Biomedical subjects
L Levin
Publications and source records attributed to L Levin.
Two phase I studies of carboplatin dose escalation in chemotherapy-naive ovarian cancer patients supported with granulocyte-macrophage colony-stimulating factor.
Recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) may reduce myelosuppression and, thus, allow dose escalation of certain chemotherapeutic agents. We conducted two sequential phase I trials of escalating doses of carboplatin and a fixed dose and schedule of rHuGM-CSF in ovarian cancer patients who had not previously had chemotherapy, i.e., chemotherapy-naive patients. In the first trial, patients were assigned to regimens of increasing dose levels of carboplatin (starting at 400 mg/m2) and fixed doses and schedules of cyclophosphamide (600 mg/m2) and rHuGM-CSF (10 micrograms/kg given subcutaneously once daily on days 2-11). Chemotherapy was given every 3 weeks (regimen A). In the subsequent trial, the design was the same except that cyclophosphamide was omitted (regimen B). Fifteen patients received regimen A, and seven patients received regimen B. In regimen A, all three patients treated at the first dose level tolerated five cycles at full doses. Hematologic toxicity was dose limiting at the 600-mg/m2 dose level. When 500 mg/m2 carboplatin was given, six of eight patients tolerated three or four cycles at full doses before requiring dose reductions or treatment delays. In regimen B, doses could not be escalated above the first dose level (600 mg/m2) because of severe hematological toxicity. Nonhematological toxicity was tolerable and managed with acetaminophen, antihistamines, and/or nonsteroidal, anti-inflammatory medication. Compliance was excellent. We conclude that (a) rHuGM-CSF can be given safely and reliably to chemotherapy-naive ovarian cancer patients receiving these treatment regimens, (b) early and severe thrombocytopenia was a major problem with or without cyclophosphamide with doses of carboplatin at or above 600 mg/m2, and (c) 500 mg/m2 carboplatin administered every 3 weeks is the highest dose in regimen A that can be given safely in the outpatient setting.
Functional expression of mammalian adenosine cyclic monophosphate-dependent protein kinase in Saccharomyces cerevisiae.
The heterologous expression of protein kinases in E. coli has proved difficult and unpredictable. Although the v-abl protein kinase is successfully expressed in E. coli, our experiments on expression of yeast C subunits in E. coli produced large amounts of predominantly insoluble and inactive protein. Attempts to refold the protein proved unsuccessful. In contrast, a major fraction of mouse C alpha expressed in E. coli is soluble and the enzyme in the soluble fraction is active; however, certain mutant forms have proved to be unstable, difficult to purify, or insoluble. In addition, the E. coli system cannot be used to study the biological role of posttranslational modifications specific to eukaryotic systems. Several protein kinases have been expressed in soluble form in insect cells using baculovirus, suggesting that this system is generally more reliable than E. coli. However, the presence and nature of posttranslational modifications in insect cells may be different from that found in the natural source and may affect the biochemical function. In addition, baculovirus expression is not particularly useful for studying biological questions. Mouse C alpha and C beta have been overexpressed in NIH3T3 cells. This approach is useful in characterizing the biochemical properties of C alpha versus C beta, but it may not be an ideal system for studying mutant proteins since wild-type C subunits are still expressed from the chromosomal copies in this genetic background. This small level of wild type may make it difficult to analyze weakly functional mutants, which have activities less than 10% that of wild type. Several cell lines with altered subunits of cAMP-dependent protein kinase have been identified but a strain completely devoid of C subunit has not been adequately characterized for protein structure/function studies. Disruption of the genes encoding cAMP-dependent protein kinase in mammalian cells has not yet been accomplished. This chapter describes a method to express a C subunit of mammalian cAMP-dependent kinase in yeast. We have demonstrated that the mouse C alpha subunit can substitute for its yeast counterpart. Since at least one functional C subunit is required for viability, these results suggest that the yeast substrates important for viability are recognized by the mammalian C subunit. Although the sequence conservation between yeast and mouse C subunit is only about 50%, these results demonstrate that heterologous proteins with relatively low sequence conservation with their yeast counterparts can be functional in yeast.(ABSTRACT TRUNCATED AT 400 WORDS)
Graves exophthalmos unrelated to extraocular muscle enlargement. Superior rectus muscle inflammation may induce venous obstruction.
