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M E Anderson

Publications and source records attributed to M E Anderson.

At least 109 records · Page 6Linked to original sources
PubMed1993

p53 domains: identification and characterization of two autonomous DNA-binding regions.

We have investigated the DNA-binding, oligomerization, and trans-activation functions of isolated segments of murine p53. We find that p53 has two autonomous DNA-binding regions. One domain, from amino acid 280 to 390, forms stable tetramers and binds DNA nonspecifically. The biological significance, if any, of this DNA-binding activity is not known. A second domain, from amino acid 80 to 290, does not form stable tetramers under stringent conditions but binds DNA both specifically and nonspecifically. The specific DNA-binding function of p53, therefore, resides in the highly conserved central region of the protein and does not require stable tetramerization. Amino acids 1-290, which include both the specific DNA-binding domain and the amino-terminal acidic region, activate a p53-specific promoter in vivo. This finding strongly argues that the DNA-binding activity of p53 segment 80-290 is physiologically significant. The role of tetramerization in p53 function remains to be determined.

Amino Acid Sequence↗
PubMed1993

Inorganic phosphate compartmentation in the normal isolated canine brain.

In vivo 31P magnetic resonance spectra of 16 isolated dog brains were studied by using a 9.4-T wide-bore superconducting magnet. The observed Pi peak had an irregular shape, which implied that it represented more than one single homogeneous pool of Pi. To evaluate our ability to discriminate between single and multiple peaks and determine peak areas, we designed studies of simulated 31Pi spectra with the signal-to-noise (S/N) ratios ranging from infinity to 4.4 with reference to the simulated Pi peak. For the analysis we used computer programs with a linear prediction algorithm (NMR-Fit) and a Marquardt-Levenberg nonlinear curve-fit algorithm (Peak-Fit). When the simulated data had very high S/N levels, both methods located the peak centers precisely; however, the Marquardt-Levenberg algorithm (M-L algorithm) was the more reliable at low S/N levels. The linear prediction method was poor at determining peak areas; at comparable S/N levels, the M-L algorithm determined all peak areas relatively accurately. Application of the M-L algorithm to the individual experimental in vivo dog brain data resolved the Pi peak into seven or more separate components. A composite spectrum obtained by averaging all spectral data from six of the brains with normal O2 utilization was fitted using the M-L algorithm. The results suggested that there were eight significant peaks with the following chemical shifts: 4.07, 4.29, 4.45, 4.62, 4.75, 4.84, 4.99, and 5.17 parts per million (ppm). Although linear prediction demonstrated the presence of only three peaks, all corresponded to values obtained using the M-L algorithm. The peak indicating a compartment at 5.17 ppm (pH 7.34) was assigned to venous pH on the basis of direct simultaneous electrode-based measurements. On the basis of earlier electrode studies of brain compartmental pH, the peaks at 4.99 ppm (pH 7.16) and 4.84 ppm (pH 7.04) were thought to represent interstitial fluid and the astrocyte cytoplasm, respectively.

Adenosine Triphosphate↗
PubMed1993

Acute effects of nitroglycerin depend on both plasma and intracellular sulfhydryl compound levels in vivo. Effect of agents with different sulfhydryl-modulating properties.

BACKGROUND: Changes in sulfhydryl (SH) compound availability may alter the hemodynamic effect of nitroglycerin (NTG). Data on the relation between NTG effect and thiol levels are, however, limited to in vitro experiments. The present study investigates how intracellular and extracellular changes in SH group concentrations (cysteine and glutathione [GSH]) affect the responsiveness to NTG in vivo. METHODS AND RESULTS: GSH and cysteine levels in plasma, vena cava, and aorta were measured after administration of N-acetylserine (placebo, n = 6), N-acetylcysteine (NAC, extracellular and intracellular SH donor, n = 6), oxothiazolidine (OXO, intracellular SH donor, n = 6), buthionine sulfoximine (BSO, intracellular GSH-depleting agent, n = 6), BSO+NAC (n = 6), and BSO+OXO (n = 6) in chronically catheterized conscious rats. In addition, the effect of 2.5 mg NTG/kg i.v. on mean arterial pressure (MAP) was determined before and after the same treatment. NAC (5 mmol/kg i.v. for 2 hours) significantly (p < 0.05) increased extracellular cysteine and GSH levels and potentiated the hypotensive effect of NTG (from 26 +/- 3 to 31 +/- 4 mm Hg [mean +/- SEM], p < 0.05). OXO (5 mmol.kg-1 x hr-1 i.v. for 2 hours) significantly increased intracellular cysteine and GSH levels but had no effect on NTG responsiveness (p > 0.05). BSO (1 g i.p. three times within 24 hours) significantly decreased intracellular GSH levels (p < 0.05) and attenuated the effect of NTG (from 28 +/- 3 to 16 +/- 2 mm Hg). CONCLUSIONS: The results suggest that the acute hypotensive effect of NTG in vivo is: 1) increased by high extracellular GSH and/or cysteine levels (NAC), 2) decreased by low intracellular GSH levels (BSO), and 3) unaffected by high intracellular levels of cysteine and GSH (OXO).

