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Biomedical subjects

R A Kramer

Publications and source records attributed to R A Kramer.

At least 19 recordsLinked to original sources
PubMed1992

Distribution of etanidazole into human brain tumors: implications for treating high grade gliomas.

Etanidazole was developed as an oxygen-mimetic radiosensitizer less lipophilic than misonidazole. Sensitization depends on an adequate concentration of drug in the tumor at the time of irradiation. Therefore, due to the presence of the blood-brain barrier, brain tumors may theoretically be difficult to radiosensitize due to the hydrophilic characteristics of etanidazole. Based on previous reports of loss of BBB integrity in brain tumors, we investigated the ability of etanidazole to penetrate into malignant gliomas of patients receiving etanidazole as part of a Phase I continuous infusion protocol. The patients had completed previous external beam irradiation. Twenty-two patients were studied and their etanidazole plasma and biopsy data were compared to the 2-compartment model derived from a second group of 19 patients with bolus etanidazole. Etanidazole concentration in brain tumor biopsies varied widely and appeared to be clustered into a higher and a lower pharmacokinetic group having mean tumor to well-perfused second compartment ratios of 1 and 0.25, respectively. Both high and low etanidazole concentrations were evident in different biopsies obtained from the same patient. Correlations between histology and tissue concentrations suggested that the higher level correspond to malignant tissue. These data indicate that the blood brain barrier is disrupted to varying degrees by the brain tumor and/or prior irradiation and that etanidazole penetrates into brain tumors.

Brain Neoplasms↗
PubMed1992

Final report of the phase I trial of continuous infusion etanidazole (SR 2508): a Radiation Therapy Oncology Group study.

Seventy-eight patients have been treated on a Phase I trial using continuous infusion etanidazole while undergoing brachytherapy for locally advanced tumors. There were two sequential schemata, the first treated 63 patients with doses ranging from 8-23 g/m2 over 48 hr and the second treated 15 patients with doses ranging from 20-23 g/m2 over 96 hr. The tumor sites were: brain (n = 42), cervix (n = 22), and breast (n = 14). Patients received a loading dose of etanidazole of 2 g/m2 followed by a continuous infusion for a total of 48 or 96 hr while radioactive implants were in place. Of the 63 patients in the 48-hr study, 52 were entered at doses of less than or equal to 21 g/m2 and there were no definite neuropathies but two patients with the cramping/arthralgia syndrome. Of the 11 patients entered at 22-23 g/m2, 1 patient had symptoms of peripheral neuropathy (Grade II) and 6 had the cramping/arthralgia syndrome. This is a new syndrome, distinct from the peripheral neuropathy, characterized by transient alterations in sensations consisting of cramping, arthralgias, or tingling that resolved completely at intervals varying from a few hours to about 1 week post-treatment. The cramping/arthralgia syndrome limited dose escalation; therefore, the maximum tolerated dose over 48 hr was determined to be 20-21 g/m2. The 96-hr infusion was limited to patients with recurrent gliomas undergoing stereotactic implantation. To date, 15 patients have been treated with doses of 20-23 g/m2. No toxicity was encountered at doses less than or equal to 22 g/m2. At 23 g/m2, one patient developed Grade III neuropathy and three patients had mild cramping/arthralgia syndrome, for whom the drug was discontinued. Therefore, it appears the maximum tolerated dose at 96 hr will be approximately 23 g/m2, which is 10-15% higher than for the 48-hr infusion.

Brachytherapy↗
PubMed1992

Age of onset in childhood asthma: data from a national cohort.

There is controversy over the role of age of asthma onset in childhood asthma. Data collected on self-reported physician-diagnosed asthmatic children and young adults aged 6-24 years (N = 352), who participated in the second National Health and Nutritional Examination, 1976-80 (NHANES II), a national sample, were examined to see whether reported age at onset was associated with the future course of the asthma. Three definitions were used for early-onset asthma: asthma beginning before the second birthday, before the third birthday, and before the fourth birthday. Late-onset asthma was defined as asthma beginning on or after the second birthday, the third birthday, and the fourth birthday, respectively. Among 6-14 year olds, late-onset asthmatic subjects as compared with early-onset asthmatic subjects using the three definitions reported more allergic rhinitis OR = 3.79 (95% CI 1.53, 9.41), 3.06 (1.33, 7.07), 2.71 (1.18, 6.22), and were more likely to have at least one positive allergen skin test OR = 2.21 (95% CI 1.02, 4.79), 2.90 (1.29, 6.49), 3.41 (1.50, 7.75). Late-onset asthmatic subjects tended to report that their asthma was active, have more problems during the past 12 months with wheezing, and have lower values for predicted FVC and FEV1. No difference was found in reported chronic rhinitis, sinusitis, other allergies, problems within the last 12 months with cough attacks, or during the past 3 years a period of cough and phlegm lasting more than 3 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

Expression of growth hormone-releasing factor analog Leu27GRF(1-44)OH in Escherichia coli: purification and characterization of the expressed protein.

