Biomedical subjects
Publications and source records attributed to D E Graves.
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OBJECTIVES: To observe the trends in vital capacity (VC) over time in tetraplegics 20 years and more after injury, the effects of age at injury, severity of injury and gender on this trend. METHODS: The medical records of all spinal cord injured persons admitted to a regional spinal injury center from January 1960 to December 1996 were reviewed. Fifty-seven patients had documented post-rehabilitation VC (mean 1.3+/-1.1 years) and VC at 10 (mean 11.8+/-2.69) and 20 (20.60+/-2.67) years post injury and beyond. RESULTS: The mean age at injury was 23.2+/-9.1 years. Severity of injury when classified according the system proposed by Coll et al were: Group 1: C1-4 Frankel A injury: 11.6%, Group 2: C5-8 Frankel A injury: 55.6%, Group 3: C2-8 Frankel B and C: 29.8% and Group 4: C2-8 Frankel D: 3.5% respectively. The mean VC at initial, 10 and 20 years post injury was 2586+/-948, 2803+/-940 and 2525+/-818 cc respectively. Multivariate analysis of variance revealed that there was significant difference in VC over a 20 year period, (F(2,54)=8.43, P<0.05). The difference between VC at 10 years and VC at 20 years accounted for the 19.8% of the variance in VC over time (F(1,55)=12.35, P<0.05). Age at injury, gender and severity of injury did not have a significant influence on the rate of decline in VC. Analysis of a subset of 26 patients who were followed up more than 20 years post injury (range 22 to 34.5 years) revealed similar, with a greater drop in the VC from 10 years post injury (F(1,23)=6.52, P<0.05). In this subset of patients, the mean VC at initial injury was 2840.9+/-847.3 cc, at 10 years was 2549.6+/-750.3 cc, at 20 years was 2400.9+/-724.1 cc and beyond 20 years was 2194.2+/-738.7 cc. There was no significant difference in mean VC between non smokers and ex/current smokers at initial, 10 and 20 years post injury, using the independent t-test (P>0.05). CONCLUSION: Vital capacity in tetraplegics declines significantly over the years, with a greater decline occurring at more than 20 years post injury.
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STUDY DESIGN: A prospective double blind cross over trial of intravenous 4-Aminopyridine (4-AP). OBJECTIVE: To determine the efficacy of this drug in the treatment of spinal cord injured (SCI) patients for neurologic impairment, pain and spasticity. SETTING: The post anesthesia care unit (PACU) of a tertiary care acute hospital. METHODS: Twelve paraplegic patients were enrolled in a double blind cross over intravenous trial of 4-Aminopyridine (4-AP). Thirty milligrams of 4-AP or placebo were administered over a 2 h period. Patients were serially examined during and after the infusion clinically for pain, sensorimotor function, hypertonicity and motor control using electromyography (EMG). Samples of blood and cerebrospinal fluid (CSF) were also analyzed at similar intervals. RESULTS: Despite penetration of 4-AP into the CSF, no significant differences were noted in the clinical and EMG parameters at the times measured. Individual changes in sensory function were reported by some patients in both the placebo and 4-AP trials, however mean values were not robust. Frequently, patients complained of unpleasant symptoms during the 4-AP infusion. CONCLUSION: The intravenous route may not be the best way to administer this drug as no short term benefits were observed.
TAS-103 is a novel anticancer drug that kills cells by increasing levels of DNA cleavage mediated by topoisomerase II. While most drugs that stimulate topoisomerase II-mediated DNA scission (i.e., topoisomerase II poisons) also inhibit the catalytic activity of the enzyme, they typically do so only at concentrations above the clinical range. TAS-103 is unusual in that it reportedly inhibits the catalytic activity of both topoisomerase I and II and does so at physiologically relevant concentrations [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. Without a topoisomerase activity to relieve accumulating torsional stress, the DNA tracking systems that promote the action of TAS-103 as a topoisomerase II poison would be undermined. Therefore, the effects of TAS-103 on the catalytic activity of topoisomerase I and II were characterized. DNA binding and unwinding assays indicate that the drug intercalates into DNA with an apparent dissociation constant of approximately 2.2 microM. Furthermore, DNA strand passage assays with mammalian topoisomerase I indicate that TAS-103 does not inhibit the catalytic activity of the type I enzyme. Rather, the previously reported inhibition of topoisomerase I-catalyzed DNA relaxation results from a drug-induced alteration in the apparent topology of the nucleic acid substrate. TAS-103 does inhibit the catalytic activity of human topoisomerase IIalpha, apparently by blocking the DNA religation reaction of the enzyme. The lack of inhibition of topoisomerase I catalytic activity by TAS-103 explains how the drug is able to function as a topoisomerase II poison in treated cells.