Animals↗
PubMed1993

Casualty rates among naval forces ashore.

Casualty rates for naval forces ashore across varying battle intensities are needed as input to medical and manpower planning models. Casualty data of medical and construction battalions participating in World War II amphibious assaults, Korea, and Vietnam were extracted from archival sources. Casualty rates among naval ashore forces fluctuated with battle intensity. The rate during intense combat was 15 per 1,000 per day among hospital corpsmen organic to infantry units, 7.5 for medical battalion personnel, and 2.1 among construction forces. Casualty rates dropped dramatically with decreasing battle intensity, especially among construction and medical battalions.

Humans↗
PubMed1993

p53 domains: suppression, transformation, and transactivation.

We investigated the suppression, transformation, and transactivation functions of isolated segments of wild-type murine p53. Intact p53, but no segment of p53, inhibited cellular transformation by the activated ras and adenovirus E1A proteins. We conclude that most of p53 is needed for suppression of cellular proliferation. Nevertheless, the transactivating domain of herpesvirus protein VP16 was able to substitute for the N-terminal transactivating domain of p53 in cellular suppression. Thus, unless the interchanged p53 and VP16 acidic segments share additional functions, transactivation is required for suppression by p53. Interestingly, we found that all p53 segments containing amino acids 320-360 enhanced transformation by ras and E1A. This region has been associated with the oligomerization of p53 (Milner et al., 1991; Sturzbecher et al., 1992). Furthermore, no p53 segment lacking amino acids 320-360 transformed cells. Amino acids 320-360, therefore, may account for the major transforming activity of p53. Intact p53 and chimeric VP16-p53 transactivated the CAT gene under control of a p53-specific promoter, while transforming segments of p53 interfered with transactivation by wild-type p53. Our findings argue that transactivation by p53 is required for cellular suppression and that any nontransactivating p53 that retains the capacity to oligomerize with wild-type p53 would have transformation potential.

Amino Acid Sequence↗
PubMed1992

High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis.

Exposure of human ovarian tumor cell lines to cisplatin led to development of cell lines that exhibited increasing degrees of drug resistance, which were closely correlated with increase of the levels of cellular glutathione. Cell lines were obtained that showed 30- to 1000-fold increases in resistance; these cells also had strikingly increased (13- to 50-fold) levels of glutathione as compared with the drug-sensitive cells of origin. These levels of resistance to cisplatin and the cellular glutathione levels are substantially greater than previously reported. Very high cisplatin resistance was associated with enhanced expression of mRNAs for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase; immunoblots showed increase of gamma-glutamylcysteine synthetase but not of glutathione synthetase. Glutathione S-transferase activity was unaffected, as determined with chlorodinitrobenzene as a substrate. These studies suggest the potential value of examining regulation of glutathione synthesis as an indicator of clinical prognosis. The highly resistant cell lines are proving useful for studying the multiple mechanisms by which tumor cells acquire drug- and radiation-resistance.

Cisplatin↗
PubMed1992

Characterization of tailshock elicited withdrawal reflexes in intact and spinal rats.

The tail flick withdrawal reflex (TFR) was generated by applying graded electric current to the tail of intact and spinally transected rats. In Experiment 1, separate groups of rats were tested 1, 3, 7, 10, 14, or 21 days after spinal transection. The latency, amplitude, and magnitude of the TFR was highly related to current intensity in both intact and spinal animals. However, the TFR changed dramatically as a function of the number of days between spinalization and TFR measurement. Compared to intact controls, the current intensity at which TFR was initiated (threshold) in spinal rats was elevated 1 and 3 days after transection, did not differ at 7 and 10 days, and was reduced at 14 and 21 days. Latency of TFR in spinal rats did not differ from controls 1 day after transection, but decreased steadily thereafter. Amplitude and magnitude of TFR in spinal rats remained depressed, but did show recovery toward control levels as the interval between transection and testing increased. Changes in the TFR of spinal rats was correlated with recovery of tailpinch-elicited hindlimb withdrawal. Experiment 2 demonstrated that the dose-response curve relating systemic morphine treatment to increases in TFR thresholds was shifted to the right in chronic spinal rats. Threshold increases in both spinal and intact rats were not necessarily accompanied by changes in TFR performance. These experiments establish the segmental organization of tailshock-elicited TFR and supports its use as a measure of nociceptive transmission at spinal levels.

