Prognostic factors in follicular lymphoma: a single institution study.

Recently developed prognostic models for follicular lymphoma have proven useful in predicting overall survival (OS), but most have used data from multiple centers. Our goal was to look at prognostic factors within a single institution. We conducted a retrospective study on 77 newly diagnosed patients with follicular lymphoma, focussing on clinical characteristics, symptom duration before diagnosis, pathologic findings, including grade, laboratory data, imaging studies at initial presentation and management. The study population was 53% male. Ages ranged from 25 to 87 years (median 51). By Ann Arbor classification 4% were stage I, 8% stage II, 18% stage III and 69% stage IV. Initial therapy was deferred in 39%. The remaining patients received stage-appropriate therapy. Survival was measured from time of diagnosis to death. Prognostic factors at initial diagnosis that were statistically significant in univariate log-rank comparisons of Kaplan-Meier survival curves were used to build a multi-variate proportional hazard regression model of OS. Median OS for these patients was 10.3 years. OS differed only with high (>12 g/dl) versus low (<12 g/dl) hemoglobin (p=0.001) and in younger (<60 years) versus older (>60 years) patients (p=0.05), as indicated by univariate log-rank tests. Both hemoglobin and age were also significant in a multivariate proportional hazards analysis. Low hemoglobin and increased age were independent predictors of lower OS with hazard ratios of 6.6 (95% CI, 2.2-20.1) and 3.7 (95% CI, 1.2-11.7), respectively. Median survival for older patients who also had anemia was only 3.1 years. A test for interaction between age and hemoglobin was negative (p=0.35). The estimated hazard ratio for an older individual with low hemoglobin was 24.7 (95% CI, 4.0-153.3). To assess the proportional hazards assumption we tested for an interaction between time and both age (p=0.92) and hemoglobin (p=0.66) and found no evidence against proportionality. A hemoglobin <12 g/dl and age >60 years at diagnosis are significant predictors of worse OS.

Molecular and cellular actions of platelet-activating factor in rat heart cells.

Platelet-activating factor (PAF) is a phospholipid with cardiovascular actions at low concentrations (1-100 nM) but with uncertain direct myocardial actions. We investigated the cellular and molecular effects of PAF on heart cells using isolated adult and neonatal rat myocytes. Addition of PAF, in the superfusion solution, decreased twitch amplitude and contractile velocity in both systems. Concentrations of PAF below 1 nM stimulated reproducible responses with maximal effects seen at 100 nM. These functional actions of PAF could be blocked by the known PAF antagonist, BN 50739, in a dose-dependent manner. Parallel biochemical studies showed that nanomolar PAF rapidly stimulated the phosphoinositide pathway in cultured myocytes, evidenced by the accumulation of [3H]inositol phosphates in prelabeled cultured myocytes. The potency and specificity of PAF, as well as the time course, for the response were nearly identical in the biochemical and functional assays. PAF produced no functional changes in protein kinase C-depleted myocytes, but it did stimulate inositol trisphosphate accumulation in such cells. We conclude that: (a) PAF exerts a direct negative inotropic effect on myocardial tissue; (b) the effects of PAF are mediated by a specific, high affinity cardiac receptor; (c) an underlying biochemical mechanism for the action of PAF includes the activation of the phospholipase C/phosphatidylinositol intracellular signaling pathway, which leads to activation of protein kinase C.

Ca(2+) channel modulation by recombinant auxiliary beta subunits expressed in young adult heart cells.

L-type Ca(2+) channels contribute importantly to the normal excitation-contraction coupling of physiological hearts, and to the functional derangement seen in heart failure. Although Ca(2+) channel auxiliary beta(1-4) subunits are among the strongest modulators of channel properties, little is known about their role in regulating channel behavior in actual heart cells. Current understanding draws almost exclusively from heterologous expression of recombinant subunits in model systems, which may differ from cardiocytes. To study beta-subunit effects in the cardiac setting, we here used an adenoviral-component gene-delivery strategy to express recombinant beta subunits in young adult ventricular myocytes cultured from 4- to 6-week-old rats. The main results were the following. (1) A component system of replication-deficient adenovirus, poly-L-lysine, and expression plasmids encoding beta subunits could be optimized to transfect young adult myocytes with 1% to 10% efficiency. (2) A reporter gene strategy based on green fluorescent protein (GFP) could be used to identify successfully transfected cells. Because fusion of GFP to beta subunits altered intrinsic beta-subunit properties, we favored the use of a bicistronic expression plasmid encoding both GFP and a beta subunit. (3) Despite the heteromultimeric composition of L-type channels (composed of alpha(1C), beta, and alpha(2)delta), expression of recombinant beta subunits alone enhanced Ca(2+) channel current density up to 3- to 4-fold, which argues that beta subunits are "rate limiting" for expression of current in heart. (4) Overexpression of the putative "cardiac" beta(2a) subunit more than halved the rate of voltage-dependent inactivation at +10 mV. This result demonstrates that beta subunits can tune inactivation in the myocardium and suggests that other beta subunits may be functionally dominant in the heart. Overall, this study points to the possible therapeutic potential of beta subunits to ameliorate contractile dysfunction and excitability in heart failure.