A recombinant plasmid has been constructed to direct the synthesis of Leu27GRF(1-44)OH in Escherichia coli as a fusion protein containing a hexa-His tail followed by amino acids 1-99 of interferon-gamma and a methionine residue at the N-terminal. The expression of the 18-kDa fusion protein (H6GAMGRF) was induced by isopropylthiogalactoside treatment and the protein accumulated as insoluble aggregates in inclusion bodies. The protein aggregates were solubilized in 6 M guanidine-HCl and purified directly by affinity chromatography on a Nichelate column. The growth hormone-releasing factor (GRF) moiety was released from the fusion protein by cyanogen bromide cleavage and purified to homogeneity by anion-exchange chromatography followed by reverse-phase chromatography. The identity of the GRF peak was determined by comparing its retention time with that of synthetic Leu27GRF(1-44)OH. The purified material was further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and amino-acid analysis. The recombinant-derived product and the synthetic compound showed identical reactivities toward anti-GRF polyclonal antibodies and were essentially equipotent as determined by an in vitro biological assay for growth hormone-releasing activity.

Amino Acid Sequence↗
PubMed1990

Expression of the HTLV-I tax transactivator in yeast: correlation between phenotypic alterations and tax function in higher eukaryotes.

The transcriptional activator protein (Tax) from human T-cell leukemia virus type 1 was expressed in yeast using several different promoters in several strains: In all instances, expression of Tax resulted in very strong aggregation of the yeast cells. This phenotype appears to be identical by all criteria tested to the flocculation phenotype of the dominant mutation flo 1. Of most significance, mutations in Tax that affect transactivation of the IL-2R alpha regulatory sequences, but retain their ability to activate the viral long terminal repeat also fail to yield the aggregation phenotype. Based on these findings, expression of Tax in yeast may prove to be a simple primordial system for examining the regulatory mechanisms and cellular functions involved in regulation of gene expression.

Gene Products, tax↗
PubMed1990

Access to care for children and adolescents with disabilities.

A survey of Connecticut pediatricians indicated that 90% of the respondents intended to provide care to children with disabilities. However, only 72% provided wheelchair access and only 68% accepted Medicaid reimbursement. Details of barrier-free design are presented and systems of support for the special needs of disabled patients are described.

Adolescent↗
PubMed1989

Cytochrome P-450-dependent formation of alkylating metabolites of the 2-chloroethylnitrosoureas MeCCNU and CCNU.

Rat liver microsomes catalyzed the biotransformation of the clinically important nitrosourea anticancer agents 1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea (MeCCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) to alkylating metabolites that bound covalently to microsomal protein and to DNA. The enzyme-mediated microsomal alkylation required NADPH and oxygen and was inhibited by carbon monoxide, indicating the participation of a cytochrome P-450-dependent monooxygenase. Additional studies with inhibitors such as piperonyl butoxide and with the inducers 3-methylcholanthrene and phenobarbital were consistent with this view. In contrast to these observations on the formation of alkylating metabolites, carbamylation reactions were not affected significantly by microsomal metabolism. Reduced glutathione, cysteine or N-acetylcysteine decreased the microsomal alkylation by MeCCNU and produced a corresponding increase in the formation of polar metabolites that was resolved by HPLC as three distinct N-acetylcysteine-MeCCNU adducts. The addition of semicarbazide to the reaction decreased microsomal alkylation by 30%, indicating that the formation of the alkylating species may proceed via an aldehyde intermediate. Renal microsomes were not found to catalyze the alkylation reaction. Moreover, MeCCNU inhibited the renal slice accumulation of p-aminohippuric acid only in the presence of liver microsomes and NADPH, suggesting that a liver metabolite may be responsible for the renal toxicity of the parent nitrosourea.

Acetylcysteine↗
PubMed1989

Misplacement of the amino-terminal positive charge in the prepro-alpha-factor signal peptide disrupts membrane translocation in vivo.

