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D W Hoffman

Publications and source records attributed to D W Hoffman.

At least 37 records · Page 2Linked to original sources
PubMed1994

The DNA-binding domain of the MotA transcription factor from bacteriophage T4 shows structural similarity to the TATA-binding protein.

The bacteriophage T4 middle-mode transcription factor MotA consists of two domains of approximately equal size. The C-terminal domain has been shown to contain the DNA-binding elements of the molecule, and the N-terminal domain appears to interact with RNA polymerase. A 12.5-kDa fragment of the C-terminal domain (MotCF), comprising residues 105-211 of MotA, was found to be suitable for structural studies by NMR. The 1H and 15N assignments have been made for MotCF by using two-dimensional homonuclear and heteronuclear experiments. A secondary structure has been determined which consists of a six-stranded antiparallel beta-pleated sheet with three alpha-helical segments. The secondary structure of MotCF has a clear similarity to one half of the eukaryotic TATA-binding protein (TBP), which is an intramolecular dimer. Therefore, MotCF may be related to a monomeric ancestral protein of TBP. TBP binds its target DNA in the minor groove by specific interactions with hydrophobic and aromatic residues on the exposed sheet surface of the protein. Similar residues are also present on the beta-sheet surface of MotCF, suggesting that it too binds DNA in the minor groove.

Amino Acid Sequence↗
PubMed1994

Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein.

The crystal structure of protein L9 from the Bacillus stearothermophilus ribosome has been determined at 2.8 A resolution using X-ray diffraction methods. This primary RNA-binding protein has a highly elongated and unusual structure consisting of two separated domains joined by a long exposed alpha-helix. Conserved, positively charged and aromatic amino acids on the surfaces of both domains probably represent the sites of specific interactions with 23S rRNA. Comparisons with other prokaryotic L9 sequences show that while the length of the connecting alpha-helix is invariant, the sequence within the exposed central region is not conserved. This suggests that the alpha-helix has an architectural role and serves to fix the relative separation and orientation of the N- and C-terminal domains within the ribosome. The N-terminal domain has structural homology to the smaller ribosomal proteins L7/L12 and L30, and the eukaryotic RNA recognition motif (RRM).

Amino Acid Sequence↗
PubMed1994

Solid-phase extraction and high-performance liquid chromatography for therapeutic monitoring of haloperidol levels.

This laboratory has developed a simple and efficient solid-phase extraction method that is combined with a high-performance liquid chromatographic method for rapid and precise therapeutic monitoring of haloperidol (Haldol) blood levels. The solid-phase extraction utilizes a mixed bed column. Sensitivity of the chromatographic method is 0.5 ng/ml (1.3 nM) of drug in serum, and separations can be performed in a 15-min chromatographic run. Advantages of this approach include enhanced speed, sensitivity, and efficiency. A high level of sensitivity may be achieved because of the absence of interferences from other drugs, metabolites, or serum components.

Animals↗
PubMed1993

Ribosomal protein S17: characterization of the three-dimensional structure by 1H and 15N NMR.

The structure of ribosomal protein S17 from Bacillus stearothermophilus was investigated by two-dimensional homonuclear and heteronuclear magnetic resonance spectroscopy. The 1H and 15N chemical shift assignments are largely complete, and a preliminary structural characterization is presented. The protein consists of five beta-strands that form a single antiparallel beta-sheet with Greek-key topology. The beta-strands are connected by several extended loops, and two of these contain residue types that are frequently seen in the RNA-binding sites of proteins. Additionally, two point mutations that affect antibiotic resistance, translational fidelity, and ribosome assembly are located in these two regions of the protein. Since these potential RNA-binding sites are distributed over a large surface of the protein, it appears that the molecule may interact with several regions of 16S rRNA.

Amino Acid Sequence↗
PubMed1993

The MotA protein from bacteriophage T4 contains two domains. Preliminary structural analysis by X-ray diffraction and nuclear magnetic resonance.

Controlled protease cleavage experiments and N-terminal sequence analyses were used to show that the transcriptional activator MotA from bacteriophage T4 has a two-domain structure. The N and C-terminal domains have M(r) values of 10,300 and 11,800, respectively, and were separately cloned and overexpressed in Escherichia coli. One and two-dimensional NMR spectroscopy indicate that both domains have stably folded structures and contain extensive secondary structure. The N-terminal domain is substantially alpha-helical, whereas the C-terminal domain has a high content of beta-strand. The N-terminal domain has been crystallized under three different conditions, all with the space group P3(1(2))21 and similar unit cell dimensions. The best crystals are grown from ammonium sulfate, have cell dimensions a = b = 46.7 A, c = 139.6 A, and diffract to beyond 2.4 A. The high quality of the NMR and diffraction data will allow a complete structural analysis of MotA by a combination of these techniques.

Bacteriophage T4↗
PubMed1993

Structural features of the trans-activation response RNA element of equine infectious anemia virus.