Angiotensin II signal transduction pathways in the cardiovascular system.

The renin-angiotensin system in mammals represents a complex cascade of tightly regulated proteolytic enzymes and peptide products. One important product, angiotensin II (Ang-II), is a circulating hormone that displays a wide range of physiologic effects in many tissues, including those of the cardiovascular system. It is well known that Ang-II increases the contractile state of vascular smooth muscle and cardiac myocytes by triggering multiple intracellular responses following its binding to specific receptor sites. Some of the signal transduction mechanisms that underlie these responses are understood, while others are not defined at present. Further, there has been much recent interest in Ang-II-evoked signaling, since the renin-angiotensin system has been associated with the development of cardiovascular disease. Thus, the identification of Ang-II-stimulated signal transduction mechanisms will illuminate the underlying principles of normal hormone-regulated cardiovascular homeostasis as well as those involved in pathologic processes such as human hypertension, cardiac arrhythmias, and the cellular remodeling that follows vascular injury or sustained pressure overload. This review examines the current status and direction of our knowledge regarding Ang-II-stimulated signal transduction mechanisms in vascular smooth muscle and cardiac myocytes.

Effect of methylated bepridil on slow action potentials in cardiac muscle and vascular smooth muscle.

The anti-anginal agent bepridil blocks slow Ca2+ channels in a variety of tissues. Since bepridil accumulates inside cells, the possibility exists that bepridil acts, at least partially, from inside the cell. To test this possibility, we examined the effects of a quaternary ammonium analog of bepridil, methylated bepridil, which presumably would enter the cells less readily, on the Ca2+-dependent slow action potentials of guinea pig papillary muscles (in 25 mM [K+]0) and rabbit pulmonary arteries (in tetraethylammonium chloride). In cardiac muscle, methylated bepridil had little effect on the slow action potentials at low stimulation frequencies (0.5 Hz), but at higher frequencies (1.0 and 2.0 Hz) the slow action potentials were depressed and/or the muscle was unable to follow each stimulation. These effects are similar to those obtained with bepridil, but bepridil was more potent than methylated bepridil. In vascular smooth muscle cells, methylated bepridil inhibited the slow action potentials at a somewhat lower dose than bepridil. We conclude that, in cardiac muscle, bepridil probably has two sites of action, one outside the cell (presumably on or associated with the slow Ca2+ channel) and a second site inside the cell. On the other hand, in vascular smooth muscle cells, bepridil may act only on an external site.

Synthesis and biological activity of carboxy verapamil, a new derivative of verapamil.

New analogues of the calcium antagonist verapamil (alpha-isopropyl-alpha-[(N-methyl-N-homoveratryl)-gamma-aminopropy l] -3,4-dimethylphenyl acetonitrile) were synthesized. Carboxy verapamil (alpha-isopropyl-alpha-[(N-methyl-N-homoveratryl)-gamma -aminopropyl]-3,4-dimethylphenyl acetic acid) was prepared by hydrolysis of the nitrile group of verapamil. Derivatives of carboxy verapamil could be synthesized by coupling substituents onto the carboxylic acid moiety. All syntheses can be prepared on a microscale. The biological activity of carboxy verapamil was compared with that of verapamil by testing their ability to block the isoproterenol-induced slow action potential (APs) in guinea pig papillary muscle. Carboxy verapamil was about 10-fold less potent than verapamil in blocking the slow APs. The IC50 for carboxy verapamil was about 6 X 10(-6) M. Furthermore both drugs had a similar frequency dependency. The effects of carboxy verapamil were rapidly reversed upon washout, suggesting that this drug may act on the external surface of the myocardial plasma membrane. In contrast, verapamil-induced effects are slowly reversed upon washout and it may exert is primary effects on the inner surface of the plasma membrane. Carboxy verapamil, because of its dipolar charge character may not readily penetrate into myocardial cells.

Self-reference and the encoding of personal information.

The degree to which the self is implicated in processing personal information was investigated. Subjects rated adjectives on four tasks designed to force varying kinds of encoding: structural, phonemic, semantic, and self-reference. In two experiments, incidental recall of the rated words indicated that adjectives rates under the self-reference task were recalled the best. These results indicate that self-reference is a rich and powerful encoding process. As an aspect of the human information-processing system, the self appears to function as a superordinate schema that is deeply involved in the processing, interpretation, and memory of personal information.

