by severe aortic stenosis and regurgitation. The Vf attack was resistant to cardioversion and adrenaline for lack of left ventricular support by PCPS. She was transported to the operation theater with PCPS in situ and emergency aortic valve replacement was performed. Although preoperative cardiac resuscitation time was long (35 minutes), she was discharged from the hospital on foot without any neurological complications on 84th postoperative day. Because PCPS does not decrease left ventricular systolic stress in poorly contracting dilated heart, early surgical treatment is needed in patients with severely damaged heart.     

Protein turnover in the hypertrophying kidney

To establish whether altered proteolysis contributes to the increase in protein content in hypertrophying kidneys, we studied protein turnover in proximal renal tubules isolated from rats with three forms of renal hypertrophy, diabetes mellitus (DM), ammonium chloride-induced acidosis and compensatory renal growth (CRG). We found that in DM and in chronic acidosis the normal balance in protein turnover is altered due to attenuated proteolysis and accelerated protein synthesis. Together this favors an increase in kidney protein content. In contrast, in CRG, the increase in protein content is entirely due to increased protein synthesis. Thus, the changes in protein turnover leading to the net gain in kidney protein content in renal hypertrophy depends on the cause of hypertrophy.     

Thioether adducts of a new imine reactive intermediate of the pneumotoxin 3-methylindole

Cytochrome P450 enzymes can potentially oxygenate 3-methylindole to form 2,3-epoxy-3-methylindoline which could rearrange to the stable metabolite 3-methyloxindole or open to form 3-hydroxy-3-methylindolenine, a putative electrophilic imine. The purpose of the current work was to determine if the imine was formed, and to characterize it via its adducts with thiol nucleophiles. Thiols were added to incubations of goat lung microsomes with 3-methylindole and deuterated analogues of 3-methylindole to trap the imine intermediate as its thioether conjugates. The N-acetylcysteine conjugate of 3-hydroxy-3-methylindolenine was detectable by LC/MS, but a molecular ion was not observed because the adduct rapidly dehydrated to form the 2-substituted indole. However, the imine was S-alkylated, and the intermediate carbinol was intramolecularly trapped using thioglycolic acid as a trapping agent that induced cyclocondensation to a lactone. The retention of one atom of deuterium from [2-2H]-3-methylindole and three from 3-[2H3-methyl]indole substantiated the mechanism in which the lactone adduct was produced by sulfur addition to either 3-hydroxy-3-methylindolenine or the epoxide. Tandem mass spectrometry of the lactone adduct produced a daughter ion spectrum consistent with this adduct. These studies demonstrated the existence of a new reactive intermediate of 3-methylindole, 3-hydroxy-3-methylindolenine, which may play a role in the pneumotoxicity of this chemical.