Biomedical subjects
Publications and source records attributed to R Frank.
A clone harbouring the entire urocanase gene (hutU) was obtained from a genomic library of Pseudomonas putida using oligonucleotide probes synthesised on the basis of known flanking sequences. One subunit of urocanase consists of 556 amino acids and has a molecular mass of 60,771 Da.
Overexpression of native human parathyroid hormone in Escherichia coli was achieved by a modification of the 5' end of the genomic gene sequence, thereby adapting this part of the translation initiation region to the bacterial host. Some simple rules abstracted from optimization studies of translation initiation of a beta-interferon gene were applied. These included (a) extending complementarity of the mRNA to the anticodon loop of tRNAfMet by use of a codon with a purine nucleotide directly following the ATG, (b) avoidance of stable secondary structure in the mRNA by use of synonymous A/U-rich codons, (c) elimination of a potential second Shine-Dalgarno sequence. The appropriate silent changes led to a 20-fold increase in parathyroid hormone production resulting in 4.3% of total soluble protein. This result proves the validity of our simple approach for optimization of foreign gene expression in E. coli.
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DNA synthesis of human hepatoblastoma HepG2 cells is reversibly inhibited by butyrate. When butyrate is removed from the culture medium, cells re-enter the cell cycle, synthesizing DNA with a time lag of about 12 h. HepG2 cells, growth-inhibited for 30 h with butyrate, synthesize and accumulate a nuclear protein, called D. Protein D synthesis is inhibited in cells which, released from the butyrate block, have resumed DNA synthesis as well as in growing cells never exposed to butyrate. Protein D has been purified from growth-arrested cells and partially sequenced. The amino acid sequences of five internal trypsin peptides indicate that protein D is a novel nuclear protein.
The three-dimensional structure of the self-complementary DNA octamer d(GCCCGpGGC) has been determined in the crystalline state using X-ray diffraction data to a nominal resolutoin of 2.12 measured from a very small crystal at DESY, Hamburg. The structure was refined with stereochemical restraints to an R value of 17.1%. d(GCCCGpGGC), containing one single 3'-methylene phosphonate linkage (denoted p), forms an A-DNA double helix with strict dyad symmetry, that is distinct from canonical A-DNA by a wide open major groove and a small average base-pair inclination against the helix axis. The conformation of the unmodified control d(GCCCGGGC) is known from an X-ray analysis of isomorphous crystals (Heinemann et al. (1987) Nucleic Acids Res. 15, 9531-9550). Comparison of the two structures reveals only minor conformational differences, most notably in the pucker of the reduced deoxyribose. It is suggested that oligonucleotides with charged 3'-methylene phosphonate groups may form stable duplexes with complementary DNA or RNA strands rendering them candidates for use as gene-regulatory antisense probes.
We have cloned and sequenced beta-COP, a peripheral 110 kd Golgi membrane protein. beta-COP shows significant homology to beta-adaptin. It is present in a membrane-bound form and in a cytosolic complex of 13-14S, with a Stokes radius of approximately 10 nm and an estimated Mr of approximately 550,000. By immunofluorescence labeling, beta-COP is associated with the structures of the Golgi complex. Immunoelectron microscopy has localized beta-COP to non-clathrin-coated vesicles and cisternae of the Golgi complex. These coated vesicles accumulate in rat liver Golgi fractions treated with GTP gamma S and strongly label for beta-COP. Our data suggest that beta-COP is a component of a coat associated with vesicles and cisternae of the Golgi complex.
The structure of the basic region (i.e., the region responsible for sequence-specific binding to DNA) of the transcriptional activator GCN4 was studied. Two peptide fragments containing either the basic region alone (residues 240-280) or the basic and the dimerization leucine zipper domains (220-280) were synthesized and investigated by nuclear magnetic resonance and circular dichroic spectroscopy. The basic region in the absence of DNA appears as a mobile flexible segment folded into a loose helix. The helical stability increases upon addition of trifluoroethanol and/or lowering of the temperature. Dimerization via the leucine zipper does not affect the three-dimensional structure of the basic region. Possible consequences for the binding to DNA are discussed.