Use of thapsigargin to study Ca2+ homeostasis in cardiac cells.

Several reports have documented that thapsigargin is a potent inhibitor of the SR Ca2+ ATPase isolated from cardiac or skeletal muscle. We have characterized the specificity of this agent in intact rat cardiac myocytes using cells maintained in the whole cell voltage clamp configuration. We have shown that thapsigargin decreases the magnitude of the Ca2+ transient and the twitch by about 80% while it slows the decay rate for these responses. These changes were not accompanied by any alterations in sarcolemmal currents or in the trigger Ca2+ generated by the inward calcium current. Taken together these results reveal that the action of thapsigargin is restricted to the SR Ca2+ ATPase in intact cardiac myocytes. Furthermore, it is demonstrated unambiguously that SR intracellular Ca2+ stores are an absolute requirement for the development of contractile tension in rat heart myocytes. It is shown that thapsigargin is a valuable probe to examine the importance of SR pools of Ca2+ and the role of the Ca2+ ATPase in intact myocytes as well as in genetically altered heart cells.

The structure of habilitation.

Biographic, program and background variables traditionally employed in the habilitation program and accountability literature were assembled for 75 mentally handicapped adults who had completed a three year comprehensive program of training and up to three years of subsequent community placement. Factor analysis treatment yielded 12 input and 4 output factors, the nature of which suggests a greater role for cognitive and clinical variables in the overall structure of habilitation than has been supported by the habilitation services accountability literature to date.

High affinity angiotensin II receptors in myocardial sarcolemmal membranes. Characterization of receptors and covalent linkage of 125I-angiotensin II to a membrane component of 116,000 daltons.

High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis of carboxy-nifedipine and its use in the preparation of an affinity resin for the 1,4-dihydropyridine receptor.

Affinity chromatography represents a potentially valuable approach to study the calcium antagonist receptor in many tissues. Methods have been developed to synthesize carboxy-analogues of the 1,4-dihydropyridine calcium antagonists. Carboxy-nifedipine ([+/-]1,4-dihydro-2,6 dimethyl-4-[3-nitrophenyl] pyridine-3-carboxylic acid-5-carboxylic methyl ester) was prepared with a yield of 50% and its structure has been thoroughly characterized. Carboxy-nifedipine has been coupled to a hexamine-agarose resin through an acid chloride intermediate producing an affinity resin (1.6 mumol of drug/ml). Experiments have shown that this affinity resin is capable of binding the [3H]nitrendipine receptor solubilized from transverse tubule membranes.

Angiotensin II activates the Na+/HCO3- symport through a phosphoinositide-independent mechanism in cardiac cells.

Angiotensin II (AngII) is a hormone that alters contractility as well as myocyte growth in heart. Since many hormones that regulate cardiac contractility have also been found to modulate intracellular pH (pHi) the goal of this study was to determine if AngII altered pHi in cultured neonatal rat ventricular myocytes. Changes in pHi were monitored in single cells using the fluorescent pH indicator carboxy-seminaphthorhodafluor-1. Application of 100 nM AngII resulted in a rapid, receptor-mediated alkalinization of 0.08 +/- 0.02 pH unit. The Na+/H+ exchanger was not involved since the response was HCO3(-)-dependent and amiloride-insensitive. Ammonia rebound experiments showed AngII increased the initial rate of recovery from an imposed acid load by 3.15-fold and showed that the hormone led to the selective activation of the Na+/HCO3- symport. In contrast, phorbol ester activation of protein kinase C led to the selective activation of Na+/H+ antiport in these cells. Pharmacological studies showed that the alkalinization was independent of the AngII receptor subtype 1 (AT1) phosphoinositide signaling path. In contrast, AngII activation of the symport was blocked by nanomolar AT2 receptor antagonist PD 123319. Superfusion of the myocytes with exogenous arachidonic acid (5 microM) mimicked the AngII-mediated alkalinization, further suggesting that the AT2 signaling pathway underlies the response. In summary, while most of the known actions of AngII in heart are mediated through AT1 receptors, activation of the Na+/HCO3- symport occurs through a distinct alternative path that is likely related to fatty acid production.

Use of a PCR-based method to characterize protein kinase C isoform expression in cardiac cells.