We have identified a series of mutations in the signal peptide of yeast prepro-alpha-factor which specifically attenuate translocation across the endoplasmic reticulum membrane in vivo. In prepro-alpha-factor-somatostatin hybrids, transposition of the amino-terminal tripeptide from wild-type NH2-Met-Arg-Phe to NH2-Met-Phe-Lys or NH2-Met-Phe-Arg causes a 45-75% reduction in the efficiency of membrane translocation. This is evidenced by the intracellular accumulation of unglycosylated, signal-containing precursors which are membrane-associated and are exposed to the cytosol. Surprisingly, abolition of the single positive charge by replacing arginine with phenylalanine has little effect on translocation into the endoplasmic reticulum. We conclude that the presence of an amino-terminal positive charge is not necessary for efficient targeting or translocation; however, misplacement by one position markedly disrupts translocation without affecting targeting. These mutations thus define an early stage of membrane interaction that is sensitive to local charge effects. Furthermore, our data suggest that post-translational translocation, signal cleavage, and core glycosylation of these polypeptides may occur to a significant extent in vivo.

Cell Membrane↗
PubMed1989

Protection against cisplatin nephrotoxicity by prochlorperazine.

Prochlorperazine (Compazine; PCPZ) is often used to limit cisplatin (CDDP)-induced emesis. However, recent studies in mice have shown that PCPZ protects against renal injury produced by treatment with various nephrotoxicants (e.g., MethylCCNU, mercuric chloride). Because renal toxicity remains a serious limitation to the effective use of CDDP, we conducted the present study to determine whether PCPZ could also protect against CDDP-induced renal injury. PCPZ treatment was shown to ameliorate CDDP-induced renal lesions in both rats and mice at doses and treatment schedules that were comparable with those used for alleviating chemotherapy-induced emesis. A PCPZ dose of 10 mg/kg x 2 offered complete protection against CDDP-induced increases in blood urea nitrogen (BUN) levels in mice, with significant protection occurring at a PCPZ dose as low as 5 mg/kg. Similarly, PCPZ ameliorated CDDP-induced increases in BUN, glucosouria, and enzymuria in F344 rats. PCPZ treatment did not affect the urinary excretion or renal tissue levels of total platinum or the plasma pharmacokinetics of free platinum. However, it did cause a marked reduction in the concentration of total plasma platinum (free platinum + protein-bound platinum). PCPZ was not found to affect the in vivo antitumor activity of CDDP against P388 leukemia. The present study suggests that PCPZ may be of therapeutic benefit when used with CDDP and provides a rational basis for the selection of antiemetic therapy.

Animals↗
PubMed1989

Nephrotoxicity of 5-(N-phenylcarboxamido)-2-thiobarbituric acid in the Fischer 344 rat.

In the present investigation, administration of a single i.p. dose of the anticancer drug merbarone [5-(N-phenylcarboxamido)-2-thiobarbituric acid] produced an acute and reversible decrease in renal function in female but not male Fischer 344 rats. The renal lesion in female rats was biochemically characterized as a decrease in p-aminohippuric acid accumulation by renal slices along with polyuria, glucosuria, proteinuria, and enzymuria. These functional changes were accompanied by histopathologic changes of focal tubular necrosis that was confined to the deep cortex and outer stripe of the outer medulla. The changes in these parameters were dose-dependent and were observed at doses as low as 0.2 x MELD(10) (12 mg/kg). This low merbarone dose increased urinary glucose and protein excretion by 26- and 9-fold, respectively, in the initial 16-h urine collection in female rats. This increase was accompanied by a 2- to 15-fold increase in the excretion of N-acetyl-beta-D-glucosaminidase (NAG), gamma-glutamyl transpeptidase (gamma-GTP), and lactate dehydrogenase (LDH) activities. No significant changes in renal function were observed in male rats apart from mild enzymuria after a high dose of merbarone (36 mg/kg). The drug did not increase urea nitrogen levels in male or female rats, reflecting the focal nature of this tubular lesion. Merbarone produced small elevations in serum transaminase activities [i.e., glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT)] at doses that produced marked alterations in renal function in female rats, suggesting only mild hepatotoxicity. The present study establishes the kidney as a possible dose-limiting target organ for merbarone toxicity.

Alanine Transaminase↗
PubMed1989

The effect of glutathione (GSH) depletion in vivo by buthionine sulfoximine (BSO) on the radiosensitization of SR 2508.