Complexes of the type II restriction endonuclease EcoRV with a variety of short, selfcomplementary deoxyoligonucleotides have been crystallized. The best crystals diffract to about 2.7 A resolution and consist of 1:1 complexes between endonuclease dimers and duplexes of the cognate decamer GGGATATCCC containing the hexameric RV recognition sequence GATATC. Crystals with the non-cognate DNA octamer duplexes CGAGCTCG and CGAATTCG diffract to 3.0 and 3.5 A resolution, respectively, and contain two DNA duplexes per enzyme dimer.
Delineation of major T helper cell recognition sites of human immunodeficiency virus (HIV-1) proteins represents one important step in the design of an efficient acquired immune deficiency syndrome (AIDS) vaccine. Towards this end, we have explored the immunogenicity of HIV-1BRU proteins in the mouse model. Preliminary experiments revealed that inbred mice primed with whole inactivated HIV-1 developed strong CD4+ T cell proliferative responses to a variety of recombinant viral proteins including reverse transcriptase (RT). To characterize further the mouse T cell responses to this protein, several Ad- or Ed-restricted T hybridoma cells (THC) were established from BALB/c or DBA/2 mice. These THC were tested for their capacity to recognize a series of 15-mer synthetic overlapping peptides spanning three segments of HIV-1 RT that had been preselected on the basis of either alpha-helicity, amphipaticity, and/or for containing rare amino acid sequence patterns. Peptides corresponding to a C-terminal region (residues 528-560) of RT were recognized by several of the THC established from RT-primed mice. Furthermore, a non-alpha-helical peptide from this region (A3, 528-543) was capable of priming mice with different H-2 haplotypes for both peptide A3 and native RT CD4+ T cell recognition. In addition to the recently identified RT determinant 203-219 capable of triggering both mouse and human CD8+ CTL, the present results identify a good candidate for an immunodominant RT epitope capable of eliciting RT-specific T helper cell responses.
A large number of human immunodeficiency virus type 1 (HIV-1) specific HLA-restricted cytotoxic T cell (CTL) epitopes have been mapped, including an HLA-B27-restricted immunodominant epitope within p25gag. Accordingly, this segment of the HIV-1 provirus was amplified by the polymerase chain reaction from DNA derived from fresh uncultured peripheral blood mononuclear cells (PBMC) of four HLA-B27 HIV-1-infected individuals. In all cases the majority of infected PBMC bore sequences encoding the HLA-B27-restricted peptide. CTL escape mutants had not accumulated in vivo 8 and 14 months later despite demonstrable CTL activity in vitro. These data emphasize the importance of silently infected lymphocytes in evading immune surveillance.
Intracellular alphavirus nucleocapsids express a binding site for the cytoplasmic domain of the viral E2 spike glycoprotein. This binding site is recognized by the anti-idiotype monoclonal antibody, F13. The monoclonal anti-anti-idiotype antibody, raised against F13 and designated 3G10, recognizes the carboxy-terminal eight residues of the E2 cytoplasmic domain in Semliki Forest virus (SFV), identifying this as the signal for nucleocapsid interaction. F13 binding to cells infected with SFV or a second alphavirus, Sindbis virus, is inhibited by a synthetic peptide corresponding to the entire 31 residue cytoplasmic domain (E2c), and also by a synthetic peptide corresponding to the eight residue epitope recognized by 3G10. Both E2c and the eight residue peptide inhibited viral budding in microinjection experiments and when conjugated to colloidal gold are bound specifically to nucleocapsids in infected cells. These results identify a short linear signal in the E2 cytoplasmic domain required for the interaction with nucleocapsids which leads to budding of at least two alphaviruses from infected cells.