OBJECTIVE: To determine the incremental value of neurologic and rehabilitation process indices in predicting gain in functional abilities during rehabilitation after spinal cord injury (SCI) and to describe a model for program evaluation that provides unbiased comparisons of rehabilitation process and normative comparison of individual patient gains in functional ability. STUDY DESIGN: Multiple regression including variables in a prespecified hierarchical fashion. Linear models are formulated to gauge the incremental value of neurologic measures and rehabilitation process indices when investigating the rehabilitation process and the outcome of medical rehabilitation. RESULTS: All measures and indices vary in predictable and expected manners across individual centers and injury groups; moreover, each has demonstrated the capacity to provide unique information to the investigation of the rehabilitation process. The comprehensive set of variables accounts for 52.8% of the variance in self-care gain and 53.3% of the variance in mobility gain. The rehabilitation process indices together contribute 15% of the variance of self-care gain and over 18% of the explained variance in mobility gain. CONCLUSION: Forecasting gain in functional ability of patients in the domains of self-care and mobility may be enhanced when measures of neurologic impairment are supplemented with rehabilitation process indices. In addition, technical enhancements in measurement of rehabilitation process indices and gain in functional ability provide objective comparison of individual center differences and individual patient gains.
OBJECTIVE: To determine the life circumstances and psychosocial status of individuals with respirator-assisted and respirator-independent high tetraplegia an average of 19 years after spinal cord injury. DESIGN: Survey data were analyzed separately for ventilator-assisted and ventilator-independent groups. SETTING: Three spinal cord injury rehabilitation centers in California, Colorado, and Texas. SUBJECTS: Eighty-two individuals with CI-C4 tetraplegia between 14 and 24 years postinjury who had received acute inpatient rehabilitation. MAIN OUTCOME MEASURES: Demographics, health care utilization patterns, activities of daily living (Katz Level of Free Time Activities Scale), self esteem (Rosenberg Self Esteem Scale), quality of life, and employment. RESULTS: Self esteem and quality of life were, reported as high. Most subjects had some form of health care insurance. More than 90% lived in private homes. Approximately one third of cases had at least a college degree, yet only one quarter reported being employed. One fifth of individuals were married. Almost half of ventilator-independent cases and one quarter of ventilator-assisted cases lived in households with income of less than $20,000 per year. Mean hospital days in the past year were 11 for the ventilator-independent group and 6 for the ventilator-assisted group. The latter group required more nursing level care, significantly more hours of care, and more paid attendants over the year. Ninety-five percent of individuals reported being "glad to be alive." CONCLUSIONS: Assistance in the areas of socialization, financial status, personal assistance services, transportation, and entry into competitive employment were defined as needed. Quality of life was higher than expected, considering the substantial physical limitations of the group. The sample was almost unanimously glad to be alive, including all ventilator-assisted individuals.
