Utilization of mismatched initiator termini by avian myeloblastosis virus DNA polymerase.

DNA synthesis by avian myeloblastosis virus was studied using poly(C) as template and modified oligo(dG) as primer. The addition of one noncomplementary base to the 3'-end of the primer has no important effect on synthesis. The mispaired base is incorporated into the product and the apparent Km (for primer) and the V of the reaction remain unchanged. This confirms the absence of a 3' leads to 5'-exodeoxynuclease activity using a template that is transcribed faithfully rather than one that can undergo a slippage reaction.

Effect of alternating combination chemotherapy on survival of ambulatory patients with metastatic large-cell and adenocarcinoma of the lung. A Southwest Oncology Group Study.

Using a randomized prospective trial design, chemotherapy with 5-fluorouracil, vincristine, and mitomycin C (FOMi) was compared with cyclophosphamide, doxorubicin, and cisplatin (CAP) and with FOMi alternating with CAP (FOMi/CAP) in 452 eligible patients with metastatic large-cell undifferentiated and adenocarcinoma of the lung. Objective responses were obtained in 26%, 17%, and 22% of patients treated with FOMi, CAP, and FOMi/CAP, respectively. The median survival was similar for FOMi, CAP, and FOMi/CAP therapies (20, 24, and 23 weeks, respectively), but the overall survival (log rank test), 1-year survival, and remission duration were longer for FOMi/CAP-treated patients. Survival was significantly longer for fully ambulatory FOMi/CAP-treated patients compared with either FOMi (P = .01) or CAP (P = .04). Younger patients treated with full doses of therapy responded more often than older patients receiving reduced drug doses (26% and 11%, respectively; P = .003). A prognostic factor regression analysis of all eligible patients indicates that sex, performance status, stage, and treatment assigned were important independent variables determining survival (P less than .05). Toxicity was comparable in each treatment group.

Concurrent chemotherapy/radiotherapy for limited small-cell lung carcinoma: a Southwest Oncology Group Study.

The Southwest Oncology Group (SWOG) has conducted a phase II study to explore the efficacy and toxicity of initial, concurrent use of radiation therapy with cisplatin, etoposide (VP-16), and vincristine in limited-stage small-cell carcinoma of the lung. Two courses of cisplatin, VP-16, and vincristine chemotherapy were given with concurrent radiotherapy (XRT) to the primary tumor to a total dose of 4,500 cGy. Elective brain XRT was given to all patients concurrent with a third course of cisplatin/VP-16 therapy. Consolidation chemotherapy consisting of vincristine, methotrexate, and VP-16 alternating with Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH) and cyclophosphamide, was given for 12 weeks following the initial induction chemotherapy/XRT program. Patients with a complete response had all therapy discontinued. Among 154 eligible patients treated, the complete response rate was 56%, with a partial response rate of 27%. The median survival is 17.5 months with an estimated 30% survival rate at 4 years from initiation of treatment. Combined modality toxicities were acceptable with the predominant toxicity being moderate to severe leukopenia and mild radiation esophagitis. The results of this treatment program appear superior to any previously reported by our group and compare favorably to those in the literature at large.

Traumatic cardiac hemolytic anemia: a late complication of a Starr-Edwards mitral valve prosthesis.

Severe, traumatic, cardiac, hemolytic anemia developed in a patient nine years after mitral valve replacement with a Starr-Edwards model 6120 prosthesis. Cardiac catheterization failed to demonstrate a perivalvular leak or prosthetic malfunction. Transfusion on two occasions resulted in accelerated hemolysis and failed to maintain an appreciable elevation of the hemoglobin level. At operation, a perivalvular leak was found. Replacement of the valve led to complete resolution of the hemolytic problem. The case demonstrates that cardiac hemolysis may be a good indicator of valve dysfunction.

Growth of human malignant micromegakaryocytes in vitro.