A 25-nucleotide RNA with the sequence of the trans-activation response (TAR) element of equine infectious anemia virus (EIAV) was analyzed by biochemical methods and by one- and two-dimensional NMR spectroscopy. NMR, nuclease probing, and polyacrylamide gel migration rates show that the RNA consists of an A-helical stem capped by two non-Watson-Crick U-G base pairs and a compact four-nucleotide loop. The loop is stabilized by base stacking, with loop nucleotides C12 and C15 stacked upon U11 and G16, respectively. Near the 5' end of the molecule, the stem contains a bulge at nucleotide C2, most likely a result of base pairing between G1 and C25. A method for distinguishing RNA stem-loops from palindromic dimers is described and was used to confirm that the EIAV TAR RNA has a stem-loop structure at conditions used for NMR spectroscopy. RNase A and RNase T1 cleavage patterns are consistent with the structural features derived from the NMR data.

Base Composition↗
PubMed1993

Structural features of an RNA containing the CUGGGA loop of the human immunodeficiency virus type 1 trans-activation response element.

A 19-nucleotide RNA containing the CUGGGA loop sequence corresponding to nucleotides 30-35 of the HIV-1 trans-activation response element (TAR) was synthesized in vitro and analyzed by biochemical methods and one- and two-dimensional NMR spectroscopy. Diagnostic RNase cleavage patterns were similar for the loops in the full-length HIV-1 TAR and the 19-nucleotide RNA, indicating that they are similar in structure. NMR data showed that the loop is stabilized by base-stacking interactions. The first loop nucleotide is stacked upon the A-helical stem, and the loop uridine is stacked upon this cytosine. On the opposite side of the loop, the third loop guanosine is stacked upon the adenosine, which is stacked upon the stem. No specific Watson-Crick or non-Watson-Crick base pairing across the loop was identified. Unusually short interribose distances indicate a significant distortion of the sugar-phosphate backbone centered at the adenosine. Relatively short NMR relaxation times for protons of the adenosine and its adjacent guanosine, as well as rapidly exchanging imino protons, provide evidence for dynamic processes occurring in the loop.

Base Sequence↗
PubMed1993

Localization of preproenkephalin mRNA-expressing cells in rat auditory brainstem with in situ hybridization.

Hair cells and auditory nerve dendrites in the inner ear are innervated by pontine neurons that have been demonstrated by immunochemical techniques to contain several neurotransmitters, including acetylcholine and the opioid peptide enkephalins and dynorphins. The functions of these nerve fibers are not known, but may involve modifying auditory sensitivity to low intensity stimuli. In the guinea pig the opioid pathways originate in the lateral superior olivary region. A recent study in the gerbil has reported cells expressing preproenkephalin mRNA present only in the ventral nucleus of the trapezoid body, and not in the superior olivary region. In the present study, a non-radioisotopically labeled in situ hybridization method was used to identify cells expressing mRNA coding for preproenkephalin in rat pontine neurons, specifically in the ventral nucleus of the trapezoid body. These cells may represent an enkephalin-containing medial olivocochlear system in the rat, the origin of the lateral system in the rat that differs markedly from the better-studied guinea pig and cat, or a non-olivocochlear enkephalin-containing system.

Animals↗
PubMed1992

Crystallization and preliminary structural analysis of the replication terminator protein of Bacillus subtilis.

The replication terminator protein (RTP) is a dimeric molecule that binds specific sequences within the replication terminus of the Bacillus subtilis chromosome and prevents the passage of replication forks. The gene for RTP has been expressed in Escherichia coli, and the protein has been purified in amounts sufficient for structural studies by nuclear magnetic resonance (NMR) and x-ray crystallography. One-dimensional NMR experiments show that the protein has a well-folded compact tertiary structure, as well as a high alpha-helical content. Circular dichroism (CD) studies confirm this finding and show that approximately 32% of the protein is alpha-helical. The terminator protein has been crystallized as monoclinic plates that diffract to better than 2.5 A and are suitable for high resolution structural analysis. Precession photographs show the space group to be C2 with unit cell dimensions a = 77 A, b = 53 A, c = 70 A, and beta = 90 degrees, and two molecules occupy the asymmetric unit. With a view to producing crystals of an RTP.DNA complex, gel-shift assays were performed to establish the shortest sequence of DNA that is required for tight binding to RTP. These clearly show that two turns of DNA are required, centered on an 8-base pair consensus sequence, to elicit relatively stable binding.

Autoradiography↗
PubMed1992

Comparison of cyclosporine and FK506 effects on glutathione levels in rat cochlea, brain, liver and kidney.

Cyclosporine treatment (50 mg/kg/day, p.o.) caused increases in rat renal reduced glutathione (GSH) levels of 205 and 673%, respectively, after 5 and 10 days. No changes were seen in liver GSH with either dose of cyclosporine. FK506 (2.5 mg/kg/day, p.o., for 7 days) caused an approximately 200% increase in kidney GSH, and an approximately 250% increase in hepatic GSH levels. Oxidized glutathione (GSSG) was never more than 1-2% of the level of the reduced form in any tissue from control animals. Small increases in the ratios of oxidized to reduced glutathione were seen in livers and kidneys from both cyclosporine- and FK506-treated animals. No changes in GSH or GSSG levels were seen in brains or cochleas from any animal.

