False-positive pregnancy test associated with gonadoblastoma.

Gonadoblastomas are known to be hormonally active tumors that occur in streak or dysgenetic gonads of patients with intersex abnormalities. Several reports document their ability to produce beta-human chorionic gonadotropin (HCG), but none have documented an elevated peripheral serum beta-HCG. We report on the case of a patient with pure gonadal dysgenesis with XY karyotype who was found to have an elevated peripheral serum beta-HCG after a positive pregnancy test. Knowledge of gonadoblastoma's potential to elevate serum beta-HCG levels may prevent unnecessary searches for other causes.

Radical prostatectomy in patients with indwelling inflatable penile prosthesis.

Patients with impotence who have undergone placement of an inflatable penile prosthesis (IPP) and then subsequently are diagnosed with carcinoma of the prostate (CaP) present a surgical dilemma. We performed radical retropubic prostatectomy on 3 patients with clinically localized CaP and an indwelling IPP. At laparotomy all 3 patients had the IPP reservoir relocated to facilitate dissection. In each case the reservoir was relocated to the left hypogastrium within the extraperitoneal space without disrupting the vacuum tubing system. There were no complications related to IPP, no IPP was injured, and each IPP was reactivated successfully 6 weeks after surgery.

Transurethral biopsy of the prostate for persistently elevated or increasing prostate specific antigen following multiple negative transrectal biopsies.

PURPOSE: Treatment of the patient with persistently elevated prostate specific antigen (PSA) levels after pathologically negative transrectal or manually directed prostate needle biopsy is unclear. We retrospectively evaluated the use of transurethral biopsy of the prostate as an adjunctive study for the diagnosis of prostate cancer in these patients. MATERIALS AND METHODS: From January 1993 through February 1996, 71 patients underwent transurethral biopsy in conjunction with repeat prostatic needle biopsy for a persistently elevated PSA (greater than 4 ng./ml.) after previously negative needle biopsy. All patients had at least 1 previous ultrasound guided sextant prostatic needle biopsy (mean 1.85, range 1 to 7) with or without manually directed biopsies. Following negative prostatic needle biopsy these patients subsequently underwent a minimum of a 4-quadrant transurethral sampling of the prostatic fossa followed by repeat sextant prostatic needle biopsy. A subset of patients underwent sampling of the anterior prostatic tissue or transition zone using transrectal ultrasound guided prostatic needle biopsy at transurethral biopsy. RESULTS: Of the 71 patients with elevated PSA (mean 16.2 ng./ml., range 4.2 to 171) 17 (24%) had prostate cancer on the repeat prostatic needle biopsy. Both patients who had prostate cancer on the transurethral biopsy specimens also had prostate cancer on the repeat prostatic needle biopsy specimens. A total of 68 patients had benign prostatic tissue and 1 had high grade prostatic intraepithelial neoplasia on transurethral biopsy specimens. Of 19 patients with high grade prostatic intraepithelial neoplasia on the initial prostatic needle biopsy, transurethral biopsy specimens revealed no prostate cancer or prostatic intraepithelial neoplasia. Repeat prostatic needle biopsy in these patients with high grade prostatic intraepithelial neoplasia revealed prostate cancer in 6 and high grade prostatic intraepithelial neoplasia in 4. CONCLUSIONS: In patients with persistently elevated or increasing serum PSA after a previously negative prostatic needle biopsy, transurethral biopsy is not a useful adjunct in diagnosing prostate cancer. In this high risk group of patients transurethral biopsy adds little or no diagnostic value to prostatic needle biopsy even in those with high grade prostatic intraepithelial neoplasia.

Lead induced rise in intracellular free calcium is mediated through activation of protein kinase C in osteoblastic bone cells.