Mononuclear cells from the peripheral blood of a patient with megakaryoblastic transformation of Philadelphia chromosome-positive chronic myelogenous leukemia were cultured. Morphological and cytochemical studies and cell ploidy determinations were done daily for 4 days. PAS staining of the cells increased progressively during culture. Ultrastructural study of circulating and cultured cells revealed demarcation membranes and alpha granules indicating the cells were micromegakaryocytes. Deoxyribonucleic acid synthesis, determined by 3H-thymidine uptake, peaked at 72 hours. The DNA content of cultured cells was diploid at all times. All 15 metaphases analyzed at 72 hours were Ph1-positive. Malignant (Ph1-positive) megakaryoblasts and micromegakaryocytes grown successfully were capable of partial cytoplasmic maturation as demonstrated by glycogen deposition and increase in subcellular organelles, while endoreduplication was impaired. Malignant megakaryoblasts and micromegakaryocytes can be grown successfully in short term liquid culture and have more complete maturation in vitro than observed in vivo.

Concurrent chemotherapy and radiotherapy for limited small-cell carcinoma of the lung: a Southwest Oncology Group Study.

This pilot study in limited-stage small-cell carcinoma of the lung using concurrent cisplatin (Platinol) and etoposide (VePesid) chemotherapy with radiotherapy has yielded a high complete response rate in 23 of 40 patients evaluable for response. Five of these responders have survived greater than 2 years off all therapy with a stable, high performance status. Median survival of all patients is 18 months. Toxicity has been acceptable, the most common being neutropenia. Radiation toxicities include 17 of 40 patients experiencing mild to moderate esophagitis, with one severe toxicity; and three of 40 patients developing mild to moderate radiation pneumonitis. The high complete remission observed with this program and the long tumor-free interval seen off maintenance therapy deserve further exploration. Toxicities appear only moderately greater than with other programs currently utilized.

Effects of the calcium channel facilitator, CGP 28,392, on different modes of contraction in smooth muscle of rabbit and rat aortae and guinea-pig taenia caeci.

Effects of a Ca2+ channel facilitator, CGP 28,392, on smooth muscle contractions were examined in order to delineate characteristics of Ca2+ channels in rabbit and rat aortae and guinea-pig taenia caeci. Application of increasing concentrations of KCl induced contractile responses in these smooth muscles and CGP 28,392 shifted the concentration-response curve for KCl to the left. The maximum response was also increased in rat aorta and guinea-pig taenia. CGP 28,392 also shifted the concentration-response curves for noradrenaline in rat aorta and for histamine in taenia to the left and increased the maximum response in rat aorta. However, the corresponding curve for noradrenaline in rabbit aorta was not affected by CGP 28,392. The sustained contractions induced by KCl were inhibited by cumulative application of verapamil in these smooth muscles. Pretreatment of the muscle with CGP 28,392 decreased the inhibitory effect of verapamil. The noradrenaline-induced contraction in rat aorta and the histamine-induced contraction in taenia were also inhibited by verapamil, and CGP 28,392 antagonized the effect of verapamil. The noradrenaline-induced contraction in rabbit aorta was only slightly inhibited by verapamil, and CGP 28,392 did not modify the effect of verapamil. In these smooth muscles, cumulative application of Ca2+ to the Ca2+-depleted, KCl-treated muscle induced contraction, and the concentration-response curve for Ca2+ was shifted to the left by CGP 28,392 and to the right by verapamil. The concentration-response curves for Ca2+ in Ca2+-depleted, noradrenaline-treated rabbit and rat aortae and in Ca2+-depleted, histamine-treated taenia were also shifted to the left by CGP 28,392 and to the right by verapamil. In some contractions, CGP 28,392 increased and verapamil decreased the maximum responses. CGP 28,392 antagonized the inhibitory effect of verapamil. 5 These results suggest that the Ca2 channel facilitator, CGP 28,392, has a relatively selective activating effect on voltage-dependent Ca2+ channels in rabbit aorta. However, it also activates receptor-linked Ca2+ channels in rabbit aorta when Ca2+ concentrations are low. In rat aorta and guinea-pig taenia this facilitator activates both types of Ca2+ channels.

Calcium kinetics in vascular smooth muscle.

The accumulation, binding, and mobilization of Ca++ in vascular smooth muscle directly affects intracellular free Ca++ levels and contractility. Techniques have been developed to delineate Ca++ uptake and efflux parameters in isolated vessels. Similar Ca++-related components are present in different types of vessels, but their relative importance for induction and maintenance of tension differ.

Comparison of direct harvest and cultures for karyotyping EDTA anticoagulated marrow.