Animals↗
PubMed1991

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins.

An RNA recognition motif (RRM) of approximately 80 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing. The U1 RNA-binding domain of the A protein component of the human U1 small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy. The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel beta-strands and two alpha-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent beta-strands. Conserved aromatic side chains that are critical for RNA binding are clustered on the surface of the molecule adjacent to a variable loop that influences recognition of specific RNA sequences. The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins.

Amino Acid Sequence↗
PubMed1991

Effects of opioid be drugs on auditory evoked potentials suggest a role of lateral olivocochlear dynorphins in auditory function.

Multiple gene products of opioid peptide families (e.g., enkephalins, dynorphins) with differing opioid receptor specificities are present within olivocochlear efferent terminals. Enkephalins activate mu- and delta-opioid receptors, and are generally inhibitory in the nervous system, and dynorphins are kappa-receptor agonists, which may be excitatory to postsynaptic neurons. We have examined the effects of intravenously administered opioid agonists and antagonists on click-evoked N1 and N2 amplitudes and latencies of the compound action potential in the chinchilla recorded at the round window. Parenteral administration of the opioid receptor antagonist naloxone or the potent mu-receptor agonist fentanyl did not alter N1 and N2 amplitudes or latencies. The kappa-receptor agonist, mu-receptor antagonist pentazocine caused marked increases in N1 and N2 amplitudes over baseline values at threshold intensities. These effects were not abolished by naloxone. No effects were seen on the cochlear microphonic, supporting a site of action of these effects at the lateral olivocochlear efferent terminals on auditory nerve dendrites under inner hair cells. Similar results were obtained when far field auditory evoked responses were recorded. Results were obtained under ketamine/pentobarbital anesthesia, which provided stable recording baselines in contrast to tiletamine/zolezepam/pentobarbital, with which an upward drift in auditory potentials was observed. This stimulatory action of kappa-agonists on auditory-evoked potential amplitudes appears to represent a physiological role of the lateral olivocochlear efferent innervation. The different neurotransmitters of the olivocochlear efferents (e.g. enkephalins, dynorphins, acetylcholine) may have antagonistic actions on auditory potentials, as may the lateral and medial systems themselves.

Animals↗
PubMed1990

Use of gel filtration in the preparation of biological fluids for magnetic resonance spectroscopy.

Analysis of biological fluids by proton magnetic resonance spectroscopy is often complicated by dynamic range problems created from the large water resonance. Gel filtration chromatography is found to be a simple and nondestructive method for exchanging D2O for H2O and for removing low molecular weight molecules from both plasma and urine, significantly improving subsequent one- and two-dimensional MRS spectra.

Body Water↗
PubMed1990

An introduction to the functional neurochemistry of the auditory system.

Neurochemistry of the auditory system has become one of the fastest growing areas in the study of audition. Many recent advances have implications for not only the basic auditory researcher but also the clinician. This article introduces some of the fundamentals of auditory neurochemistry as well as some recent developments. Also highlighted are some of the studies that appear to have current and future clinical significance.

Animals↗
PubMed1989

Measurement of blood levels of neuroleptics and metabolites by combined high performance liquid chromatography-radioreceptor assay for D2 and sigma sites.

The dopamine (D2) receptor blocking property of antipsychotic medications has been proposed as the mechanism of the therapeutic activity of this class of drugs. This property has also been exploited as a method to quantify therapeutic levels of these drugs in patients. However, the lack of correlation among dosage, blood levels and clinical response has resulted in a contradictory literature on both mechanism and quantification of these drugs. Bioactivity and chemical identity of the commonly prescribed neuroleptic drug fluphenazine and its metabolites in human plasma were determined by a new method which combines the selectivity of chemical methods with the sensitivity and bioassay of the radioreceptor assay (RRA) method. Fluphenazine and its metabolites were separated and identified in human plasma by an ion-pairing reverse phase high performance liquid chromatographic method with electrochemical detection. A volatile buffer system was employed which was compatible with facile sample preparation for post-column analyses, and which provided sharp, symmetrical chromatographic peaks of parent compound and metabolites. Post chromatography, HPLC fractions were assayed by RRA for D2, alpha 1 and sigma receptors. More than one pattern of metabolism of the drug was seen, including biosynthesis of drug metabolites with biological activities at these receptor types. The individual differences with which this occurs may contribute to the variabilities seen in clinical response to neuroleptics, and to difficulties in neuroleptic blood level determinations.

Animals↗
PubMed1988

Putative neurotransmitters in the rat cochlea at several ages.

We have performed a longitudinal study of the content of the putative neurotransmitter substances, enkephalin, dynorphin, and acetylcholine (ACh), in the cochlea of the Fischer 344 rat. It is the first study of transmitters in the rat cochlea over an extended time span. This study also provides biochemical verification of the presence of ACh in cochlear tissues. No change was seen in the cochlear content of these transmitter candidates up to 24 months of age.

Acetylcholine↗