Lead characteristically perturbs processes linked to the calcium messenger system. This study was undertaken to determine the role of PKC in the Pb2+ induced rise of [Ca2+]i. [Ca2+]i was measured using the divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy) ethane N, N,N',N'-tetraacetic acid (5F-BAPTA) and 19F-NMR in the osteoblast cell line, ROS 17/2.8. Treatment of cells with Pb2+ at 1 and 5 microM produced a rise in [Ca2+]i from a basal level of 125 nM to 170 nM and 230 nM, respectively, while treatment with phorbol 12-myristate 13-acetate (PMA) (10 microM), an activator of PKC, produced a rise in [Ca2+]i to 210 nM. Pretreatment with calphostin C, a potent and highly selective inhibitor of PKC activation failed to produce a change in basal [Ca2+]i and prevented any rise in [Ca2+]i in response to Pb2+. To determine whether Pb2+ acts directly on PKC, we measured the Pb2(+)-dependent activation of phosphatidylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous TCA precipitable proteins in the 100,000 X g supernatant from homogenized ROS 17/2.8 cells. The free concentrations of Pb2+ and Ca2+ were set using 5F-BAPTA; and [Ca2+] and [Pb2+] in the PKC reaction mixtures were confirmed by 19F-NMR. We found that Pb2+ activates PKC in the range of 10(-11)-10(-7) M, with an activation constant of 1.1 X 10(-10) M, whereas Ca2+ activates PKC in the range from 10(-8) to 10(-3) M, with an activation constant of 3.6 X 10(-7) M. These data suggest that Pb2+ activates PKC in ROS 17/2.8 cells and that Pb2+ activation of PKC mediates the documented rise in [Ca2+]i and, perhaps, other toxic effects of Pb2+.

Sequential penile ultrasound monitoring of patients treated with chronic intracavernous prostaglandin E1.

OBJECTIVES: Clinical outcome studies of prostaglandin E1 (PGE1) have shown a markedly decreased rate of palpable fibrosis and plaque formation. In this prospective study we investigate the potential of this agent to produce subclinical fibrotic changes. METHODS: Real-time high-resolution ultrasound scanning of the corpora was performed using a 7.5 to 10 MHz linear array transducer in 80 men on initiation of treatment with self-administered PGE1 and at quarterly intervals during the course of following (3 to 28 months). The dorsal portion of the penile shaft was scanned in the transverse and sagittal planes from base to glans for a side by side comparison of the cavernosal tissue, evaluating local abnormalities of tissue echogenicity. RESULTS: Palpable lesions were not detected in any men on quarterly follow-up examination. Thirteen (16.5%) men developed new echogenic foci not present on pretreatment scanning at the following locations: proximal corpus cavernosum, subcutaneous tissues, and corpus spongiosum. These changes were observed both as single and multiple lesions ranging in size from 1 to 10 mm. The presence of these findings was independent of the etiology of impotence, dose frequency, and duration of intracavernous therapy. CONCLUSIONS: The significance of these subclinical changes is unknown but their low incidence should be recognized when considering long-term intracavernous therapy.

Testicular cancer in blacks. A multicenter experience.

BACKGROUND: The rarity of testis tumor in black patients has made the study of a large series difficult. Much of the epidemiologic and clinical information regarding this neoplasm in this population is in dispute, including data on incidence, prognosis, histologic distribution, age and stage at presentation, and side distribution. METHODS: A retrospective review of 66 blacks with testicular tumors from seven military medical centers was performed. RESULTS: Similar results were found for blacks with testis tumor to those of the general testis cancer population regarding prognosis, side distribution, and age of onset for nonseminoma and interstitial tumors. There is a slight increase in the expected number of interstitial tumors in blacks, but the distribution between seminoma and nonseminoma is similar to the general population. The mean age of presentation for seminoma in blacks was younger than that of the general testis cancer population. For testis tumor treated at the same institution, there was an increased delay of diagnosis in blacks compared with whites. The number of new cases of testicular cancer between 1979 and 1991 at one major center was increased for whites but not for blacks. The availability of cisplatin-based combination chemotherapy has resulted in an improved prognosis for blacks, as has already been demonstrated for white populations. CONCLUSIONS: Testis tumor in blacks behaves similarly to testis tumor in the general population except that in blacks there are more interstitial tumors and the mean age of presentation for seminoma is younger. Further, there is an increased delay in diagnosis for blacks compared with whites, but the incidence of this tumor in this population does not appear to be increasing. Cisplatin-based chemotherapy has significantly improved survival in this population.

Lead activation of protein kinase C from rat brain. Determination of free calcium, lead, and zinc by 19F NMR.