Studies were undertaken to determine whether EDTA was a satisfactory anticoagulant for tissue specimens for cytogenetic analysis and to investigate a modification of a currently used culture technique for obtaining metaphases. The latter involved to prolonged exposure to very low-dose colcemid and was successful in qualitative or quantitative enhancement, or both, of the temperature yield over that obtained from direct harvest in 53% of the patients studied. EDTA is a suitable anticoagulant for cytogenetic studies of specimens from either direct harvest or short-term culture if the specimen is either processed within 24 hr after collection or diluted 1:1 with Eagles minimal essential media, supplemented with fetal bovine serum and refrigerated until processed. Success has been obtained with specimens stored up to 144 hr.

Dissociation of actions of BRL 34915 in the rat portal vein.

In rat portal vein, 0.5 and 5.0 microM BRL 34915 [(+/-)-6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1-pyrrolidyl++ +)-2H- benzo[b]pyran-3-ol] abolished spontaneous rhythmic movements and norepinephrine (NE)-induced tension responses, respectively. Only the higher (5 microM) concentration of BRL 34915 increased 42K efflux and inhibited the NE-induced increase in 42K efflux. These results suggest that BRL 34915 inhibits spontaneous rhythmic movements and NE-induced tension responses by differing mechanisms of action and that only the block of the NE-induced tension response is related to K+ permeability or conductance changes.

Differential calcium movements induced by agonists in guinea pig tracheal muscle.

The effects of high potassium, carbachol and histamine on tension responses and 45Ca fluxes in tracheal smooth muscle were examined. Calcium depletion or nitrendipine (10(-8) M) inhibited potassium-induced contractile responses more than those obtained with either histamine or carbachol, whereas Sr2+ inhibited mainly responses to histamine or carbachol. The Ca2+ entry facilitator, CGP 28392 (3 X 10(-6) M), potentiated contractions induced only by potassium. Uptake of 45Ca in guinea pig tracheal muscle can be separated into high and low affinity components. The 45Ca efflux rate from tracheal muscle into a La3+-substituted solution was over four-fold higher than in other smooth muscles. Potassium, carbachol and histamine induced sustained increases in 45Ca efflux into solutions containing 1.5 mM Ca2+; only transient increases in 45Ca efflux with carbachol and histamine were obtained after Ca2+ depletion. These agonists elicit contractile responses in tracheal muscle by selectively mobilizing different cellular and extracellular Ca2+ components.

Comparative effects of a selective adenosine A2 receptor agonist, CGS 21680, and nitroprusside in vascular smooth muscle.

CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethyolcarboxamidoa denosine) is an adenosine agonist that has been reported recently to bind selectively to adenosine A2 receptors in rat brain. This adenosine agonist, and the parent compound NECA (5'-N-ethylcarboxamidoadenosine), were found to be potent vasorelaxants of prostaglandin F2 alpha (PGF2 alpha) precontracted porcine coronary smooth muscle with EC50s of 4.5 and 9.7 nM, respectively. Schild analysis of the inhibition of CGS 21680, NECA and 2-chloroadenosine induced relaxation of the porcine coronary artery by CGS 15943 (9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-C]quinazolin-5-amine), an A2 receptor antagonist, yielded identical pA2 values for the antagonist (approximately 9.3). This indicates that the same receptor mediates the effects of these three adenosine agonists. NECA and CGS 21680 were equipotent in most vascular preparations except in the canine coronary artery. Porcine coronary arterial rings contracted with PGF2 alpha were relaxed by NECA or CGS 21680 as well as by nitroprusside; those contracted with KCl (40 mM) were relaxed only by nitroprusside. In rabbit aorta, contractions induced by phenylephrine or PGF2 alpha were inhibited by nitroprusside but not by NECA or CGS 21680. Thus, the adenosine A2 receptor agonists, NECA and CGS 21680, are potent vasorelaxants that display regional vascular and species variations that differ from those of nitroprusside.

Evidence that BKCa channel activation contributes to K+ channel opener induced relaxation of the porcine coronary artery.

