Phospholamban-mediated stimulation of Ca2+ uptake in sarcoplasmic reticulum from normal and failing hearts.

Studies in animal models have suggested that alterations affecting phospholamban-mediated stimulation of Ca2+ uptake by sarcoplasmic reticulum are involved in the pathophysiology of heart disease. A monoclonal antibody that binds to phospholamban and stimulates Ca2+ uptake was used to characterize phospholamban-mediated effects in human cardiac sarcoplasmic reticulum and to compare these effects in tissue from normal and failing hearts. Stimulation of Ca2+ uptake by anti-phospholamban monoclonal antibody simulated the effect of phosphorylation of phospholamban by cAMP-dependent protein kinase. Binding of anti-phospholamban antibody reduced the K0.5 of the Ca2(+)-transporting ATPase from 0.53 microM [( Ca2+]) to 0.29 microM [( Ca2+]), without affecting Vmax or nHill. At 0.2 microM Ca2+, stimulation was 1.93-fold in sarcoplasmic reticulum prepared from normal human left ventricular myocardium and 1.94-fold in sarcoplasmic reticulum prepared from the left ventricular myocardium of patients with heart failure resulting from idiopathic dilated cardiomyopathy. Stimulation of Ca2+ uptake in canine cardiac sarcoplasmic reticulum under identical conditions was 1.89-fold. Phospholamban-mediated stimulation of Ca2+ uptake in human cardiac sarcoplasmic reticulum is thus comparable in magnitude to that observed in other species and results from an increase in the apparent affinity of the Ca2(+)-transporting ATPase for Ca2+. The pathogenesis of heart failure in idiopathic dilated cardiomyopathy does not, however, appear to involve intrinsic alterations of this mechanism.

Sarcoplasmic reticulum-associated cyclic adenosine 5'-monophosphate phosphodiesterase activity in normal and failing human hearts.

Sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was examined in microsomes prepared from the left ventricular myocardium of eight heart transplant recipients with end-stage idiopathic dilated cardiomyopathy and six unmatched organ donors with normal cardiac function. At cAMP concentrations less than or equal to 1.0 microM, sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was functionally homogeneous. cAMP phosphodiesterase activity was inhibited competitively by cGMP (Ki = 0.031 +/- 0.008 microM) and the cilostamide derivative OPC 3911 (Ki = 0.018 +/- 0.004 microM), but was essentially insensitive to rolipram. Vmax and Km were 781.7 +/- 109.2 nmol/mg per min and 0.188 +/- 0.031 microM, respectively, in microsomes prepared from nonfailing hearts and 793.9 +/- 68.9 nmol/mg per min and 0.150 +/- 0.027 microM in microsomes prepared from failing hearts. Microsomes prepared from nonfailing and failing hearts did not differ with respect to either the ratio of cAMP phosphodiesterase activity to ATP-dependent Ca2+ accumulation activity or the sensitivity of cAMP phosphodiesterase activity to inhibition by OPC 3911. These data suggest that the diminished inotropic efficacy of phosphodiesterase inhibitors in failing human hearts does not result from changes in the level, kinetic properties, or pharmacologic sensitivity of sarcoplasmic reticulum-associated cAMP phosphodiesterase activity.

Definitive irradiation and chemotherapy for radiosensitization in management of anal carcinoma: interim report on Radiation Therapy Oncology Group study no. 8314.

Following documented evidence of the synergism of 5-fluorouracil (5-FU) and radiation therapy and an additive effect with mitomycin and irradiation, pilot studies have demonstrated the potential for definitive radiation therapy in the management of squamous cell and basaloid carcinomas of the anal canal, allowing sphincter preservation. Our study explored the long-term effectiveness of combined therapy at this disease site and examined the feasibility of a Radiation Therapy Oncology Group study involving concomitant radiation therapy and chemotherapy. Between 1983 and 1987, 79 assessable patients with any primary tumor stage of anal canal carcinoma were treated by external-beam irradiation combined with mitomycin given by bolus iv injection and 5-FU given by continuous infusion. Radiation was delivered to the perineum and pelvis to a total dose of 4,080 cGy in 4.5-5 weeks. The inguinal nodal areas received 4,080 cGy, calculated at a 3-cm depth in the center of the nodal area. A 96-hour infusion of 5-FU was started on days 2 and 28 of the irradiation at a dose of 1,000 mg/m2 over 24 hours, and a bolus injection of mitomycin was administered on day 2 at a dose of 10 mg/m2. The overall survival rates are 97% at 1 year and 73% at 3 years. Patients with lesions less than 3 cm in diameter and those treated strictly according to the protocol did significantly better than those with larger lesions and those whose treatment did not comply with the protocol. The interim outcome of the study demonstrates that this combined therapy is effective for patients with anal cancer and allows preservation of the sphincter and of sexual function.