Exophthalmos is the most commonly measured sign of Graves ophthalmopathy, whereas enlargement of the extraocular muscles is the principal pathologic abnormality. The purpose of this article is to explore possible etiologies of increased volume of orbital fat and of proptosis in patients with no substantially increased total extraocular muscle volume. Computed tomographic scans of the 13 orbits reviewed in this study had the following characteristics in common: a fine, reticular pattern within the orbital fat, a prominent superior ophthalmic vein, and an enlarged superior rectus muscle. Quantitative analysis revealed that superior rectus muscle volume showed a statistically significant correlation with proptosis, whereas medial, lateral, and inferior rectus muscle volumes did not correlate with proptosis. Based on anatomic considerations, the authors postulate that superior rectus muscle enlargement alone may produce reduced venous outflow from the orbit, thereby expanding the apparent orbital fat volume and producing proptosis.
PNA member Lee Levin: an entrepreneurial profile. Interview by Kimberley Jackson.
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Postprandial levels of prolactin and gut hormones in breast cancer patients: association with stage of disease, but not dietary fat.
Previous studies of the relationship between dietary fat and breast cancer have produced conflicting results and have provided no definitive evidence of a mechanistic link between fat and breast tumorigenesis. We conducted a study to compare postprandial levels of prolactin (Prl), a hormone suspected of promoting the growth of some human breast cancer, and several gut hormones, i.e., gastrin (Gs), vasoactive intestinal polypeptide (VIP), neurotensin (Nt), and cholecystokinin (CCK), following high- and low-fat isocaloric test meals. Data were obtained in the posttreatment period from 13 patients with breast cancer (nine stage I and four stage II), who were disease free clinically, and nine healthy controls. Subjects admitted to the research unit on 2 days were given the high-fat meal on day 1 and the low-fat meal on day 2. Blood samples were drawn before (i.e., fasting) and after test meal consumption. All hormone analyses were performed by radioimmunoassay. Results indicated a significant rise in postprandial Prl levels for stage II patients, but not for stage I patients or the controls. Postprandial Gs levels were also elevated, whereas VIP levels were markedly reduced in patients versus controls; these differences were most marked in stage II patients. No significant intergroup differences were noted in postprandial levels of Nt and CCK. Hormone levels of patients and controls did not differ between the test meal situations, which indicated that some other component of the test meals might have been responsible for altered Prl and Gs levels. The differences observed between the stage I and II patients indicated that diet may influence the aggressiveness of tumor behavior and development through alterations in postprandial hormone release.
Graves' ophthalmopathy. Correlation of saccadic eye movements with age, presence of optic neuropathy, and extraocular muscle volume.
Quantitative infrared oculography was used to record saccadic eye movements of 49 patients with Graves' ophthalmopathy. Peak saccadic velocities were decreased in those patients who developed or presented with optic neuropathy. This effect was more pronounced for larger eye movements. Peak saccadic velocity also decreased as total extraocular muscle volume and limitation of ocular motility increased. For any given extraocular muscle volume, peak saccadic velocity was 40 degrees/s slower in patients 40 years or older than in younger patients. The relationship between velocity and motility limitation was most pronounced for intermediate muscle volumes (8% to 15% of total orbital volume). Saccadic velocities in those patients with optic nerve compression often improved following treatment. This study demonstrated that eye movement recording was a useful adjunct in evaluation of patients with Graves' ophthalmopathy. Furthermore, age-related lowering of peak saccadic velocities implicated changes of extraocular muscle structure as a factor in the development of optic neuropathy.
Frequency of use, knowledge, and attitudes toward the contraceptive sponge among inner-city black and Hispanic adolescent females.