P23, a 23 kDa pathogenesis-related (PR) protein, was purified from citrus exocortis viroid (CEVd)-infected tomato leaves. Partial amino acid sequencing of this protein including the N-terminal and nine additional tryptic fragments covering about 50% of its primary structure revealed extensive homologies to the members of the family of plant thaumatin-like proteins. Sequence alignment revealed that tomato P23 is the previously described NP24 protein found to be associated to osmotic stress in tomato. In view of this fact the possible role of pathogenesis-related P23 protein as a component of a general mechanism of response of the plant is discussed.
Ten patients (44 y), 6 with the Wolff-Parkinson-White syndrome, and none with hypertensive disease, underwent electrophysiological studies before and after intravenous infusion of a single dose of 1 mg rilmenidine administered over 15 min. The regimen produced a mean plasma rilmenidine concentration of 3.16 ng.ml-1 at the end of the infusion. There was no significant change in sinus cycle length, PR interval, QRS, QT duration or in PA, AH and HV intervals. Estimated sinoatrial conduction time and corrected sinus node recovery time did not significantly change. In one patient, however, an abnormal pause was noted after termination of rapid atrial pacing. The right atrial effective refractory period decreased from 209 to 194 ms. There was no significant change in the anterograde and retrograde block cycle length or in the refractoriness of the nodal, ventricular and accessory pathways. The cycle length of induced reciprocating tachycardia decreased slightly from 374 to 351 ms. No patient exhibited an abnormal response to the carotid sinus massage. The findings indicate that intravenous administration of 1 mg rilmenidine exerts modest effects on the electrophysiological parameters of the human heart.
A captopril challenge test (CCT) for renovascular disease in adults was described recently. We used it in 20 consecutive, untreated hypertensive children and adolescents. All had a normal urinalysis and glomerular filtration rate and non-diagnostic renal sonograms or intravenous urograms. Plasma renin activity (PRA) was measured before and 1 h after administration of captopril (0.76 +/- 0.17 mg/kg). The CCT was positive in 10 patients. Renal arteriograms were performed in 7 patients with a positive CCT and in 2 with a negative CCT. Renovascular disease was found in 4 patients, 1 of whom had a negative CCT. The PRA response to captopril was the same in patients with true- and the false-positive tests. The predictive value of the positive test was 43%. In conclusion, we did not find the CCT, as described for adults, to be of value in children and adolescents.
Water samples were collected from the mouths of the three major agricultural watersheds, the Grand, the Saugeen, and the Thames (Ontario, Canada), between January 1986 and December 1990. Analyses were performed for 18 herbicides, 26 insecticides, and 4 fungicides in use in the basins. A total of between 425 and 474 samples were analyzed for each of the major groups of pesticides. Six herbicides, two insecticides and polychlorinated biphenyls (PCBs) were identified in surface water. Atrazine and its metabolite desethylatrazine were the most frequently found pesticide present in 340 of 474 samples or 72%; the metabolite was not always present with the parent compound. The second most frequently found pesticide was metolachlor which was identified in 30 of 474 samples or 6.3%. 2,4-D and cyanazine were present in 3.3% and 1.5% of the samples, respectively; alachlor, mecoprop, and simazine were present in 0.5% of the samples. Dicamba and metribuzin were present in single samples (0.2%). DDT, heptachlor epoxide, and PCB were identified in only single samples over the 5-year period. Between 342 and 2959 kg/annum of total atrazine were found passing the mouth of the three rivers and entering Lakes Erie or St. Clair between 1986 and 1990. The greatest loss was from the Thames River and the least from the Saugeen River. Between 1% and 2% of that applied in the watershed was lost at the mouth. Loadings of only two other pesticides to the rivers exceeded 5 kg in any one year, namely, metolachlor and 2,4-D. In the case of metolachlor, loadings ranged from less than 5 to 1,726 kg/annum, the highest being in the Thames and the lowest in the Saugeen River. 2,4-D exceeded a loading of 5 kg/annum in 1988 in the Grand River. Atrazine, cyanazine, and metolachlor were tracked across Lake St. Clair from the mouth of the Thames to the mouth of the Detroit River in 1987.
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