Afferent Pathways↗
PubMed1992

Periosteal reaction induced by prostaglandins.

Prostaglandin administration is the main therapy for ductal-dependent congenital heart disease prior to definitive therapy. We report a case in which tetralogy of Fallot and Noonan syndrome were diagnosed, complicated by small pulmonary arteries, which occasioned a delay in surgery. Treatment was started with prostaglandins, which were given for a total of 2 months, and a diffuse periosteal reaction occurred secondary to the administration of intravenous prostaglandin E1.

Alprostadil↗
PubMed1992

Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen.

We investigated the molecular properties of eight temperature-sensitive mutants of simian virus 40 large T antigen (tsA mutants). The mutants have single amino acid substitutions that block DNA replication at 39 to 41 degrees C in vivo. In vitro, five of the mutant proteins were highly sensitive to a brief heat shock at 39 degrees C, while the three remaining proteins were only partially sensitive at 41 degrees C. We characterized the five most defective mutant proteins, using a variety of biochemical assays for replication functions of T antigen. Heat shock of purified T antigen with a mutation at amino acid 422 significantly impaired the oligomerization, origin-binding, origin-unwinding, ATPase, and helicase functions of T antigen. In contrast, substitution of amino acid 186, 357, 427, or 438 had more selective, temperature-sensitive effects on T-antigen functions. Our findings are consistent with the conclusion that T antigen functions via a hierarchy of interrelated domains. Only the ATPase activity remained intact in the absence of all other functions. Hexamer formation appears to be necessary for core origin-unwinding and helicase activities; the helicase function also requires ATPase activity. All five tsA mutants were impaired in functions important for the initiation of DNA replication, but three mutants retained significant elongation functions.

Adenosine Triphosphatases↗
PubMed1991

Suppression of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine.

The effects of glutathione (GSH), glutathione ester (GSE), and N-acetyl-L-cysteine (NAC) on the induction of human immunodeficiency virus (HIV) expression were investigated in the chronically infected monocytic U1 cell line, a previously described cellular model for HIV latency. U1 cells constitutively express low levels of virus, which can be increased by phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and other inducers. GSH, GSE, and NAC suppressed in a dose-dependent fashion the induction of HIV expression mediated by PMA, TNF-alpha, and IL-6, in the absence of cytotoxic or cytostatic effects. Reverse transcriptase activity, inducible by PMA, TNF-alpha, or IL-6, was decreased by 80-90% after pretreatment with GSH, GSE, or NAC. The induction of total HIV protein synthesis was also decreased appreciably after pretreatment with GSH, GSE, or NAC. The accumulation of HIV mRNA was substantially suppressed after pretreatment with NAC but to a lesser extent after pretreatment with GSH or GSE. Although PMA induces the expression of TNF-alpha in U1 cells, the suppressive effect of GSH, GSE, and NAC on PMA-induced HIV expression in U1 cells was not associated with the inhibition of TNF-alpha expression. The present findings, which elucidate relationships between cellular GSH and HIV expression, suggest that therapy with thiols may be of value in the treatment of HIV infection.

Acetylcysteine↗
PubMed1991

A quantitative analysis of pallidal discharge during targeted reaching movement in the monkey.

Neurons in the globus pallidus have been studied during reaching movements of the arm made at varying speeds. The reaching task is similar to one used in earlier experiments, in which disruption of normal pallidal output caused changes in movement time. The pallidal cells studied were those that showed task-related changes in activity and a modification of discharge when the arm was manipulated outside of the task. Neuronal discharge was assessed to evaluate two possible models, one in which the timing of task-related discharge varied as a function of movement time and the other in which the amplitude (mean firing rate) of the change in discharge varied as movement time varied. The relation between neuronal discharge and movement time was examined quantitatively on a trial-by-trial basis using a statistical algorithm that identified each phase of the change in neuronal discharge on each trial. A non-parametric statistic was used to determine the correlation between movement time and the duration or latency of changes in neural firing or the mean discharge during each phase of the cell's response. For fifty-five percent of the 40 neurons studied, the timing of the cell's response (duration or latency) varied as a function of movement time. For only 10 cells (25%) was there a significant correlation between movement time and the mean firing rate during one or more phases of the cell's response. Both timing and frequency modulation with movement time were limited to cells responsive to manipulation at the wrist or the shoulder.