The rank order of potency of a series of benzopyran and cyanoguanidine K+ channel openers (KCOs) for causing relaxation of the PGF2 alpha-precontracted porcine coronary artery was determined. Glyburide, an inhibitor of KATP channels, showed an apparent competitive inhibition of the vasorelaxant activity of the KCOs. The pA2 values of glyburide when cromakalim and CGP 14877 (P1060) were used as vasorelaxants were 7.66 and 7.83, respectively. Charybdotoxin (40 nM), an inhibitor of BKCa channels, also caused a significant inhibition of the cromakalim mediated relaxation of the porcine coronary artery. In order to clarify the site of action of these KCOs, we identified a K+ channel current in single porcine coronary arterial cells and measured channel activity in the presence of these compounds. The prominent K+ ion current in these cells had characteristics typical of the conventional large Ca(2+)-activated K+ channel (BKCa) present in other smooth muscle cells. Using symmetrical K+ concentrations, the channel had a conductance of 214 pS and was found to be sensitive to [Ca2+]i and membrane potential. The KCOs were found to reversibly increase the open probability (P(o)) of the channel without changing channel conductance. The potency of the KCOs to increase K+ channel opening was similar to the potency of these compounds to cause coronary artery relaxation. These results indicate that the porcine coronary artery contains the BKCa channel and that this channel, along with other types of K+ channels (KATP), mediate the vasorelaxant effects of K+ channel openers.

The problem of nuclease activity in nucleic acid hybridization reactions. Theoretical considerations.

The presence of nuclease during DNA-DNA or DNA-RNA hybridization reactions alters the kinetics of hybrid formation. Unfortunately, while the effect (even of small amounts of nucleas) on the Cot curve may be large, it may not be readily detectable. The effect of various types of nuclease is shown. As many nucleases cause a shift in the position rather than a change in shape of the curve, all studies involving nucleic acid hybridization should assay for the presence of nucleases and care must be taken to avoid their presence as contaminants.

Metabolism and actions of CDP-choline as an endogenous compound and administered exogenously as citicoline.

CDP-choline, supplied exogenously as citicoline, has beneficial physiological actions on cellular function that have been extensively studied and characterized in numerous model systems. As the product of the rate-limiting step in the synthesis of phosphatidylcholine from choline, CDP-choline and its hydrolysis products (cytidine and choline) play important roles in generation of phospholipids involved in membrane formation and repair. They also contribute to such critical metabolic functions as formation of nucleic acids, proteins, and acetylcholine. Orally-administered citicoline is hydrolyzed in the intestine, absorbed rapidly as choline and cytidine, resynthesized in liver and other tissues, and subsequently mobilized in CDP-choline synthetic pathways. Citicoline is efficiently utilized in brain cells for membrane lipid synthesis where it not only increases phospholipid synthesis but also inhibits phospholipid degradation. Exogenously administered citicoline prevents, reduces, or reverses effects of ischemia and/or hypoxia in most animal and cellular models studied, and acts in head trauma models to decrease and limit nerve cell membrane damage, restore intracellular regulatory enzyme sensitivity and function, and limit edema. Thus, considerable accumulated evidence supports use of citicoline to enhance membrane maintenance, membrane repair, and neuronal function in conditions such as ischemic and traumatic injuries. Beneficial effects of exogenous citicoline also have been postulated and/or reported in experimental models for dyskinesia, Parkinson's disease, cardiovascular disease, aging, Alzheimer's disease, learning and memory, and cholinergic stimulation.

Calcium release in smooth muscle.

In smooth muscle, maintenance of the contractile response is due to Ca2+ influx through two types of Ca2+ channel, a voltage-dependent Ca2+ channel and a receptor-linked Ca2+ channel. However, a more transient contraction can be obtained by release of Ca2+ from a cellular store, possibly the sarcoplasmic reticulum. In spike generating smooth muscle (e.g., guinea-pig taenia caeci), spike discharges may trigger the release of cellular Ca2+ by activating a Ca2+-induced Ca2+ release mechanism. Caffeine directly activates this mechanism in the absence of a triggered Ca2+ influx. In contrast to this, maintained depolarization may not only release but also refill the Ca2+ store. Drug-receptor interactions also release Ca2+ from a cellular store. This release may be elicited with inositol trisphosphate produced by receptor-linked phosphoinositide turnover. In non-spike generating smooth muscle (e.g., rabbit thoracic aorta), maintained membrane depolarization does not release but, instead, fills the Ca2+ store. However, caffeine and receptor-agonists release the Ca2+ store - possibly by activating the Ca2+-induced Ca2+ release mechanism and phosphoinositide turnover, respectively. The Ca2+ store in smooth muscle is filled by Ca2+ entry through voltage dependent Ca2+ channels and also by resting Ca2+ influx in the absence of receptor-agonists. The Ca2+ entering the cells through these pathways may be accumulated by the Ca2+ store and may activate the contractile filaments.