This study assessed the frequency of use, knowledge, and attitudes toward the contraceptive sponge in sexually active, inner-city, black and Hispanic adolescent females. A 54-item, self-administered questionnaire was completed by 208 adolescents (mean age 17.9 years) attending a gynecology/family planning clinic within a comprehensive adolescent health center. Twenty-nine (13.9%) had never used any birth control, and 49 (23.6%) had not used any form of birth control in the 3 months prior to the study. Fifty-three (25.5%) of the adolescents had used a less reliable method such as rhythm and/or withdrawal. The birth control method used least was the sponge. Only four (1.9%) of the adolescents had used the sponge in the 3 months prior to the study, and only ten (4.8%) had ever used it. Knowledge of the correct use of the sponge was poor. However, 69 (44.8%) of the 156 girls who knew about the sponge had a positive attitude toward it, and only 19% said they did not like it. The contraceptive sponge may be a viable option for adolescents, particularly those who are not using birth control or who are using a less reliable method.
Human papilloma virus in erosive oral lichen planus.
Several types of human papilloma viruses (HPV) have been associated with benign and malignant squamous cell tumours of mucosal epithelium. To identify HPV in erosive oral lichen planus (OLPe), considered as a premalignant lesion, tissues from 20 patients were examined by Southern blot hybridization with 32P-labeled HPV DNA probes. Type 11 was found in 6 of the lesions while HPV types 6, 16 and 18 were not detected in any of the tissues examined. Using a type-specific polymerase chain reaction (PCR) assay for HPV-6, 11, 16 and 18, HPV-11 was detected in 8 of the samples (all of those positive by Southern blot), and, in addition, HPV-6 was found in 5 samples and HPV-16 in 3 samples. Overall, by the more sensitive PCR assay, 65% of samples were positive for HPV DNA. The finding of HPV DNA in many of the samples using two different techniques indicates a high prevalence of HPV in the OLPe afflicted oral mucosa. However, the role of HPV in the pathogenesis of OLPe has yet to be determined.
Graves' ophthalmopathy: V. Aetiology of upper eyelid retraction in Graves' ophthalmopathy.
The volume of the levator superior rectus muscle complex was determined from computed tomography scans of 152 orbits from 76 patients with Graves' ophthalmopathy. Clinically observed lid retraction and lid lag did not correlate with the levator/superior rectus muscle volume and did not correlate with limitation of vertical eye movements or inferior rectus muscle volume. These results do not support previously published hypotheses that muscle hypertrophy or levator/superior rectus muscle overaction secondary to inferior rectus muscle restriction are major causes of lid retraction and lid lag. Local adhesion of the levator muscle to fixed orbital tissues is postulated to be the most likely basis for the characteristic lid retraction and lid lag of Graves' ophthalmopathy.
A randomized trial comparing 12 weeks versus 36 weeks of adjuvant chemotherapy in stage II breast cancer.
A randomized trial has been performed in which women with axillary node-positive breast cancer were allocated to either a short intensive 12-week chemohormonal treatment consisting of cyclophosphamide, methotrexate, fluorouracil, vincristine, prednisone, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), and tamoxifen (CMFVP plus AT) or 36 weeks of CMFVP. The median follow-up is 37 months. Of the 222 women randomized to the 12-week treatment, 113 (50.9%) have experienced either recurrence or death as compared with 90 patients (41.9%) in the 36-week treatment group. The corresponding 3-year relapse-free survivals are 55% and 64%, respectively, P = .003. Fifty-nine (26.6%) of the patients in the 12-week group have died compared with 46 (21.4%) of the 36-week group. The corresponding 3-year survival rates are 78% and 85%, respectively, P = .04. A Cox regression analysis showed an associated increased risk ratio for recurrence or death of 1.7, P = .003, and for death of 1.8, P = .017 in the 12-week treatment group compared with the 36-week group. Thus, this 12-week regimen of adjuvant chemohormonal therapy is inadequate treatment for women with axillary node-positive breast cancer; possible explanations for this inferiority are its shorter duration and/or a negative interaction of tamoxifen on the chemotherapy.
Regional analgesia on pediatric medical and surgical wards.