Lead (Pb2+) has been reported to activate calcium/phospholipid-dependent protein kinase C (PKC) at subnanomolar concentrations (Markovac, J., and Goldstein, G. W. (1988) Nature 334, 732-734); however, others have failed to find any Pb(2+)-induced activation of PKC (Murakami, K., Feng, G., and Chen, S. G. (1993) J. Pharmacol. Exp. Ther. 264, 757-761). In neither of these studies was the actual free Pb2+ or Ca2+ concentration measured. In this study, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA) was used to buffer Pb2+ and Ca2+ concentrations in the PKC reaction mixture. The specific free ion concentrations of Pb2+ and Ca2+, as well as Zn2+ and other divalent cations contained in the PKC reaction mixtures, were determined by 19F NMR spectroscopy. Using this approach to set and confirm the free Pb2+ and Ca2+ concentrations, we measured the Pb(2+)-dependent and the Ca(2+)-dependent activation of phosphotydylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous acid precipitable proteins in the 100,000 x g supernatant from homogenized rat brain cortex. We found that free Pb2+ activates PKC in the range from 10(-11) to 10(-8) M, Kact = 5.5 x 10(-11) M, while Ca2+ activates PKC in the range from 10(-8) to 10(-5) M, Kact = 2.56 x 10(-7) M. These findings clearly resolve the activation of PKC by subnanomolar concentrations of free Pb2+ from activation induced by Ca2+ or other divalent cations. Furthermore, it documents the utility of 5F-BAPTA as buffer and indicator when resolving the contributions of multiple divalent cations in biochemical processes.

31P-NMR study of transient ischemia in rat hippocampal slices in vitro.

Intracellular high energy phosphates (HEP) were monitored in rat hippocampal slices in vitro by 31P-NMR during continuous superfusion, no flow and reperfusion in order to model the changes which occur during cerebral ischemia and reperfusion in vivo. With continuous superfusion, stable intracellular HEP resonance signals were observed for over 4 h. When superfusion was stopped, there were rapid decreases in pH and phosphocreatine levels followed by slower loss of ATP. These changes are similar to those observed during cerebral ischemia in vivo by 31P-NMR. Upon reperfusion, the pH returned to normal, but the extent of HEP recovery depended on the length of time superfusion was halted. Following a 10 min ischemic period HEP levels returned to greater than 90% of preischemic values, while following a 16 min ischemic period there was only 60% recovery. Superfusion with low calcium, high magnesium medium significantly improved the recovery of HEP following 16 min of ischemia to 80% of preischemic levels. These data support the hypothesis that calcium influx during and following ischemia can disrupt energy metabolism in the hippocampus, and that magnesium can have a protective action on cellular energy status, perhaps by further blocking calcium influx.

Lead inhibits 1,25-dihydroxyvitamin D-3 regulation of calcium metabolism in osteoblastic osteosarcoma cells (ROS 17/2.8).

We have determined the dose-response of 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) on the intracellular free calcium-ion concentration ([Ca2+]i) in the osteoblastic osteosarcoma cells, ROS 17/2.8, using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA). The dose-response demonstrated an inverted U-shaped relationship with maximal elevation of [Ca2+]i at doses of 1 to 10 nM 1,25-(OH)2D3. At 10 nM, 1,25-(OH)2D3 elevated the [Ca2+]i from a control level of 118 +/- 4 nM to a peak value of 237 +/- 8 nM within 40 min. 1,25-(OH)2D3 also increased the initial rate of Ca2+ influx into ROS 17/2.8 cells, measured by 45Ca uptake, with a dose-response relationship which paralleled its effect on [Ca2+]i. Treatment of ROS 17/2.8 cells with Pb2+ at 1 and 5 microM significantly increased [Ca2+]i but significantly reduced the 1,25-(OH)2D3-induced elevation of [Ca2+]i. Simultaneous treatment of naive cells with 1,25-(OH)2D3 and Pb2+ produce little reduction of 1,25-(OH)2D3-induced 45Ca uptake while 40 min treatment with Pb2+ before addition of 1,25-(OH)2D3 significantly reduced the 1,25-(OH)2D3-induced increase in 45Ca influx. These findings suggest that Pb2+ acts by inhibiting 1,25-(OH)2D3-activation of Ca2+ channels and interferes with 1,25-(OH)2D3 regulation of Ca2+ metabolism in osteoblastic bone cells.

