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R L Davis

Publications and source records attributed to R L Davis.

At least 235 records · Page 13Linked to original sources
PubMed1989

Copia RNA levels are elevated in dunce mutants and modulated by cAMP.

Clones carrying sequences expressed at altered abundance levels in dunce mutants were isolated by differentially screening a genomic library with cDNA probes representing the RNA population from dunce+ flies and the RNA population from dunce mutant flies. These mutants have an elevated cAMP content, so some isolates potentially contain cAMP responsive genes. Two classes of clones were isolated. One class contains genes expressed at a higher steady state abundance level in dunce mutants compared to dunce+ flies and the other contains genes expressed at a lower steady state level in the mutants. The recovery of clones from the differential screen demonstrates that in addition to altering normal behavior, fertility, and cAMP metabolism, dunce mutation confers an alteration in the level of expression of certain genes. The class of clones carrying sequences which are overexpressed in the mutants have been characterized. These clones carry a common repetitive sequence which codes for a 5.5 kb poly(A)+ RNA - the RNA species found to be overexpressed in the mutants. Restriction analysis and hybridization experiments show these repetitive sequences to be members of the copia family of transposable elements. Administration of pharmacological agents to normal flies to increase cAMP levels leads to an increased steady state level of copia RNA. Thus, copia RNA metabolism appears to be influenced by cAMP levels.

1-Methyl-3-isobutylxanthine↗
PubMed1989

MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer.

MyoD is a skeletal muscle-specific protein that is able to induce myogenesis in a wide variety of cell types. In this report, we show that MyoD is a DNA binding protein capable of specific interaction with two regions of the mouse muscle creatine kinase gene upstream enhancer, both of which are required for full muscle-specific enhancer activity. MyoD shares antigenicity and DNA binding specificity with MEF1, a myocyte-specific DNA binding factor. The contiguous basic and myc homology regions of MyoD that are necessary and sufficient for specific DNA interaction are the same regions of the protein required to convert 10T1/2 fibroblasts into muscle. These findings suggest that the biological activity of MyoD is mediated via its capacity for specific DNA interaction.

Animals↗
PubMed1989

5-bromo-2'-deoxyuridine blocks myogenesis by extinguishing expression of MyoD1.

The pyrimidine analog 5-bromodeoxyuridine (BUdR) competes with thymidine for incorporation into DNA. Substitution of BUdR for thymidine does not significantly affect cell viability but does block cell differentiation in many different lineages. BUdR substitution in a mouse myoblast line blocked myogenic differentiation and extinguished the expression of the myogenic determination gene MyoD1. Forced expression of MyoD1 from a transfected expression vector in a BUdR-substituted myoblast overcame the block to differentiation imposed by BUdR. Activation of BUdR-substituted muscle structural genes and apparently normal differentiation were observed in transfected myoblasts. This shows that BUdR blocks myogenesis at the level of a myogenic regulatory gene, possibly MyoD1, not by directly inhibiting the activation of muscle structural genes. It is consistent with the idea that BUdR selectively blocks a class of regulatory genes, each member of which is important for the development of a different cell lineage.

Animals↗
PubMed1989

Positive autoregulation of the myogenic determination gene MyoD1.

Transfection of cDNA expression vectors encoding either MyoD1 or myogenin into 10T1/2 cells converts them to myogenic cells. We show that transfection of 10T1/2 cells with the MyoD1 cDNA activates expression of endogenous MyoD1 mRNA, indicating that MyoD1 is subject to positive autoregulation. This activation of endogenous MyoD1 mRNA was also observed in Swiss 3T6 cells, but not in several other fibroblast or adipoblast cell lines transfected with the MyoD1 cDNA. In addition, transfection of the MyoD1 cDNA leads to activation of myogenin expression, and transfection of the myogenin cDNA leads to activation of MyoD1 expression. Thus, MyoD1 and myogenin appear to function in a positive autoregulatory loop that could either: account for or contribute to the stability of myogenic commitment; or amplify the level of expression of both MyoD1 and myogenin above a critical threshold that is required for activation of the myogenic program.

Animals↗
PubMed1989

Variability in the proliferative potential of human gliomas.