Regional anesthetic approaches to pain management were examined in 72 children and young adults (ages 3 weeks to 31 years) who were observed on the surgical or medical wards of a children's hospital separate from intensive care areas. A protocol was devised to permit safe conduct of these techniques on the ward. Full resuscitation supplies were kept at each bedside. All patients receiving epidural narcotics had an apnea monitor, hourly counting of respiratory rates, and restriction of systemic analgesics. All bolus re-injections into the catheters were performed by an anesthesiologist who monitored the patient for 20 min. Minor side-effects, including pruritus, nausea, and urinary retention were common, but manageable. Significant complications included: one case of decubitus ulcers requiring skin-grafting, one episode of mild hypotension in a patient with terminal malignancy, requiring ephedrine and phenylephrine, and one mild toxic reaction on test dosing due to presumed intravascular migration of a lumber sympathetic catheter. Regional analgesic techniques can provide excellent analgesia on the wards for selected children and young adults, provided precautions are taken. Further study is required to define specific indications, risks and benefits relative to simpler techniques.
Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1.
In the yeast Saccharomyces cerevisiae, three genes TPK1, TPK2, and TPK3 encode catalytic subunits of cAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPK1 gene. In order to purify C1 completely free of C2 and C3, a strain was constructed that contained only the TPK1 gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPK1) and yeast regulatory subunit (BCY1) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-200, then bound to an anti-regulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of cAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. Kinetic analysis showed that the apparent Km for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 microM, respectively. The kcat was determined to be 640 min-1. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked.
cAMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae.
Genes encoding the regulatory (BCY1) and catalytic (TPK1, TPK2, and TPK3) subunits of the cAMP-dependent protein kinase (cAPK) are found in S. cerevisiae. bcy1- yeast strains do not respond properly to nutrient conditions. Unlike wild type, bcy1- strains do not accumulate glycogen, form spores, or become resistant to heat shock when nutrient limited. We have isolated mutant TPK genes that suppress all of the bcy1- defects. The mutant TPK genes appear to encode functionally attenuated catalytic subunits of the cAPK. bcy1- yeast strains containing the mutant TPK genes respond appropriately to nutrient conditions, even in the absence of CDC25, both RAS genes, or CYR1. Together, these genes encode the known components of the cAMP-generating machinery. The results indicate that cAMP-independent mechanisms must exist for regulating glycogen accumulation, sporulation, and the acquisition of thermotolerance in S. cerevisiae.
The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway.
The gene corresponding to the S. cerevisiae cell division cycle mutant cdc25 has been cloned and sequenced, revealing an open reading frame encoding a protein of 1589 amino acids that contains no significant homologies with other known proteins. Cells lacking CDC25 have low levels of cyclic AMP and decreased levels of Mg2+-dependent adenylate cyclase activity. The lethality resulting from disruption of the CDC25 gene can be suppressed by the presence of the activated RAS2val19 gene, but not by high copy plasmids expressing a normal RAS2 or RAS1 gene. These results suggest that normal RAS is dependent on CDC25 function. Furthermore, mutationally activated alleles of CDC25 are capable of inducing a set of phenotypes similar to those observed in strains containing a genetically activated RAS/adenylate cyclase pathway, suggesting that CDC25 encodes a regulatory protein. We propose that CDC25 regulates adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins.
Dose intensity analysis of chemotherapy regimens in ovarian carcinoma.
The relationship between outcome and dose intensity was analyzed for first-line chemotherapy of advanced ovarian cancer using a particular CHAP (cyclophosphamide, hexamethylmelamine, Adriamycin [Adria Laboratories, Columbus, OH], cisplatin) regimen as the standard. Previously described techniques were used to calculate the average dose intensity of regimens containing one, two, three, or all four drugs of CHAP, relative to the standard. The average relative dose intensity, especially the relative dose intensity of cisplatin, correlated significantly with clinical response and with median survival time (MST) of the entire group (not just the remitters). There was a distinct advantage for multiagent regimens over single alkylating agents and especially for multiagent regimens containing cisplatin. Survival correlated with response rate (of multiagent regimens). This analysis suggests that dose intensity is a determinant of treatment outcome. Prospective randomized trials would be required to test whether, and to what extent, dose intensity determines outcome independently of total amount of drug given, performance status, or other factors. If dose intensity does determine outcome, methods of increasing it should be tested in an attempt to improve treatment results.