Smooth muscle cell sarcolemma. Purification and properties of plasma membranes from the rat uterus.

A membrane fraction with sarcolemmal properties was purified from the smooth muscle layers (myometrium) of rat uterus by successive differential and equilibrium centrifugation in sucrose. The putative sarcolemmal fraction was identified by iodination with [125I]iodosulfanilic acid, had an equilibrium density of 1.15, and was enriched in enzyme activities usually associated with the plasma membrane including 5'-nucleotidase (EC 3.1.3.5) and (Na+ + K+) ATPase (EC 3.6.1.3). These membranes were free of mitochondrial or nuclear membrane contamination, suggesting the relative enrichment of sarcolemmal membranes in the fraction. Proteins of the membranes were heterogeneous with respect to molecular weight, but only a few were labelled when intact muscle was radioiodinated. Uniform resistance of sarcolemmal proteins to trypsin digestion and salt extraction suggested many are tightly bound or intrinsic membrane proteins and was a further indication of the homogeneity of membranes in this fraction.

Hormonal control of uterine contraction. Characterization od cyclic AMP-dependent membrane properties in the myometrium.

A mitochondria-free membrane fraction prepared from rat myometrium accumulated 45Ca2+ in the presence of oxalic acid and ATP. The rate of transport of Ca2+ into the membranous vesicles was increased by greater than 50% in the presence of 3',5'-cyclic AMP, but not by 2',3'-cyclic AMP or 5'AMP. Membrane ATPase activity was stimulated by Mg2+; slight additional stimulation was obtained in the presence of Na+ and K+ but not in the presence of Ca+2. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca2+-transport suggest it has little to do with Ca2+ accumulation by the membranes. Cyclic AMP-induced increase in Ca2+-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10(-4)M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca2+-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility.

Concurrent Measurements of Oxygen and Carbon Dioxide Exchange during Lightflecks in Maize (Zea mays L.).

Leaves of maize (Zea mays L.) were enclosed in a temperature-controlled cuvette under 35 Pa (350 [mu]bars) CO2 and 0.2 kPa (0.2%)O2 and exposed to short periods (1-30 s) of illumination (light-flecks). The rate and total amount of CO2 assimilated and O2 evolved were measured. The O2 evolution rate was taken as an indicator of the rate of photosynthetic noncyclic electron transport (NCET). In this C4 species, the response of electron transport during the lightflecks qualitatively mimicked that of C3 species previously tested, whereas the response of CO2 assimilation differed. Under short-duration lightflecks at high photon flux density (PFD), the mean rate of O2 evolution was greater than the steady-state rate of O2 evolution under the same PFD due to a burst of O2 evolution at the beginning of the lightfleck. This O2 burst was taken as indicating a high level of NCET involved in the buildup of assimilatory charge via ATP, NADPH, and reduced or phosphorylated metabolites. However, as lightfleck duration decreased, the amount of CO2 assimilated per unit time of the lightfleck (the mean rate of CO2 assimilation) decreased. There was also a burst of CO2 from the leaf at the beginning of low-PFD lightflecks that further reduced the assimilation during these lightflecks. The results are discussed in terms of the buildup of assimilatory charge through the synthesis of high-energy metabolites specific to C4 metabolism. It is speculated that the inefficiency of carbon uptake during brief light transients in the C4 species, relative to C3 species, is due to the futile synthesis of C4 cycle intermediates.

Regulation of Photosynthetic Induction State in High- and Low-Light-Grown Soybean and Alocasia macrorrhiza (L.) G. Don.