One hundred forty-three patients with gliomas of astrocytic origin (61 with glioblastomas multiforme (GM) and 82 with astrocytomas) received an intravenous infusion of bromodeoxyuridine (BUdR), 150-200 mg/m2 at the time of surgery, to label tumor cells undergoing DNA synthesis. BUdR-labeled cells were identified by the indirect immunoperoxidase method using anti-BUdR monoclonal antibodies. The percentage of BUdR-labeled cells, or BUdR labeling index (LI), was calculated by microscopic examination of selected viable areas of the tissue sections. The GMs had a median LI of 7.5%, and three quarters of these tumors had LIs greater than 5%. Highly anaplastic astrocytomas (HAAs) and moderately anaplastic astrocytomas (MoAAs) had median LIs of 2.3% and less than 1%, respectively. Among the HAAs, the LI was 1% to 5% in 56% of tumors, greater than 5% in 26%, and less than 1% in 18%. More than 60% of MoAAs had LIs less than 1%, which agrees with the slow clinical progression of this type of tumor, and the remainder had LIs of 1.4% to 9.3%. These results show that histopathologically similar tumors may have different proliferative potentials. Measuring the proliferative potential of individual gliomas is therefore crucial for predicting the prognosis more accurately and for devising more tumor-specific treatment regimens for individual patients.

Astrocytoma↗
PubMed1989

Cloning and characterization of mammalian homologs of the Drosophila dunce+ gene.

A probe representing the Drosophila dunce+ (dnc+) gene, the structural gene for a cAMP phosphodiesterase (PDEase), detects homologous sequences in many different organisms, including mouse, rat, and human. Genomic and cDNA clones representing a homolog of the Drosophila dnc+ gene were isolated from rat libraries and characterized. This gene has been named ratdnc-1. One cDNA clone defines a large open reading frame of approximately 1.8 kilobases (kb), predicting a protein sequence of 610 amino acids with significant homology to a conserved domain of approximately 275 residues found in most other PDEases. The amino acid identity value to the Drosophila cAMP PDEase within this domain is a striking 75%. Other cDNA clones show blocks of sequence divergence from this cDNA clone close to the predicted N terminus, indicating the potential existence of a family of related enzymes encoded by alternatively spliced messenger RNAs from ratdnc-1. Genomic blotting experiments suggest the existence of at least one other rat gene with homology to ratdnc-1. RNAs homologous to ratdnc-1 are heterogeneous in size between tissues, with heart containing a major transcript of 4.4 kb and brain one of 4.0 kb. The potential identity of the product of the ratdnc-1 gene with known PDEases is discussed.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD.

MyoD is a master regulatory gene for myogenesis. Under the control of a retroviral long terminal repeat, MyoD was expressed in a variety of differentiated cell types by using either a DNA transfection vector or a retrovirus. Expression of muscle-specific proteins was observed in chicken, human, and rat primary fibroblasts and in differentiated melanoma, neuroblastoma, liver, and adipocyte lines. The ability of MyoD to activate muscle genes in a variety of differentiated cell lines suggests that no additional tissue-specific factors other than MyoD are needed to activate the downstream program for terminal muscle differentiation or that, if such factors exist, they are themselves activated by MyoD expression.

Adipose Tissue↗
PubMed1989

Voltage-dependent properties of electrical synapses formed between identified leech neurones in vitro.

1. The voltage-dependent properties of rectifying and non-rectifying electrical synapses formed between identified leech neurones were quantified during their regeneration in vitro. 2. Junctional conductance increased with time in culture. This was evaluated by making comparisons between cell pairs maintained in vitro for differing amounts of time, as well as by taking repeated measurements from a single cell pair at different time intervals. 3. Non-rectifying electrical synapses were formed between certain identified neurones of the same type. Thus, Leydig cells cultured with Leydig cells established non-rectifying electrical connections, as did Retzius cells, longitudinal motoneurones (L cells) and anterior pagoda (AP) cells, each paired with its own cell type. 4. Rectifying synapses developed when sensory neurones (P cells or N cells) were paired with the other neurones mentioned above that form non-rectifying connections between themselves. The cell combinations examined were L cell-P cell. Leydig cell-N cell, and AP cell-P cell. The direction of current flow across these rectifying synapses was consistently from the sensory neurone to the other cell in the pair. 5. Non-rectifying connections early in the process of synapse regeneration (1-3 days) showed non-linearities greater than those observed in established non-rectifying synapses. There was a subtle, but clear, voltage dependence even at the later stages of synapse formation (4-18 days). 6. In contrast to non-rectifying connections, rectifying synapses formed between cells at early times in culture showed less voltage dependence than those observed at later times. 7. The marked non-linearities of the non-rectifying connections at early stages in synapse formation along with the reduced voltage dependence of the rectifying connections within the same time period revealed unexpected similarities between the two. At the early stages of synapse formation, the two types of electrical synapse were essentially indistinguishable for one direction of junctional current.

Animals↗
PubMed1989

Effect of norfloxacin on theophylline metabolism.