The effect of glutathione depletion (GSH) on the efficacy of SR 2508 was evaluated in two murine tumor models with single large doses of radiation or with low doses administered in an accelerated fractionated schedule. To deplete tumor GSH, buthionine sulfoximine (BSO) was administered in the animals drinking water (10 mM) following two i.p. injections of 450 mg/kg. This treatment decreased RIF and MCA tumor GSH concentrations by 95% and 80%, respectively. Mice (C3H/Sed) received BSO for 48-72 hr before the first dose of radiation, and were maintained on BSO drinking water for the duration of the fractionated course of therapy. SR 2508 (200-1000 mg/kg) was injected 45 min prior to each fraction of radiation. Radiation was administered as a single dose of 15 Gy or 20 Gy, for RIF and MCA tumors, respectively. Alternatively, animals received a fractionated course of radiotherapy which consisted of 2.5 Gy/fraction for the RIF, and 3 Gy/fraction for the MCA tumors, b.i.d. for five days (total of 10 fractions). Tumor response with and without BSO, and with and without SR 2508, was determined by regrowth delay. BSO pretreatment increased the efficacy of SR 2508 with single dose radiation in the MCA but not RIF tumor. SR 2508 administered with fractionated radiation produced lower enhancement ratios (SER) than with a single radiation dose. However, BSO significantly enhanced the efficacy of SR 2508 with fractionated radiation. BSO increased the maximum SER for SR 2508 (3 mM/fraction) from 1.2 to 1.4 in the RIF tumor, and from 1.4 to 1.8 in the MCA tumor. BSO also increased the toxicity of SR 2508 by a factor of 2. However, the ability of BSO to increase the efficacy of low doses of sensitizer at clinically relevant doses of radiation suggests that this combined modifier treatment may be of clinical benefit.

Animals↗
PubMed1989

Initial results of a phase I trial of continuous infusion SR 2508 (etanidazole): a radiation therapy oncology group study.

To exploit both the oxygen-mimetic and "pre-incubation" or continuous exposure effects of the 2-nitroimidazole radiosensitizers, we are conducting a Phase I trial of continuous infusion SR 2508 for patients receiving brachytherapy. Following the administration of a loading dose of 2 g/m2, SR 2508 is administered by continuous infusion for 48 hr. Twenty-one patients have completed treatment. The initial total dose was 8 g/m2 with patients currently receiving 15 g/m2. No toxicity has been observed. At the higher doses the steady-state plasma concentrations have been between 50 and 70 micrograms/ml. It is not yet known whether or not hypoxic sensitizers will be of benefit clinically, and if so, when during a course of treatment is the optimal time to use them. Given the lack of toxicity and plasma concentrations achievable with continuous infusion, future studies will be conducted using SR 2508 during both the external beam and brachytherapy aspects of treatment.

Brachytherapy↗
PubMed1988

Role of the glutathione redox cycle in acquired and de novo multidrug resistance.

Drug resistance represents a major obstacle to successful cancer chemotherapy. However, the specific biochemical mechanisms responsible for clinical drug resistance are unknown. In these studies resistance to the antitumor agent adriamycin was found to involve two mechanisms, one that decreased drug accumulation by the P170 mechanism and another that altered the glutathione redox cycle, an important pathway in the detoxification of reactive oxygen. This dual mechanism of drug resistance was demonstrated in cell lines that had acquired the multidrug-resistant phenotype and in human colorectal cancer cells with de novo resistance. These studies support a model of acquired and de novo multidrug resistance that includes alterations in both drug accumulation and the glutathione redox cycle.

Animals↗
PubMed1988

An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.

In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation.

Cloning, Molecular↗
PubMed1988

Chemical modifiers of cancer treatment.

Chemical modification is a concept in cancer therapy in which the state of tumor cells or normal tissues is modified such that a therapeutic gain can be achieved using conventional therapeutic modalities. Hypoxic zones targeted as cells within them may be radiation resistant, poorly perfused by chemotherapeutic agents, and possibly drug resistant due to hypoxia-related gene amplification. Nitroimidazoles have gained particular attention as chemical modifiers because they can increase the radiation sensitivity of hypoxic cells, are cytotoxic to hypoxic cells, can increase sensitivity to chemotherapeutic agents, and are useful for imaging hypoxic cells. While both radiosensitization and chemosensitization require hypoxia, the mechanism of the enhancement of each of the modalities is different. The 2-nitroimidazole hypoxic sensitizers SR 2508 and Ro-03-8799, which are less toxic than the prototype misonidazole (Miso), are in clinical trials, and dual function molecules that include a hypoxic sensitizer and alkylating function are being developed. The presence of both acutely and chronically hypoxic cells in animal tumors has been demonstrated by new imaging techniques. Oxygen delivery to tumors is being altered by the use of perfluorocarbons, and agents that alter hemoglobin affinity for oxygen. Compounds that are selectively toxic to hypoxic cells are being developed. Nonhypoxic modifiers are also being investigated. Thiol modification, particularly the alteration of glutathione concentration, has complex effects on the cell's biochemistry, in addition to affecting the competition between oxygen and thiol groups for the restoration and fixation of radiation-induced radicals. WR-2721 is being studied as a means of reducing the normal tissue toxicity of radiation and chemotherapy. Increased thiol concentration may be a mechanism of cross-resistance between certain chemotherapeutic agents and radiation.

Antineoplastic Agents↗
PubMed1987

Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae.

We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

Acid Phosphatase↗