Alocasia (Alocasia macrorrhiza [L.] G. Don) and soybean (Glycine max [L.]) were grown under high or low photon flux density (PFD) conditions to achieve a range of photosynthetic capacities and light-adaptation modes. The CO2 assimilation rate and in vivo linear electron transport rate (Jf) were determined over a range of PFDs and under saturating 1-s-duration lightflecks applied at a range of frequencies. At the same mean PFD, the assimilation rate and the Jf were lower under the lightfleck regimes than under constant light. The activation state of two, key enzymes of the photosynthetic carbon reduction cycle pathway, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and fructose-1,6-bisphosphatase, and the photosynthetic induction states (ISs) were also found to be lower under flashing as compared to continuous PFD. Under all conditions, the IS measured 120 s after an increase in PFD to constant and saturating values was highly correlated with the Rubisco activation state and stomatal conductances established in the light regime before the increase. Both the fructose-1,6-bisphosphatase and Rubisco activities established in a particular light regime were highly correlated with the mean Jf in that regime. The relationships between enzyme activation state and Jf and between IS and enzyme activation state were similar in soybean and Alocasia and were not affected either by growth-light regime, and hence photosynthetic capacity, or by flashing versus constant PFD. The common relationship between the linear Jf and the activation state of key enzymes suggests that electron transport may be the determinant of the signal regulating IS, at least to the extent that the IS is controlled by the activation state of key stromal enzymes.

Distribution binding sites for the progesterone receptor within chick oviduct chromatin.

Chromatin from the oviducts of estrogen-treated chicks was sheared and fractionated on sucrose gradients. This resulted in the production of several chromatin fractions which differ in their sedimentation properties, protein composition, the number of acceptor sites for the progesterone-receptor complex, and the ability to serve as a template for RNA synthesis. The pellet chromatin fraction shows an enhanced ability to bind the progesterone-receptor complex in vitro and in cell-free systems. Kinetic analysis indicates that the majority of the acceptor sites for the progesterone-receptor complex are located in the pellet chromatin fraction which is lowest in template activity. The sites may be important loci for initiating the changes in RNA synthesis following the exposure of target cells to steroid hormones.

Dopaminergic binding and inhibitory effect in the bovine adrenal zona glomerulosa.

Dopaminergic mechanisms may be involved in the regulation of aldosterone secretion in humans and in the rat. Whether these effects are indirect or are exerted directly at the adrenal level has not yet been resolved. We now report the identification of dopaminergic binding sites in the bovine adrenal zone glomerulosa using [3H]spiperone, a butyrophenone with high affinity for D2 dopamine receptors. Specific [3H]spiperone binding (defined as binding displaceable by 10 microns (+)-butaclamol) reached equilibrium within 20 minutes at 22 degrees C, was reversible, and was heat labile (60 degrees C). Binding was of high affinity and saturable with a Kd of 1.8 +/- 0.2 nM and maximal specific binding of 38 +/- 8 fmol/mg (means +/- SEM; n = 18). [3H]Spiperone binding was unaffected by coincubation with angiotensin II, adrenocorticotropic hormone, or KCl. Binding characteristics, including a dissociation constant at the nanomolar range, greater potency of the D2-agonist LY 171555 relative to the D1-agonist SKF 38393 in inhibiting [3H]spiperone binding, and lack of stimulation of cyclic adenosine 3',5'-monophosphate by dopamine (10(-4) M), were consistent with a predominantly D2-receptor. In vitro studies with collagenase-dispersed adrenal zona glomerulosa cells showed that dopamine (10(-4) M) attenuated angiotensin II-stimulated aldosterone secretion. These observations are consistent with a direct inhibitory effect of dopamine on aldosterone secretion in the adrenal zona glomerulosa.

The relationship of birth size to the rate of growth in infancy and childhood.

To assess the relationship between birth weight or birth length and the corresponding velocities of growth in infancy and childhood, 3995 subjects, followed longitudinally, were studied. Pearson correlations indicated no significant relationship between birth size and velocity in infancy, between birth size and velocity in childhood, or between velocities of growth in infancy and childhood. The only correlation that approached any significance suggested a tendency for subjects who gained weight faster in infancy to gain weight faster in childhood.