The purpose of this study was to investigate the effect of norfloxacin on theophylline elimination. Ten normal volunteers were studied. In a randomized crossover sequence, each subject received 6 mg of aminophylline per kg of body weight by a 30-min intravenous infusion on day 4 of taking norfloxacin (400 mg every 12 h) or while drug free. Mean theophylline clearance decreased and mean elimination half-life increased after norfloxacin administration (from 0.036 +/- 0.006 to 0.033 +/- 0.004 liter/h per kg and from 8.7 +/- 1.2 to 9.5 +/- 1.5 h, respectively; P less than 0.05, Wilcoxon signed-ranks test). We conclude that norfloxacin taken in recommended doses for 3 days has a small inhibitory effect on theophylline metabolism that would probably not cause clinically important elevations in theophylline concentrations in most patients.

Adult↗
PubMed1989

Levels of RNA from a family of putative serine protease genes are reduced in Drosophila melanogaster dunce mutants and are regulated by cyclic AMP.

We have isolated several genes expressed at abnormal levels in the memory mutant, dunce (dnc), of Drosophila melanogaster. These mutants have an elevated cyclic AMP (cAMP) content due to a mutation in the structural gene for cAMP phosphodiesterase, so the isolated genes are potentially ones regulated by cAMP. Here, we describe the characterization of a genomic clone and corresponding cDNA clones which contain sequences that are underexpressed in dnc mutants. Sequence analysis of portions of the genomic clone and representative cDNAs revealed the presence of two uninterrupted and complete open reading frames (SER1 and SER2) and part of a third (SER3). The predicted amino acid sequences of all of these were found to be homologous to the serine protease family of enzymes. The genomic clone was localized to the polytene chromosome region 99C-D, although genome-blotting experiments indicated the existence of several other genes related to the cloned serine protease-like genes. Hybridization experiments with probes representing each of the three sequenced genes showed that only the SER1-related genes were differentially expressed in dnc mutants. The putative serine protease genes were abundantly expressed in the larval gut, suggesting a major function in digestion. Feeding normal flies cAMP, isobutylmethylxanthine, or forskolin resulted in a decreased RNA level of the SER1-related genes. Thus, RNA levels of this serine protease gene family are negatively regulated by cAMP.

Amino Acid Sequence↗
PubMed1989

Correlation of histopathological features and proliferative potential of gliomas.

One hundred fifty-two intracranial gliomas of various types were reviewed in order to correlate the histopathological features with the proliferative potential of each tumor as reflected by the bromodeoxyuridine (BUdR) labeling index (LI). Patients undergoing surgical removal of gliomas were given a 30-minute intravenous infusion of BUdR (150 to 200 mg/sq m) to label S-phase tumor cells. The tumor specimens were stained immunohistochemically for BUdR and processed for routine histopathological diagnosis. The BUdR LI was calculated as the percentage of labeled cells among cells analyzed. Twenty-seven histological features in three categories (degenerative, vascular, and cellular changes) were considered. A significantly higher BUdR LI (p less than 0.05) was found in tumors with necrosis than in those without this feature; tumors with both coagulative and liquefactive necrosis had the highest BUdR LI (p less than 0.05). Increased vascularity was also associated with a higher BUdR LI (p less than 0.05). Although tumors with abnormal mitotic figures had a significantly higher BUdR LI than those without, the number of mitoses did not correlate with a higher BUdR LI. These results suggest that the number of mitoses is not a good indicator of tumor growth rate. Necrosis and increased vascularity should be heavily weighted in predicting the proliferative potential of individual gliomas.

Adult↗
PubMed1989

Prognostic implications of the bromodeoxyuridine labeling index of human gliomas.

This study includes 182 patients with intracranial gliomas who received bromodeoxyuridine (BUdR), 200 mg/sq m intravenously, at the time of craniotomy but before tumor biopsy. The tumor specimens were stained for BUdR using the immunoperoxidase method; the BUdR labeling index (LI), or S-phase fraction, was calculated as the percentage of BUdR-positive cells. The median BUdR LI's for 127 primary moderately anaplastic astrocytomas, highly anaplastic astrocytomas, and glioblastomas (less than 1%, 2.7%, and 7.3%, respectively; range 0% to 38.1%) were not significantly different from those of 55 similar recurrent tumors (less than 1%, 4.3%, and 7.4%, respectively; range 0% to 30.5%). The mean LI was significantly higher in tumors from patients over 50 years of age than in tumors from younger patients (p less than 0.001). The age-related difference in LI's was found in both groups of patients with astrocytomas but not in those with glioblastomas. Kaplan-Meier survival curves showed a significantly greater probability of survival among patients whose tumors had LI's of less than 1% than among those with LI's greater than 5%; survival probability of patients with tumor LI's of 1% to 5% was intermediate between the two extremes. Thus, the BUdR LI appears to reflect the proliferative potential more accurately than the histopathological diagnosis and should therefore be considered an important factor in determining the prognosis of individual patients with intracranial gliomas and in selecting their treatment.