Effects of potassium or potassium/magnesium supplementation on potassium content of body tissues and fluids in furosemide-treated rats on magnesium-deficient or magnesium-sufficient diet.

Persistent Mg2+ deficiency may interfere with restoration of normal tissue K+ levels. This study examined: a) the effects of chronic furosemide treatment on K+ of sartorius, aorta and ventricle of rats fed Mg2(+)-deficient (100 ppm) or Mg2(+)-sufficient (400 ppm) diet and deionized water; b) whether normal tissue K+ is restored by oral K+ or K+/Mg2+ supplementation with continued furosemide therapy. Levels of Mg2+ were also measured. Furosemide (20 mg/kg i.p.) decreased K+ in sartorius, aorta and ventricle by 5.5, 4.3 and 19.9 microEq/gm (p less than .05), respectively, in rats fed 100 ppm Mg2+ diet. Furosemide did not alter K+ levels in rats fed 400 ppm Mg2+ diet. K+ supplementation (1 mEq/kg for 7 days) restored K+ to normal in sartorius but the addition of Mg2+ supplementation was necessary to restore K+ levels to normal in ventricle and aorta. These data indicate that furosemide can decrease tissue K+ in rats on a Mg2(+)-deficient diet. This decrease can be reversed during diuretic administration by K+ supplementation in sartorius, or K+ plus Mg2+ supplementation in ventricle and aorta.

Concurrent chemotherapy and radiation therapy for limited unresectable non-small cell carcinoma of the lung. A phase I study.

We treated nine patients diagnosed as inoperable, but localized non-small cell lung cancer with aggressive high-dose radiation therapy and two cycles of concomitant cisplatin and 5-fluorouracil (5-FU) to determine the feasibility of this approach for this disease. This combined modality program was well tolerated by seven of our nine patients. One who had a poor initial performance status died of sepsis. Another could not tolerate the nausea and vomiting. Only one patient has suffered a local failure inside the irradiated areas. Eight have died, and 5 of 9 survived at least 1 year. The survival is at least consistent with that associated with radiation therapy alone.

Cellular Mg++ accumulation is altered by extracellular Na+ and directly affects agonist-induced mobilization of Ca++ in vascular smooth muscle.

Effects of altered Na+, Ca++ and Mg++ concentrations on 45Ca and 28Mg distribution and binding as well as of changes in cellular Mg++ on mobilization of Ca++ by added norepinephrine (NE) were examined in the rabbit aortic media-intimal layer. Uptake of 45Ca at cellular high affinity sites was decreased by Mg++ much more than 28Mg uptake was altered by Ca++. Substitution of Na+ affects 45Ca uptake primarily at extracellular (La( )-accessible) binding sites. Muscles were pre-loaded with Mg++ by incubation in a low-Na+ solution (75% Na+ replaced isosmotically with sucrose) for 30 min followed by a 90 min exposure to a similar solution also containing 15 mM MgCl2. These tissues, upon examination in normal (154 mM) Na(+)-containing solution, indicated decreased retention of that cellular, high-affinity Ca++ fraction important for NE-induced contractile response. Accordingly, release of 45Ca from this site and associated tension responses to added NE were attenuated in these muscles. These results suggest that variations in extracellular Na+ concentration modulate binding and subsequent mobilization of activator Ca++ by agonists through alterations in cellular Mg++ content in vascular smooth muscle.

False data & the therapeutic misconception: two urgent problems in research ethics. False data and last hopes: enrolling ineligible patients in clinical trials.

KIE: A persistent ethical dilemma in research is the potential conflict between the patient-subject's best interests and the principles of scientific methodology. Vanderpool and Weiss discuss the problem in cancer research when a physician falsifies data in order to increase a patient's chances of being selected to participate in a clinical trial. While raising the question of whether such lying is justified if it may prolong the life of a cancer patient for whom there are no other therapeutic options, the authors point out that the inclusion of ineligible subjects may render trial results invalid.^ieng