Effect of test meals of varying dietary fiber content on plasma insulin and glucose response.

To assess the effect of dietary fiber on glucose tolerance four different meals of varying fiber content but identical protein fat and carbohydrate content were fed to eight healthy men aged 22 to 45. Each meal provided 75 g of carbohydrate as liquid glucose formula, as brown rice, pinto beans, or All Bran. The mean plasma glucose and insulin responses were highest following the formula, and least for All Bran and pinto beans. Rice produced nearly as great a rise in insulin and glucose as did the formula. The rank of each meal by content of neutral detergent fiber was nearly the inverse of the rank by magnitude of the insulin response evoked, fiber content being greatest in All Bran (18 g) and pinto beans (16.2 g), low in rice (2.8 g) and absent from the formula. It was concluded that dietary fiber dampened the insulin response to a high carbohydrate meal.

Patterns of care studies: dose-response observations for local control of adenocarcinoma of the prostate.

Five hundred seventy-four patients with prostate cancer treated by external beam radiation therapy in the United States in 1973 to 1975 have been analyzed comparing radiation dose with in-field recurrence. Dose-response effects are observed for all cases (p = less than .05) and T-2 and T-3 tumors, but not for T-0, T-1 and T-4 tumors. For doses calculated at the center of the prostate, these observations suggest optimal control is obtained at no more than 6000 rad for T-0 and T-1 tumors; 6000-6500 rad for T-2 tumors; 6500-7000 rad for T-3 tumors; and that greater than 7000 rad is required only for T-4 tumors. The paraprostatic dose calculated at a point 4 cm lateral to the center of the prostate also shows a correlation of dose with infield failure for all cases (p = .01). Observations in individual T states suggest optimal control is obtained at no more than 6000 rad for T-0, T-1 and T-2 tumors, 6500-6999 rad for T-3 and greater than or equal to 7000 rad for T-4. These data suggest that for T-2 and T-3 cancers, extension in the periprostatic region must be treated. A comparison of central dose vs. stage indicates institutional policy rather than cancer volume determines the radiation dose used in treating prostate cancer. A change in institutional policies to treat with optimal doses as indicated by this study would result in an overall increase in local control and a decrease in complications.

Improvements observed in care and outcome in carcinoma of the larynx.

The 1978 Patterns of Care Studies (PCS) survey of carcinoma of the larynx reviewed the process (pre-treatment evaluation and treatment) and outcome of 521 patients. When compared to results from the 1973 PCS survey, several important changes have been observed. The use of surgery in conjunction with radiation therapy (RT) increased in Stage III cases from less than 30% to greater than 60% and in Stage IV cases from 48% to greater than 70%. This change in therapy was associated with a decline in locoregional failure in this patient group. Among Stage I and II supra- and subglottic carcinomas, an improvement in 3-year local tumor control (Stage I: 78 to 100% and Stage II: 54 to 74%) and overall freedom from recurrence (Stage I: 78 to 100% and Stage II: 45 to 73%) was seen over this 5-year period with no identifiable change in process for this subgroup. Also noted was an improvement in the freedom from recurrence rate for Stage III and IV patients receiving treatment at facilities with low process scores. The 1978 PCS survey confirmed the presence of superior patient outcome in several subgroups and the relationship of this improvement to patient process.

A ten year follow-up of 682 patients treated for prostate cancer with radiation therapy in the United States.

This report extends the follow-up of patients studied in the Patterns of Care Survey of Prostate Cancer treated in the United States between 1973 and 1975 from a maximum of 5 years to a maximum of 10 years. Survival for 60 Stage A patients was the same as expected for their age distribution (83% at 5 years and 62% at 10 years). Survival for 312 Stage B patients was 73% at 5 years and 46% at 10 years and for 296 Stage C patients was 58% at 5 years and 38% at 10 years. Infield recurrence was determined by clinical means, at 5 years 97% of Stage A patients, 86% of Stage B patients, and 74% of Stage C patients were free of local recurrence. At 10 years 97% of Stage A patients, 74% of Stage B patients, and 69% of Stage C patients remained free of local recurrence. Patients with Stage B and C cancer who developed their first failure infield show a long-term survivorship after recurrence of 40% and 20% respectively. This is in contrast to Stage B and C patients who develop a first recurrence at a metastatic site where the rate of progress to death was slower in Stage B patients than for those with Stage C disease (mean survival 32 months versus 19 months), but eventually all are dead by 7 years after recurrence. Complications were infrequent, actuarial analysis shows 93% of patients free of serious complications at 5 years and 89% free at 10 years. There were 14 patients (2%) whose complications required surgical correction and 2 of the 682 patients died of complications.