BACKGROUND: Only a few antitumor drugs inhibit the DNA breakage-reunion reaction catalyzed by topoisomerase. One is the camptothecin derivative topotecan that has recently been used clinically. Others are the glycosylated antibiotic rebeccamycin and its synthetic analog NB-506, which is presently in phase I of clinical trials. Unlike the camptothecins, rebeccamycin-type compounds bind to DNA. We set out to elucidate the molecular basis of their interaction with duplex DNA, with particular emphasis on the role of the carbohydrate residue. RESULTS: We compared the DNA-binding and topoisomerase-I-inhibition activities of two isomers of rebeccamycin that contain a galactose residue attached to the indolocarbazole chromophore via an alpha (axial) or a beta (equatorial) glycosidic linkage. The modification of the stereochemistry of the chromophore-sugar linkage results in a marked change of the DNA-binding and topoisomerase-I- poisoning activities. The inverted configuration at the C-1' of the carbohydrate residue abolishes intercalative binding of the drug to DNA thereby drastically reducing the binding affinity. Consequently, the alpha isomer has lost the capacity to induce topoisomerase-I-mediated cleavage of DNA. Comparison with the aglycone allowed us to determine the energetic contribution of the sugar residue. CONCLUSIONS: The optimal interaction of rebeccamycin analogs with DNA is controlled to a large extent by the stereochemistry of the sugar residue. The results clarify the role of carbohydrates in stereospecific drug-DNA interactions and provide valuable information for the rational design of new rebeccamycin-type antitumor agents.
DACA is a DNA-intercalating agent and dual topoisomerase (topo) I/II inhibitor currently in clinical trial as an anticancer drug. Substitutions in the acridine ring of DACA have significant effects on biological activity, with 5-substituted analogues being more potent but relatively less active against cell lines that underexpress topo II, and the converse for 7-substituted analogues. A small series of 5,7-disubstituted analogues was therefore prepared and evaluated. The compounds were prepared by CDI-assisted coupling of the appropriate acridine acids. When these contained no or only one halogen atom, they could be prepared by Al/Hg amalgam reduction of the corresponding acridine acids. However, this method could not be used to prepare dihalogen-substituted acridine acids due to substantial dehalogenation, and these intermediates were synthesized via cyclization of the appropriate aldehydes to give the acridines directly. These compounds showed enhanced DNA binding compared with the parent DACA, indicating that the known favourable influence of 5-substituents on DNA binding is retained. Cell line studies showed that the 5,7-disubstituted compounds retained both the broad-spectrum effectiveness of the 7-monosubstituted analogues and the higher cytotoxic potency of the 5-monosubstituted analogues. The 7-chloro-5-methyl and 5-chloro-7-methyl analogues showed comparable in vivo antitumour activity to DACA in the subcutaneous colon 38 model, but were substantially more potent (optimal doses of 60 mg/kg compared with 200 mg/kg for DACA).
The classic DNA intercalator, ethidium, was used to probe the effects of (i) intercalation and (ii) covalent modification of the DNA on the catalytic activity of topoisomerase II. Ethidium bromide, which binds reversibly to DNA via intercalation, does not stimulate topoisomerase II-mediated DNA cleavage at concentrations up to 100 microM, indicating that the intercalative binding of this molecule to DNA is not sufficient to alter the activity of the enzyme. In contrast, covalent attachment of the photoreactive ethidium analog to DNA resulted in marked enhancement of topoisomerase II-mediated single- and double-stranded DNA cleavage. This increase in DNA cleavage was observed at very low drug binding densities (<1 drug per 10-80 base pairs) which correspond to nanomolar concentrations, as compared with other topoisomerase II poisons such as etoposide or m-AMSA which require micromolar concentrations to elicit comparable DNA cleavage levels. Over the past decade, topoisomerase II has been an important target for a variety of clinically relevant anticancer agents due to the abilities of these agents to convert this enzyme to a cellular toxin resulting in an increase in the levels of enzyme-mediated DNA breaks. Modification of DNA by covalently attaching a DNA-targeting intercalating agent (i.e., ethidium bromide) resulted in a marked shift of the cleavage/religation equilibrium of the enzyme toward the cleaved state "poison" topoisomerase II as observed by the enhancement in single- and double-stranded cleavage; thus, key insight was gained into the mechanism(s) through which DNA binding agents may influence the catalytic properties of topoisomerase II. These data demonstrate that conversion of a reversible ethidium-DNA complex to an irreversible adduct results in the transformation of an ineffective intercalating drug into a potent topoisomerase II-targeted agent. Finally, they provide support for the recently proposed "positional poisoning model" for the actions of DNA lesions and anticancer drugs on the type II enzyme.