Effect of lead on parathyroid hormone-induced responses in rat osteoblastic osteosarcoma cells (ROS 17/2.8) using 19F-NMR.

Using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid, we have recently demonstrated that Pb2+ treatment elevates the intracellular free calcium ion concentration ([Ca2+]i) of rat osteoblastic osteosarcoma cells (ROS 17/2.8) (Proc. Natl. Acad. Sci. USA (1989) 86, 5133-5135). In this study, we have examined the effects of Pb2+ on the basal and parathyroid hormone (PTH)-stimulated levels of [Ca2+]i and cAMP in cultured ROS 17/2.8 cells. PTH treatment (400 ng/ml) stimulated a 150% elevation in [Ca2+]i from a control level of 105 +/- 25 nM to a concentration of 260 +/- 24 nM. Treatment of ROS 17/2.8 cells with Pb2+ (5 microM) alone produced a 50% elevation in the [Ca2+]i to 155 +/- 23 nM. Pb2+ treatment diminished subsequent elevation in [Ca2+]i in response to PTH administration thereby limiting the peak increase in [Ca2+]i to only 25% or 193 +/- 22 nM. In contrast to the dampening effect of Pb2+ on the peak rise in [Ca2+]i produced by PTH, Pb2+ (1 to 25 microM) had no effect on PTH-induced increments in intracellular cAMP levels. Hence, Pb2+ dissociated the PTH stimulation of adenylate cyclase from PTH effects on [Ca2+]i and shifted the regulation of [Ca2+]i beyond the control of PTH modulation. These observations further extend the hypothesis that an early toxic effect of Pb2+ at the cellular level is perturbation of [Ca2+]i homeostasis.

Development of 19F NMR for measurement of [Ca2+]i and [Pb2+]i in cultured osteoblastic bone cells.

Lead (Pb) has been shown to perturb cellular calcium (Ca) homeostasis, altering sizes and flux rates of cellular pools of exchangeable Ca and impairing Ca-mediated cell processes. To date, however, a direct effect of Pb on intracellular-free Ca2+ has not yet been demonstrated. Heavy metals bind to the commonly used fluorescent Ca ion indicators with greater affinity than does Ca and thereby interfere with the expected Ca-dependent fluorescence. In this study, the fluorinated Ca ion indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA), and 19F NMR were used to measure the free intracellular Ca ion concentration ([Ca2+]i) in the rat osteoblastic bone cell line, ROS 17/2.8. Both Pb and Ca bind to 5F-BAPTA with high affinity, but the Pb-5F-BAPTA comple produces a 19F NMR signal at a chemical shift distinct from 5F-BAPTA and the Ca-5F-BAPTA complex. The apparent dissociation constants for Pb-5F-BAPTA and Ca-5F-BAPTA are 2 X 10(-10) M and 5 X 10(-7) M, respectively, at 30 degrees C, pH 7.1, and Mg2+ (0.5 mM). Thus, this methodology allows for the simultaneous identification and quantification of free Pb and free Ca ion concentrations. Determinations of [Ca2+]i were based on 19F NMR measurements of 5F-BAPTA-loaded ROS 17/2.8 osteoblastic bone cells that were attached to collagen-coated microcarrier beads. Cells were continuously superfused with freshly oxygenated medium at 30 degrees C. Under these conditions, the [Ca2+]i of ROS 17/2.8 cells was observed to be 128 +/- 14 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of lead on intracellular free calcium ion concentration in a presynaptic neuronal model: 19F-NMR study of NG108-15 cells.

The intracellular free calcium ion concentration ([Ca2+]i) of the neuroblastoma x glioma hybrid cell line, NG108-15, was measured using the 19F-nuclear magnetic resonance divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetra-acetic acid (5F-BAPTA). The basal [Ca2+]i was measured to be 106 +/- 14 nM. Treatment with 5 microM lead (Pb) for 2 h produced a 2-fold increase in [Ca2+]i to 200 +/- 24 nM and a measurable intracellular free Pb2+ concentration ([Pb2+]i) of 30 +/- 10 pM. Intracellular free Zn2+ concentrations ([Zn2+]i) were also observed in the presence of Pb. This represents the first direct demonstration that Pb elevates the [Ca2+]i in neurons, thus providing evidence for a role of [Ca2+]i in mediating the neurotoxicity of Pb.