Molecular cloning has identified at least nine unique isozymes of protein kinase C (PKC) designated alpha, beta I, beta II, gamma, delta, epsilon, zeta, and eta/L, with the recent addition of the theta-isoform. Previous attempts to characterize PKC isoform expression in heart have been limited by low levels of protein and perhaps by the presence of novel isoforms. Thus to critically examine the diversity of PKC expression in cardiac cells, we developed a reverse transcriptase-polymerase chain reaction (RT-PCR) approach that would amplify regions of the target cDNA of all the PKC isozymes in a single reaction. Degenerate oligonucleotide primers were designed to recognize sequences in the conserved regions of the PKC sequence motif: the cysteine-rich and the ATP-binding regions. Amplification of target PKC cDNA sequences resulted in PCR products with unique sizes and restriction digestion properties. The system was validated by identifying PCR products that correspond to all of the PKC isoform transcripts, except PKC-zeta, in a single reaction with cDNA derived from hippocampus. Cardiac cDNA was RT-PCR amplified, and the products were analyzed by a combination of restriction mapping and DNA sequencing that revealed the presence of only the alpha, delta, epsilon, and eta isoforms in adult rat cardiac myocytes and cultured neonatal ventricular myocytes. A unique nondegenerate primer pair was synthesized to recognize PKC-zeta cDNA. Results with these primers show that PKC-zeta is present in both cardiac myocyte preparations as well. The RT-PCR method developed here is an efficient approach that is broadly useful to examine PKC expression in many tissues, including the identification of potentially novel isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of functional angiotensin II receptors on cultured heart myocytes.

Cultured neonatal rat myocytes have been investigated as a potential system to study the molecular mechanism of angiotensin II (All) stimulation of heart contractile behavior. Intact cultured cells and the membranes from these cells bind [125I] All in a biphasic manner. The data were analyzed as two classes of binding sites on membrane preparations: Kd1 = 0.65 nM; maximum binding (Bmax1) = 245 fmol/mg of protein and Kd2 = 5.57 nM, Bmax2 = 720 fmol/mg of protein. The receptor on intact cells is specific as All peptide analogs were potent at inhibiting the binding of [125I]All. An All antagonist, [125I]Sar1,Leu8-All, recognized a single class of high affinity receptors on intact cells: Kd = 0.63 nM, Bmax = 318 fmol/mg of protein, or 45,300 sites per cell. Guanine nucleotides regulated the binding of All to membranes by shifting the receptor from a high affinity to a low affinity form without changing Kd2. Conversely, these nucleotides altered the high affinity binding of [125I]Sar1,Leu8-All by increasing Kd without changing Bmax. All was found to stimulate the contractile frequency of spontaneously beating myocytes with maximal effects (a 50% stimulation) observed at 5 nM hormone. The responses were reversible with half-maximal effects observed at doses around 1 nM. The antagonist, Sar1,Ala8-All, could inhibit the chronotropic stimulatory responses. It is concluded that these cultured myocytes express an All receptor system with properties consistent with a true hormone receptor system. Furthermore, studies on the pharmacology of the chronotropic responses of these cells to All demonstrate that these receptors are functional.

Studies on the intoxication pathway of tetanus toxin in the rat pheochromocytoma (PC12) cell line. Binding, internalization, and inhibition of acetylcholine release.

Tetanus toxin was found to be a potent inhibitor of neurosecretion in the rat pheochromocytoma cell line PC12, a system in which biochemical and functional studies could be performed in parallel. Incubation of the cells with 10 nM tetanus toxin (3 h) led to an inhibition of acetylcholine release by 75-80% when evoked by 200 microM veratridine, 1 mM carbachol, or 2 mM Ba2+. The main characteristics of the inhibition process are: 1) the toxin is very potent, with threshold doses of 10 pM; 2) the action of toxin is blocked at low temperature (0 degrees C) and by antitoxin; 3) the effects are dose- and time-dependent; 4) a concentration-dependent lag phase precedes the onset of the inhibitory effects. Thus the PC12 cultures are a valid system for studies on the underlying molecular process in tetanus action. This system was exploited by the use of long term incubation studies to examine the processes responsible for the lag phase. When cells were incubated with 0.1 nM 125I-tetanus toxin, cell-associated toxin reached a plateau of 16 fmol of toxin/mg of protein, yet the toxic effects did not appear until 12 h. Further, PC12 cells were found to rapidly internalize tetanus toxin, with a half-life of 1-2 min, once it was bound to the surface of the cells. Thus, the lag phase results from steps that occur in the intracellular compartment after internalization. An important discovery was that the differentiation state of the PC12 cells was a critical factor in determining sensitivity to tetanus toxin. Cells that were cultured with nerve growth factor for 8-12 days were very sensitive to toxin. In contrast, acetylcholine release from nondifferentiated, autodifferentiated, or dexamethasone-treated cultures was insensitive to tetanus toxin. Since differential expression of high affinity tetanus toxin receptors cannot explain these results, it is concluded that PC12 cells are capable of expressing different forms of excitation-secretion coupling mechanisms. Tetanus toxin should prove a valuable probe to further distinguish these processes.