A diterpene hydrocarbon and diterpene ketone isolated from the paracloacal gland secretions of the Chinese alligator (alligator sinensis) were examined by GC-MS and 1H-NMR, 13C-NMR, and IR spectroscopy, and identified as 4,8,12-trimethyl-1-(1-methylethenyl)-3,7,11-cyclotetradecatrien e (cembrene A, 1) and its congeneric ketone, 4,8,12-trimethyl-1-(1-methylethenyl)-3,7-cyclotetradecadien- 10-one (2), respectively. This is the first report of cembrene A from a vertebrate; the ketone has not been described previously.
The purpose of this investigation was to study the effectiveness of gabapentin in controlling spasticity in persons with spinal cord injury (SCI) using a surface EMG-based quantitative assessment technique called the brain motor control assessment (BMCA). Six men from a Veterans Affairs Medical Center with spasticity due to traumatic SCI were studied as part of a multi-center, placebo-controlled, cross-over, clinical trial of gabapentin. Spasticity was evaluated using multi-channel surface EMG recordings of muscles in the lower extremities, abdomen and low back before and during treatment with oral gabapentin or placebo. Gabapentin or placebo was given orally in doses 400 mg three times daily for 48 h. Following a 10 day wash-out period subjects were crossed-over to receive the medication not received the first time. This was followed by an elective open-label extension. Group results during the controlled trial did not reach statistical significance at the dosage used. One subject demonstrated a dramatic improvement in spasticity that was apparent both clinically and with the BMCA. Other subjects demonstrated modest improvements which were seen in the BMCA but not recognized clinically. During the open label extension, the four subjects who participated experienced important clinical improvements with higher doses (to 3600 mg/day). These improvements were often in components of spasticity in which the BMCA had detected subclinical changes during the cross-over trial. A seventh subject was studied using the BMCA at doses of 1200 mg T.I.D. gabapentin, off gabapentin and 800 mg T.I.D. gabapentin and demonstrated quantitatively a dose-related effect with higher doses of gabapentin which matched clinical observations. Gabapentin at doses of 400 mg T.I.D. may be effective in controlling some features of spasticity in persons with SCI. Higher doses provide greater control of spasticity, and controlled studies using higher doses are needed to evaluate gabapentin's efficacy.
Absorbance spectroscopy is used to examine the thermodynamic properties associated with the interaction of the experimental antitumor agents N-[2-(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide (AAC) and N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) with nucleic acids. Placement of the amino substituent at the C9 position on the acridine ring results in marked changes to the acridine chromophore's electronic properties, with the overall charge of AAC increasing to +2 in comparison to DACA's charge of +1 at neutral pH. In comparative DNA binding studies, we examine the influence that the electrostatic properties of these ligands have on the binding energies as well as their effects on enthalpy and entropy contributions. These studies show that placement of the amino moiety at C9 results in 6 times greater DNA binding affinity as compared the deamino analog (DACA). Comparisons of ionic strength dependence for these two analogs reveal a difference in the binding energies of the compounds which can be attributed to electrostatic effects. Further dissection of the enthalpy and entropy components of the binding energy reveals the enhanced electrostatic effects are related to an increased entropy contribution upon formation of the AAC-DNA complex. Groove selectivity of these acridine analogs was probed by examining the binding profiles to native and groove-modified DNAs which included glycosylated T4 DNA and the distamycin-DNA complex. These studies are indicative of minor groove interactions for both compounds with DNA.
The DNA photoaffinity ligands, 7-azidoactinomycin D and 8-azidoethidium, form DNA adducts that cause chain cleavage upon treatment with piperidine. Chemical DNA sequencing techniques were used to detect covalent binding. The relative preferences for modifications of all possible sites defined by a base pair step (e.g. GC) were determined within all quartet contexts such as (IGCJ). These preferences are described in terms of 'effective site occupations', which express the ability of a ligand to covalently modify some base in the binding site. Ideally, the effective site occupations measured for photoaffinity agents can also be related to site-specific, non-covalent association constants of the ligand. The sites most reactive with 7-azidoactinomycin D were those preferred for non-covalent binding of unsubstituted actinomycin D. GC sites were most reactive, but next-nearest neighbors exerted significant influences on reactivity. GC sites in 5'-(pyrimidine)GC(purine)-3' contexts, particularly TGCA, were most reactive, while reactivity was strongly suppressed for GC sites with a 5'-flanking G, or a 3'-flanking C. High reactivities were also observed for bases in the first (5') GG steps in TGGT, TGGG and TGGGT sequences recently shown to bind actinomycin D with high affinity. Pyrimidine-3',5'-purine steps and GG steps flanked by a T were most preferred by 8-azidoethidium, in agreement with the behavior of unsubstituted ethidium. The good correspondence between expected and observed covalent binding preferences of these two azide analogs demonstrates that photoaffinity labeling can identify highly preferred sites of non-covalent DNA binding by small molecules.