Animals↗
PubMed1991

Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin.

The cofactor ATP stimulates the formation of T-antigen double hexamers on the simian virus 40 core origin of replication (I. A. Mastrangelo, P. V. C. Hough, J. S. Wall, M. Dodson, F. B. Dean, and J. Horwitz, Nature [London] 338:658-662, 1989). We report here the pathway for the assembly of hexamers and double hexamers on the core origin. ATP triggers the cooperative assembly of hexamers on the early and late halves of the origin even when they are completely isolated. Hexamer assembly nucleates at T-antigen recognition pentanucleotides in the early half of the origin. In intact origins, assembly of the first hexamer on the early half of the origin cooperatively stimulates the assembly of a second hexamer on the adjacent late half of the origin. Thus, monomer-monomer and hexamer-hexamer interactions of T antigen, allosterically activated by ATP, constitute two distinct types of cooperative interaction with the origin. Finally, we show that the assembly of T-antigen hexamers on isolated half origins leads to the same array of structural changes that T antigen induces in intact origins. We conclude that the origin is divided into complementary halves that each promote the assembly of functional T-antigen hexamers.

Adenosine Triphosphate↗
PubMed1991

The zinc finger region of simian virus 40 large T antigen is needed for hexamer assembly and origin melting.

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence C-302 L K C-305 I K K E Q P S H Y K Y H-317 E K H-320. Previous genetic analysis has shown that the cysteine and histidine sequences and the contiguous S H Y K Y region in the finger are important for DNA replication in vivo. We show here that representative mutations in either of these elements of the finger prevent the assembly of large T antigen into stable hexamers in vitro. These same mutations have a characteristic effect on the interaction of T antigen with the simian virus 40 core origin of replication. The mutant T antigens bind to the central pentanucleotide domain of the core origin but fail to melt the adjacent inverted repeat domain and to untwist the adenine-thymine domain. These defects would prevent the formation of a replication bubble and the initiation of DNA replication. Finger mutations have lesser effects on the helicase function of T antigen and no observable effect on binding of T antigen to the mouse p53 protein. We propose that the zinc finger region contributes to protein-protein interactions essential for the assembly of stable T-antigen hexamers at the origin of replication and that hexamers are needed for subsequent alterations in the structure of origin DNA. We cannot exclude the possibility that the zinc finger region also makes specific contacts with components of origin DNA.

Amino Acid Sequence↗
PubMed1991

Activity of neurons in cerebellar-receiving and pallidal-receiving areas of the thalamus of the behaving monkey.

1. Thalamic neurons that receive synaptic input from the globus pallidus or the cerebellar nuclei were identified in awake monkeys trained to perform an arm-reaching task. The location of electrophysiologically identified cerebellar-receiving (CR) and pallidal-receiving (PR) neurons was used to identify a total of 264 thalamic neurons in cerebellar (CB) or pallidal (GP) regions of the thalamus. 2. Stimulation in the brachium conjunctivum or white matter adjacent to the cerebellar nuclei excited 85 neurons in the thalamus at short latencies. These CR neurons were located in the oral portion of the ventral posterolateral nucleus (VPLo), in caudal portions of the ventral lateral nucleus (VLc), and in area X. 3. Stimulation in the internal globus pallidus (GPi) inhibited 10 thalamic neurons at short latency. These PR neurons were located in rostral portions of VLc, in the oral part of the ventral lateral nucleus (VLo), and in the parvicellular part of the ventral anterior nucleus (VApc). 4. There was no clear single somatotopic organization of neurons in CB and GP regions of the thalamus, as defined by "free-form" responses to passive manipulation and observation of eye movements. There was, in fact, a tendency for two representations, each, of the head/eye/mouth cells and cells with modifications of activity in response to manipulation of the arm. 5. During the hold period before illumination of a visual target, the mean firing rates and variability of discharge of arm-related CR and PR neurons did not differ significantly. This was also true for the total sample of arm-related neurons in the CB versus GP regions. 6. The activity of many neurons in both the CB and GP regions began to change before the reaching movement and, for some, before the earliest recorded changes in electromyographic (EMG) activity. The initial change was an increase in discharge for greater than 75% of the cells studied in both the CB and GP regions. 7. During the reaching task, there also was no significant difference in the time of the initial change in discharge of neurons in the CB versus GP regions of the thalamus. 8. These data are consistent with the hypothesis that the initial task-related change in discharge of PR thalamic neurons is dominated by input from the cerebral cortex and that pallidal input modulates later phases of their movement-related changes in activity.

Animals↗