An amplified mosquito dihydrofolate reductase gene: amplicon size and chromosomal distribution

Correct interpretation of conspicuous blood flow velocity waveforms cannot rely solely on the evaluation of uteroplacental vascular Doppler flow patterns by means of angle-independent indices such as the resistance or pulsatility index. In addition to the degree of pulsatility, the waveform shape between the systolic and diastolic peak values is of considerable consequence. A subdivision of the total flow waveform into orthogonal polynomial components allows both pulsatility evaluation and notching to be registered, providing a higher sensitivity in identification of pathological vascular resistance. Accurate recording and assessment of the flow waveform is therefore an important qualitative criterion for the classification of Doppler flow patterns in pregnancies with reduced uteroplacental perfusion.     

Regulation of cell growth-dependent expression of mammalian CDC6 gene by the cell cycle transcription factor E2F

CDC6 of Saccharomyces cerevisiae regulates the DNA replication initiation through the origin recognition complex (ORC). Identification of a human homolog of the CDC6 gene (HsCdc6) suggests a universal role of the gene product in DNA replication. Expression of HsCdc6 is growth-regulated. We investigated the molecular basis of growth-regulated expression of mammalian Cdc6. The promoter activity of isolated HsCdc6 upstream region was activated at late G1 and G1/S boundary in the cell cycle of rat embryonic fibroblast REF52 cells by the addition of serum. The isolated promoter was activated by exogenous expression of E2F without serum stimulation. However a mutant promoter lacking the E2F recognition sites failed to respond to serum stimulation and exogenous expression of E2F. Expression of endogenous Cdc6 was induced by exogenous expression of E2F. Therefore, we concluded that the growth-regulated expression of mammalian Cdc6 was mediated by E2F. Moreover, we demonstrated that exogenous overexpression of either HsCdc6 or HsOrc1 failed to induce DNA synthesis unlike overexpression of E2F1, even though E2F1 induced both Cdc6 and Orc1, suggesting that E2F may regulate the expression of another gene(s), besides Cdc6 and Orc1, required for induction of cellular DNA synthesis in mammalian cells.     

An AP-2 binding sequence within exon 1 of human and porcine choline acetyltransferase genes enhances transcription in neural cells

It has been established that GABAA and GABAB receptors can exist separately and/or co-exist in the membrane of dorsal root ganglion neurons. In our previous investigation it has been shown that co-existence of these two kinds of receptors is about 80% of the neurons examined (20/25). The present study was aimed to explore whether the activation of these two kinds of receptors could interact with each other using intracellular and whole-cell patch-clamp recordings. Baclofen, a specific GABAB receptor agonist, was found to exert negative modulatory effects on the responses mediated by GABAA receptor. In experiments with intracellular recording, GABA (0.3-1000 microM)- and muscimol (100-1000 microM)-induced depolarization was attenuated markedly and reversibly by preapplication of baclofen (100 microM) (15/21 and 17/21, respectively). In whole-cell patch-clamp recordings GABA (100 microM) and two specific GABAA receptor agonists, muscimol (10 microM) and isoguvacine (50 microM), activated currents were inhibited markedly by preapplication of baclofen 30 s or more and the inhibition was concentration dependent (1-100 microM baclofen) and reversible. The possible mechanisms underlying the inhibition by baclofen of the responses mediated by GABAA receptor and the physiological significance implicated are discussed.     

Ultraviolet resonance Raman study of drug binding in dihydrofolate reductase, gyrase, and catechol O-methyltransferase

This paper presents a study of the use of ultraviolet resonance Raman (UVRR) spectroscopic methods as a means of elucidating aspects of drug-protein interactions. Some of the RR vibrational bands of the aromatic amino acids tyrosine and tryptophan are sensitive to the microenvironment, and the use of UV excitation radiation allows selective enhancement of the spectral features of the aromatic amino acids, enabling observation specifically of their change in microenvironment upon drug binding. The three drug-protein systems investigated in this study are dihydrofolate reductase with its inhibitor trimethoprim, gyrase with novobiocin, and catechol O-methyltransferase with dinitrocatechol. It is demonstrated that UVRR spectroscopy has adequate sensitivity to be a useful means of detecting drug-protein interactions in those systems for which the electronic absorption of the aromatic amino acids changes because of hydrogen bonding and/or possible dipole-dipole and dipole-polarizability interactions with the ligand.     

Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta. The ratio of approximately 0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta'). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta, ori-beta', and ori-gamma), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.     

Enhanced binding of antibodies to the DTR motif of MUC1 tandem repeat peptide is mediated by site-specific glycosylation

The epithelial mucin MUC1 is an important tumor marker of breast cancer and other carcinomas. Its immunodominant DTR motif, which is the principal target for immunotherapeutic approaches, has been assumed until recently not to be glycosylated in both normal and tumor MUC1 and to acquire its immunogenic conformation by virtue of a certain number of tandem repeats. We present evidence that the antigenicity of the single repeat toward a considerable number of antibodies to the DTR motif is greatly enhanced if it is glycosylated within this motif, and only in this position. Twenty-eight monoclonal anti-MUC1 antibodies with DTR specificity were tested for binding to synthetic 21-mer (AHG21) or 20-mer (HGV20) tandem repeat peptides O-glycosylated with galactose beta1-3N-acetylgalactosamine alpha or N-acetylgalactosamine alpha at defined Thr or Ser positions. Binding was measured in ELISA experiments using the glycopeptides as plate-immobilized antigens or as inhibitors in solution. At least 12 antibodies revealed significantly enhanced binding to the peptides glycosylated at the DTR motif (Thr-10) as compared to positional isomers glycosylated at Thr-5, Ser-6, Ser-16, or Thr-17 and to the nonglycosylated peptides. Six antibodies (VU-3-C6, A76-A/C7, Ma552, VU-11-D1, VU-12-E1, and VU-11-E2) that were unreactive with the monomeric repeat peptide did bind to the DTR-glycosylated peptide. Several lines of evidence suggest that glycosylation with N-acetylgalactosamine is sufficient for the observed enhancement effect. Our results are of special interest in conjunction with the recent observation that the DTR motif of lactation-associated MUC1 is O-glycosylated in vivo (Müller et al., J. Biol. Chem., 272: 24780-24793, 1997). They may have consequences for the design of efficient tumor vaccines.     

p300 and ATF-2 are components of the DRF complex, which regulates retinoic acid- and E1A-mediated transcription of the c-jun gene in F9 cells

The effects of transferring clients from assertive community treatment to a less intensive (step-down) case management program were examined. Service use decreased significantly after transfer to the step-down program, and no negative effects of transfer on hospital use or client functioning were evident. Critical elements for successful step-down are suggested and discussed.