Astrocytoma↗
PubMed1989

Central nervous system disorders in AIDS.

AIDS is no longer a rare disease affecting only a small segment of our population. It has now been observed throughout the United States and most other countries in the world. As the current data demonstrate, the effect of AIDS on the nervous system is profound and widespread. About 10% of all AIDS patients will first present with a neurological complaint. Evaluation of this complaint will then lead to the diagnosis of AIDS. Nearly 40% of all AIDS patients will develop major neurological symptoms during their lifetime; these symptoms may be related to primary HIV infection or secondarily to any of a number of opportunistic processes. At autopsy, 75% of AIDS patients will have neuropathological abnormalities. The AIDS-related central neurological syndromes are many and varied, as are their associated signs and symptoms. As with radiologic and serologic examination, the findings resulting from clinical examination of the AIDS patient with neurological illness are nonspecific. While there are clinical findings that are suggestive of one or another class of AIDS-related neurological illness, there is such overlap in their presentations as to make specific CNS diagnosis on the basis of clinical examination virtually impossible. The differential diagnosis of AIDS-related neurological illness is made even more difficult by the frequent observation of multiple CNS pathological processes in the same AIDS patient. Nearly one-third of all histologically examined AIDS cases had multiple intracranial pathologies. Multiple treatable pathological abnormalities have been identified both within the same intracranial lesion and within different lesions, and both simultaneously and sequentially. Thus, the evaluation and treatment of the AIDS patient with central neurological illness is a difficult challenge. Close attention must be paid to subtle neurological complaints, and careful neurological examination is warranted in all AIDS patients. Once the patient complains of neurological dysfunction or a neurological abnormality is identified on clinical examination, a careful workup including MRI or CT brain scanning and cerebrospinal fluid examination is indicated. Specific diagnosis must then be made on the basis of CSF findings, response to empiric therapy or biopsy. Therapy for AIDS-related CNS diseases in not unlike that for the same disease in other patient populations. (see Chapters 18 and 19). There is no cure for HIV encephalitis; azidothymidine (AZT) appears to cross the blood-brain barrier, and trials of AZT for the treatment of HIV encephalitis show early promise.(ABSTRACT TRUNCATED AT 400 WORDS)

AIDS Dementia Complex↗
PubMed1989

MyoD and achaete-scute: 4-5 amino acids distinguishes myogenesis from neurogenesis.

The myogenic determination gene MyoD contains a 60 amino acid domain that is necessary for both sequence-specific DNA binding and myogenic conversion of transfected C3H10T1/2 mouse embryo fibroblasts. We have generated deletion, insertion, and substitution mutants to probe the structure and function of this region, both in vitro and in vivo. Our results are consistent with a previous proposal that a helix-loop-helix (HLH) motif mediates protein dimerization. A highly basic region located immediately upstream of the HLH motif is required for DNA binding, which occurs if and only if the HLH motif is capable of dimerization. All mutants of MyoD that do not bind DNA in vitro fail to activate myogenesis or expression of a co-transfected muscle-specific reporter gene in transfected C3H10T1/2 cells. Replacing either helix 1, helix 2, or the loop sequence of MyoD with the analogous sequence of the Drosophila T4 achaete-scute protein (which is required for neurogenesis) has no substantial effect on DNA binding or biological activity. However, replacing the basic region of MyoD with the analogous sequence of the kappa immunoglobulin enhancer binding protein E12, or the T4 achaete-scute protein, produces proteins still capable of specific DNA binding in vitro, yet without significant biological activity. These findings suggest that within the 13 amino acid sequence of the MyoD basic region lies a recognition code that determines muscle-specific gene expression, although specific DNA binding per se is not sufficient to activate the muscle program.

Amino Acid Sequence↗
PubMed1988

MyoD1: a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts.