The antitumour antibiotic actinomycin D normally binds to DNA by intercalation at sequences containing the CpG step, but in the presence of daunomycin it has been reported to interact with poly(dA-dT). This observation has neither been confirmed nor explained. Here we have used a photoreactive 7-azido derivative of actinomycin to study the effect of daunomycin on its binding to three DNA fragments. Daunomycin did indeed alter the binding of actinomycin to the DNA, such that the antibiotic was displaced from its primary GpC sites onto secondary sites in the DNA, though not to AT regions especially. These findings suggest a possible scientific explanation for the increased toxicity seen during combination chemotherapy with these two drugs.
Strong binding of the antitumor antibiotic actinomycin D to the sequence 5'-TGGGT-3' in double-stranded DNA was recently established by equilibrium binding studies (Bailey et al., 1993). Actinomycin D binding to this -TGGGT- containing sequence was shown to be comparable to that of an -XGCY- containing oligonucleotide (Ka approximately 10(6) M-1). Investigation of -TGGGT- as a high-affinity binding site for actinomycin D follows from our 1989 sequencing study (Rill et al., 1989) in which the photoaffinity analog of actinomycin D (7-azidoactinomycin D) was used to determine DNA base sequence specificities and neighboring base effects. The studies presented here examine the guanine requirements for actinomycin D binding to such nonclassical (non-dGpC) sites by varying the number of central guanine residues in a series of selected duplex oligonucleotides. The central -T(G)nT- motif varies from n equals 1 to 4. Actinomycin D binding to each of these undecamers is characterized and correlated with binding to oligonucleotides of identical length and similar sequences that contain classical dGpC binding sites. Binding affinities of actinomycin D to this series of oligonucleotide duplexes (10 degrees C) can be summarized as -TGGGT- > -TGGT- > TGGGGT- > -TGT. The kinetics of SDS-induced dissociation of actinomycin D from these oligonucleotides reveal single-exponential decays with duration dependent on the sequence at the binding site. With the exception of the -TGGGT- containing oligomer, dissociation times for the T(G)nT duplexes were drastically different and much shorter than times obtained for the dissociation of actinomycin D from oligonucleotides having classical dGpC sites.(ABSTRACT TRUNCATED AT 250 WORDS)
A photoreactive analog of actinomycin (7-azidoactinomycin D) has been used in experiments to probe directly the shuffling hypothesis of Fox and Waring [Fox, K. R., & Waring, M. J. (1984) Nucleic Acids Res. 12, 9271-9285]. According to this theory, actinomycin D molecules initially interact with non-sequence-specific sites on DNA and subsequently "shuffle" along the polymer in a one-dimensional migratory fashion so as to locate their preferred sequence-dependent binding sites. In the study presented here, the drug-DNA complex was allowed to equilibrate (in the dark) for various periods of time, followed by photolysis which renders the complex irreversible and traps the ligand at its instantaneous binding sites. Visualization of the piperidine-labile sites and investigation of how the intensity of reaction at each site changes with time can provide direct confirmation of the shuffling hypothesis. The data reveal that actinomycin D does indeed engage in shuffling along the DNA. After only short equilibration times (20 s) actinomycin D is observed to bind to a variety of sites on the DNA, including many which are not regarded as canonical preferred binding sites at equilibrium. However, after longer periods of equilibration the intensity of reaction is shown to drop as a function of time at nonspecific sites, with corresponding increase at sequence-specific sites. In addition, base sequences which flank the intercalation sites can be seen to play a major role in influencing the binding and sequence specificity of actinomycin.