The outcome of treatment of 313 patients with T-1 (UICC) prostate cancer treated with external beam irradiation.

Three hundred and thirteen patients with UICC T-1 N-0 M-0 prostate cancer were treated with external beam irradiation in 1973 and 1974 or in 1978, and their outcome determined 3-10 years after treatment. Survival over the first 5 years was comparable to that expected for a group of age matched normal males (77% vs. 81%), but during the second 5-year interval, there was a decrease in survival below that expected (51% vs 62%), a reflection of death in patients who developed metastasis as a first recurrence (18%). Overall, 72% of patients were free of any recurrence at 5 years and, 88% free of infield recurrence. The development of metastatic recurrence was significantly related to grade; at 5 years 87% of grade I, 79% of grade II and 69% of grade III patients were free of metastasis. There was a trend for increased local recurrence with increasing grade, but it was not statistically significant. There was a dose/response relation for complications, and radiation doses above 6500 cGy are associated with an increase in complication from 6% to 11% (p = .09). Complications requiring hospitalization for evaluation or management occurred in 30 (10%) of 313 patients. There were no deaths from complications and less than 2% of patients required surgical correction of complications. External beam radiation offers the patient with early prostate cancer a favorable opportunity for cure without the morbidity of impotence, incontinence, and occasional death experienced following LND and radical prostatectomy. Lymph node dissection does not seem necessary for most patients with T-1 prostate cancer as the positive yield in those with Grades I and II cancers is less than the complications of the procedure, and extensive involvement can be detected by non-invasive means.

Analysis of independent variables affecting survival after recurrence of prostate cancer.

Two hundred and sixty-six patients developed recurrence after external beam radiation therapy for Stage B or C prostate cancer. The median survival from the time of recurrence was 30 months, the 5-year actuarial survival was 22% and the 8-year actuarial survival was 13%. The original stage influences survival after recurrence. Median survival is 35 months for Stage B and 27 months for Stage C. Five-year actuarial survivals are 31% for Stage B and 16% for Stage C. The site of recurrence (infield or metastatic) influences survival after recurrence. Median survival is 33 months for infield and 25 months for metastatic. Five-year actuarial survival is 30% for infield and 17% for metastatic. The time of recurrence influences survival after recurrence, comparing survival in patients recurring in the first year after treatment, to patients recurring in the second or later years after treatment. Median survival after recurrence is 14 months for those who recur in the first year, 32 months for those who recur later. Histologic grade influences survival after recurrence. Median survivals are 49 months for well differentiated, 31 months for moderately differentiated, and 23 months for poorly differentiated. Cox regression analysis indicates that original stage, site of recurrence (local vs. metastatic), time of recurrence after therapy, and histologic grade are all independent variables influencing survival after recurrence (p = less than .01). This study supports the aggressive treatment of prostate cancer to prevent recurrence and provides a means of estimating prognosis for patients who suffer a recurrence of their prostate cancer after treatment.

Comparison of 1 vs 2 or more intracavitary brachytherapy applications in the management of carcinoma of the cervix, with irradiation alone.