Expression of a complementary DNA (cDNA) encoding the mouse MyoD1 protein in a variety of fibroblast and adipoblast cell lines converts them to myogenic cells. Polyclonal antisera to fusion proteins containing the MyoD1 sequence show that MyoD1 is a phosphoprotein present in the nuclei of proliferating myoblasts and differentiated myotubes but not expressed in 10T1/2 fibroblasts or other nonmuscle cell types. Functional domains of the MyoD1 protein were analyzed by site-directed deletional mutagenesis of the MyoD1 cDNA. Deletion of a highly basic region (residues 102 to 135) interferes with both nuclear localization and induction of myogenesis. Deletion of a short region (residues 143 to 162) that is similar to a conserved region in the c-Myc family of proteins eliminates the ability of the MyoD1 protein to initiate myogenesis but does not alter nuclear localization. Deletions of regions spanning the remainder of MyoD1 did not affect nuclear localization and did not inhibit myogenesis. Furthermore, expression of only 68 amino acids of MyoD1, containing the basic and the Myc similarity domains, is sufficient to activate myogenesis in stably transfected 10T1/2 cells. Genetic analysis maps the MyoD1 gene to mouse chromosome 7 and human chromosome 11.

Animals↗
PubMed1988

The proliferative potential of human ependymomas measured by in situ bromodeoxyuridine labeling.

Twelve patients with ependymomas received a 30- to 60-minute intravenous infusion of bromodeoxyuridine (BrdU), 150 to 200 mg/m2 at surgery, to label tumor cells in the DNA synthesis phase. Labeled cells were detected in excised tumor specimens by indirect immunoperoxidase staining using anti-BrdU monoclonal antibody as the first antibody. The BrdU labeling index (LI, defined as the percentage of labeled cells in relation to the total number of cells scored) was calculated for each specimen. All four spinal cord ependymomas had a BrdU LI of less than 1%, which is consistent with our clinical experience that most such tumors grow slowly and have an excellent prognosis. Five of the eight intracranial ependymomas also had a low BrdU LI of approximately 1% or less, and three had a BrdU LI of 3.2%, 3.4%, and 4.8%. The latter three tumors, only one of which was diagnosed as a malignant ependymoma at the time of study, were either recurrent or recurred within 2 years after gross or subtotal removal. Cytologic analysis of cerebrospinal fluid (CSF) was performed in five cases; CSF seeding of tumor cells was found in only one patient, who had a malignant ependymoma. A high BrdU LI did not always correlate with CSF seeding. Measurement of the LI using BrdU and anti-BrdU monoclonal antibodies can provide more accurate information on the proliferative potential of individual tumors and may lead to a more rational grading system of ependymomas. The results of such studies do not always predict the potential for CSF seeding.

Adult↗
PubMed1988

Substrate sites of the (Na+ + K+)-ATPase: pertinence of the adenine and fluorescein binding sites.

The (Na+ + K+)-activated ATPase catalyzes the K+-activated hydrolysis of 3-O-methylfluorescein phosphate (3OMFP) with a Km of 50 microM, nearly two orders of magnitude lower than the Km for nitrophenyl phosphate, 3 mM. Both ATP and nitrophenyl phosphate are competitors toward 3OMFP with Ki values corresponding to their Km values (for ATP that at the low-affinity sites of the E2 conformation). Enzyme treated with fluorescein isothiocyanate (FITC) such that 60% of the (Na+ + K+)-ATPase activity is lost still hydrolyzes both 3OMFP and nitrophenyl phosphate: the apparent Km values are increased less than 2-fold and the Vmax is unaffected. ATP still inhibits these K+-phosphatase reactions of the FITC-treated enzyme, and this inhibition can exceed the 40% of residual (Na+ + K+)-ATPase activity. Evaluation of a kinetic model indicates that the Ki for ATP is increased about an order of magnitude by FITC-binding. Similar results obtain with trinitrophenyl-ATP (TNP-ATP) as inhibitor, in this case with Ki values in the micromolar range. Finally, FITC treatment increases K+-activated ADPase activity. These observations are interpreted as the fluorescein ring of 3OMFP binding to the adenine pocket of the substrate site, thereby conferring high affinity, just as the fluorescein ring of FITC binding to the adenine pocket in the E1 conformation permits specific linkage of the isothiocyanate chain to a particular lysine, Lys-501. Then, coincident with the transition to the E2 conformation, which bears the low-affinity site for ATP and which catalyzes the K+-phosphatase reaction, the FITC molecule tethered to Lys-501 is pulled from the adenine pocket: allowing 3OMFP and ADP to bind as substrates and ATP and TNP-ATP as inhibitors, albeit in altered conformation. The E1 to E2 transition thus involves not only a change from high to low affinity for ATP, but also a distortion of the adenine pocket and the orientation between Lys-501 and Asp-369, the residue associated with catalysis.

Adenine↗