Lead increases free Ca2+ concentration in cultured osteoblastic bone cells: simultaneous detection of intracellular free Pb2+ by 19F NMR.

Lead (Pb) has been shown to perturb Ca-mediated cellular processes. However, to date, a direct effect of Pb on intracellular free Ca2+ concentration ([Ca2+]i) has not been demonstrated. 19F NMR in combination with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA) was used to simultaneously measure [Ca2+]i and intracellular free Pb2+ concentration ([Pb2+]i) in the rat osteoblastic bone cell line ROS 17/2.8. The basal concentration of [Ca2+]i in ROS 17/2.8 cells was measured to be 128 +/- 24 nM. Treatment with Pb2+ at 5 and 25 microM produced sustained 50% and 120% increases in [Ca2+]i, respectively, over a time course of 5 hr. At a medium Pb2+ concentration of 25 microM, the entry of Pb2+ into ROS 17/2.8 cells yielded measurable [Pb2+]i in cultured cells. Collectively, these findings advance the hypothesis that Pb toxicity is mediated, in part, through perturbations in [Ca2+]i.

Hippocampal long-term potentiation increases mitochondrial calcium pump activity in rat.

45Ca2+ pump activity was measured in hippocampal homogenates after the induction of long-term potentiation (LTP) of perforant path-dentate gyrus synapses in vivo. The mitochondrial poison, sodium azide, was used to separate mitochondrial from non-mitochondrial 45Ca2+ uptake. High-frequency stimulation of the perforant path input produced a selectively increased mitochondrial uptake of calcium. This increased pump activity may permit granule cells to more effectively buffer higher intracellular calcium levels associated with LTP.

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux.

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.

Alcohol-dependent liver cell necrosis in vitro: a new model.

In alcoholic liver injury, necrosis is involved in the progression from benign fatty liver to alcoholic hepatitis and cirrhosis. However, there is no practical model of alcohol-dependent liver cell necrosis. The calcium-dependent killing of cultured rat hepatocytes by two different membrane-active hepatotoxins, galactosamine and phalloidin, is potentiated by ethyl alcohol. This indicates that some general physical effect of alcohol on cellular membranes renders cells susceptible to otherwise nonlethal injuries. The in vitro model described in this report may thus be used to search for a general mechanism underlying alcohol-related tissue injury.

Galactosamine-induced cell death in primary cultures of rat hepatocytes.

Primary cultures of rat hepatocytes were exposed to 0.5 mM D-galactosamine. After 36 hours, only 10-20% of the original cells were viable, as assessed by trypan blue exclusion. In the absence of galactosamine, there was no loss of viability over this same period. The addition of 3 mM uridine to the culture medium completely prevented the cell death produced by galactosamine. Glucosamine had no effect on the viability of the hepatocytes. The extent of galactosamine-induced cell death was dependent upon the concentration of Ca++ ions in the culture medium. With the only source of Ca++ that added with the fetal calf serum, galactosamine had only a very slight effect on viability. With higher Ca++ than with the fetal calf serum, galactosamine had only a very slight effect on viability. With higher Ca++ concentrations, from 0.9 to 3.6 mM, the viability ranged from 75% to 31% 18 hours after treatment with galactosamine. The addition of 1.4 microM chlorpromazine to culture medium containing 1.8 mM Ca++ decreased the extent of the galactosamine-induced cell death. This protective effect was progressively reduced by raising the Ca++ concentration to 3.6 and 5.4 mM. Chlorpromazine given to intact rats 2 hours after treatment with 400 mg/kg galactosamine prevented the appearance of liver cell necrosis. At the same time, chlorpromazine prevented the increases in liver cell Ca++ content. These results indicate that many of the features of the effect of galactosamine on intact rat liver cells can be reproduced in primary cultures of these same cells. The data also support the hypothesis that a disturbance in intracellular Ca++ homeostasis leading to accumulations of these ions is causally related to the cell death produced by galactosamine.