Four hundred ninety-eight patients treated in the USA in 1978 for cancer of the cervix by external beam and intracavitary irradiation have been studied to determine the effect of using a single intracavitary (IC) application versus more than one. Most of the patients (46%) underwent intracavitary (IC) treatment after external radiation therapy (RT), 40% during external RT, and 14% prior to external RT. Five-year actuarial survival for all stages was 66% for greater than or equal to 2 applications versus 59% for 1 application, p less than .01. When survival was analyzed by stage, there was a trend toward improved survival with greater than or equal to 2 applications in Stage II (p .10). The rate of infield recurrence for all stages was higher in the 1 IC group (30% vs 17%, p less than .01). These were more frequent in the 1 IC group (53% vs 34%, p less than .05), in Stage III patients, but no significant difference was noted in the other stages. Distant metastases were more frequent in the 1 IC group (19% vs 11%, p .01). The incidence of major complications was the same in both groups. An analysis of patient characteristics and other variables that would explain the difference in results revealed that older patients (p .03), worse Karnofsky (p .056), and more parametrial involvement (p less than .01) accrued more in the 1 IC group. Higher paracentral point doses were administered when greater than or equal to 2 IC applications were used (p less than 0.01); this group also received higher bladder and rectal doses and lower whole pelvic doses. The use of two or more intracavitary insertions affords an opportunity to administer higher paracentral point doses, which appear to be related to higher pelvic tumor control, lower rate of distant metastasis, and better survival.

Outcome for lymph node dissection negative T-1b, T-2 (A-2,B) prostate cancer treated with external beam radiation therapy in RTOG 77-06.

One hundred four patients with stage T-1b, T-2 N-O M-O prostate cancer were treated with external beam irradiation as part of RTOG 77-06. Lymph nodes were negative by lymph node dissection in 16 patients with T-1b and 88 patients with T-2 cancers. Survival exceeds age matched expected survival for the 10 years of observation (63% vs 59% at 10 years). Patterns of failure at 10 years show 87% of patients were free of isolated local recurrence, 79% free of metastatic failure, 67% free of any failure, and cause specific survival shows 86% free of cancer death at 10 years. The outcome of this group is equal or superior to reports of radical prostatectomy in similar stage patients.

Correlation of pre-treatment transurethral resection and prognosis in patients with stage C carcinoma of the prostate treated with definitive radiotherapy--RTOG experience.

Four hundred and ninety-four patients with clinical Stage C carcinoma of the prostate, who were entered onto a phase III RTOG study, have been analyzed as to the potential effect of the pre-treatment transurethral resection (TUR) of the tumor. Treatment consisted of definitive irradiation to the prostate (6500-7000 cGy) and regional lymphatics (4500-5000 cGy). A total of 202 patients underwent pre-treatment TUR. This population was compared with the remaining 292 patients as to the rate of locoregional failure, incidence of distant metastases, disease-free survival, and survival. The TUR population fared significantly worse for all four end-points. To account for uneven distribution of recognized prognostic factors the results were then adjusted using stratified Mantel-Haenszel tests. The stratification process resulted in a reduced level of significance in the differences between the two populations. However, a trend toward a higher incidence of distant metastases could be observed within most strata. The trend was most pronounced in subpopulations characterized by Gleason score 6-7 and normal serum acid phosphatase (SAP). For the population characterized by Gleason score 6-10 and normal SAP, the differences in the incidence of distant metastases retained statistical significance. Whether these findings are secondary to tumor dissemination during TUR or are due to incompletely identified selection biases remains to be demonstrated in future (prospective) studies.

Definitive radiotherapy in resectable (stage A2 and B) carcinoma of the prostate--results of a nationwide overview.

To evaluate the efficacy of definitive radiotherapy in a population of patients with carcinoma of the prostate who satisfy the customary selection criteria for radical prostatectomy, a nation-wide search was conducted. The assessed population consists of patients with clinical Stage A2 and B carcinoma of the prostate, negative staging lymphadenectomy, negative bone scan, and normal serum acid phosphatase. The search included patients from Stanford University, Washington University in St. Louis, those participating in the Radiation Therapy Oncology Group and a broad range of radiotherapy practices surveyed by the PCS (Patterns of Care Study). A total of 209 patients satisfying the selection criteria received definitive radiotherapy during the surveyed period. The end-point of analysis was the time to progression (distant metastases). The results of the analysis indicate a very low (less than 10%) probability of progression within the first 5 years after completion of treatment. Contrary to the recent report from the VA Uro-Oncology Group the study demonstrates a comparable outcome in radiotherapeutically and surgically treated patients.