Androgens regulate the expression of proliferating cell nuclear antigen posttranscriptionally in the human prostate cancer cell line, LNCaP.

Proliferating cell nuclear antigen (PCNA) expression is required for DNA replication. Because androgens are critical for prostate cell proliferation, we investigated the effects of androgen on PCNA expression in the prostatic cancer cell line LNCaP. Flow cytometric analysis was used to measure cellular DNA content with dual labeling of PCNA. Semiconfluent LNCaP cells were grown in serum-free medium containing varying concentrations of the synthetic androgen mibolerone and processed for either fluorescence-activated cell sorting or Western analysis. Supplementation of serum-free medium with androgens resulted in dose-dependent changes in PCNA immunoreactivity, with maximum stimulation (2-fold) being achieved at 48 h with 10(-9)M mibolerone. Non-androgenic steroids did not change PCNA immunoreactivity compared with untreated controls, and the antiandrogen, casodex, inhibited the mibolerone-stimulated increase in PCNA immunoreactivity, suggesting that the androgenic induction of PCNA is mediated through the androgen receptor. The presence of a non-consensus androgen response element in the promoter region of the PCNA gene led us to investigate wether androgen responsiveness of the PCNA gene in LNCaP cells might be mediated at the transcriptional level. No change in steady-state mRNA for PCNA with androgen administration was observed. However, an investigation of the androgenic regulation of PCNA protein stability indicated that androgen treatment increased the half-life of 35S-labeled PCNA protein. In addition, polysome run-off translation assays demonstrated an increase in PCNA protein after a 6-h stimulation of LNCaP cells with 10(-9)M mibolerone. These data suggest that androgen induction of prostate cell proliferation may be mediated, at least in part, through PCNA at the posttranscriptional level.

The release of high-molecular-weight alkaline phosphatase and leucine aminopeptidase into the media of cultured human cells.

Using exclusion from Sepharose 4B as our criterion, we have found a high-molecular-weight form of alkaline phosphatase and of leucine aminopeptidase which are released into the culture media by the FL amnion cell line. A low-molecular-weight form of leucine aminopeptidase is also found to contribute to the total levels of this enzyme in the media. The levels of these enzymes increased during the growth cycle of the culture, paralleling the increase in cell density, suggesting that the two events may be related. This phenomen in culture suggests a possible explanation for the appearance of similar enzyme forms in patient serum and fluids originating from diseased tissue.

Characterization of an early growth response gene, which encodes a zinc finger transcription factor, potentially involved in cell cycle regulation.

Using differential display polymerase chain reaction, early growth response gene alpha (EGR alpha) was first isolated as a 291-base pair 3'-cDNA clone, which was highly expressed in the androgen-independent prostate carcinoma cell lines PC3 and DU145, as compared with the androgen-responsive prostate carcinoma cell line LNCaP. Full length cloning of the EGR alpha coding region revealed that EGR alpha was a new member of an important subfamily of nuclear zinc finger transcription factors (others members e.g. Sp1, EGR-2, and Wilms' tumor gene). Moreover, it was observed that EGR alpha, as with most Sp1 subfamily members, was conserved between mammalian species ranging from human to rabbit. Two hormones important for prostate development and differentiation were found to be potent regulators of EGR alpha mRNA expression. Androgens were observed to induce a down-regulation of EGR alpha mRNA expression (70% in 72 h), while epidermal growth factor induced a rapid transient up-regulation (6-fold in 100 min). The up-regulation was controlled at the transcriptional level and effectively blocked by staurosporine (which suggests the involvement of the protein kinase C pathway). Functional analysis demonstrated that EGR alpha could bind to, and stimulate transcription from, a basic transcription element (BTE) consensus sequence on DNA (BTE is a transcription-modulating sequence in the promoter region of some cytochrome P450 family members). Furthermore, in stage-synchronized prostate cells, EGR alpha mRNA was highly expressed in the early G1 phase of the cell cycle, similar to c-fos mRNA expression. These results indicated that the zinc finger transcription factor EGR alpha seems to play a role in cell cycle regulation.

Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP.

Elevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of interest to determine whether Ca2+ would affect the expression of androgen receptor messenger RNA (mRNA) and protein, thus affecting the ability of androgens to control prostate function. AR-positive human prostate cancer cells, LNCaP, were incubated with either the calcium ionophore A23187 or the intracellular endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. Subsequently, AR mRNA and protein levels were assessed by Northern and Western blot analysis. Both A23187 and thapsigargin were found to down-regulate steady state AR mRNA levels in a time- and dose-dependent manner. AR mRNA began to decrease after 6-8 h of incubation with 10(-6) M A23187 or 10(-7) M thapsigargin, reaching a nadir at 16 and 10 h of incubation, respectively. In contrast, control mRNA (glyceraldehyde 3-phosphate dehydrogenase) did not change significantly during the treatments with either A23187 or thapsigargin. AR protein levels were found to be decreased after 12 h of incubation with either 10(-6) M A23187 or 10(-7) M thapsigargin. The decrease in AR mRNA and protein seemed to precede apoptosis, since neither A23187 (24 h) nor thapsigargin (30 h) was found to alter cell morphology within the treatment time. Cycloheximide and actinomycin D were unable to change the calcium-mediated decrease in AR mRNA, ruling out the necessity for de novo protein synthesis or a change in mRNA stability. Moreover, the decrease in AR mRNA induced by calcium does not seem to involve protein kinase C- or calmodulin-dependent pathways, since inhibitors of these cellular components had no effect. Nuclear run-on assays demonstrated little or no effects of either A23187 or thapsigargin treatment on AR gene transcription (8 h and 10 h). In conclusion, these studies show that intracellular calcium seems to be a potent regulator of AR gene expression in LNCaP cells.

Endogenous cathepsin D-mediated hydrolysis of insulin-like growth factor-binding proteins in cultured human prostatic carcinoma cells.

Insulin-like growth factor I (IGF-I) seems to play an important role in prostate cell growth and its actions may be modified by IGF-binding proteins (IGFBPs) secreted by prostate epithelial cells. The IGFBP system was studied in two human prostate carcinoma cell lines, PC3 and LNCaP. Androgen receptor-negative PC3 cells secrete IGFBP-3, IGFBP-4, and IGFBP-5, as determined by immunoprecipitation of the serum-free conditioned medium with specific IGFBP antibodies. Androgen receptor-positive LNCaP cells secrete IGFBP-2 and IGFBP-3. At neutral pH, there was little or no effect of a 24-h, 37 C cell-free incubation of PC3 and LNCaP conditioned media on IGFBP. On the other hand, when media was incubated at pH 3 for 24 h, [125I]IGFBP-3 hydrolysis and the virtual elimination of endogenous IGFBP detected by Western ligand blotting were observed. This loss was not due to the acid treatment, per se, since IGFBPs remained intact if the incubation at pH 3 was carried out at 4 C. The acid-activated IGFBP protease in LNCaP and PC3 cell-conditioned media was identified as cathepsin D based on acidic pH optimum and immunoblotting. Furthermore, immunoadsorption of cathepsin D from the media attenuated the acid-activated IGFBP hydrolysis [125I]IGF-I binding to prostate cancer cells was reduced in the presence of LNCaP conditioned media that had been incubated at neutral pH for 24 h (i.e. containing intact IGFBP) but not by acid-incubated conditioned media (i.e. cathepsin D-mediated hydrolyzed IGFBP). These data indicate that prostate carcinoma cells secrete specific IGFBPs, as well as a general IGFBP protease, cathepsin D. In the proper environment, cathepsin D is capable of hydrolyzing all endogenous IGFBP and, thus, modifying IGF-I action in prostatic cells.

Subcellular distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase in bovine and murine adrenocortical tissue: species differences in the localization of activity and immunoreactivity.

Key to the production of biologically active steroids is the enzyme 3 beta-hydroxysteroid dehydrogenase-isomerase. Some controversy has arisen concerning the subcellular distribution of this enzyme within steroidogenic cells. The distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase was assessed in subcellular fractions obtained from homogenates of rat, bovine, and mouse adrenal glands in two ways. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was quantitated by measuring the conversion of radiolabeled pregnenolone to radiolabeled progesterone in an aliquot of each of the fractions obtained. The presence of the enzyme was assessed by performing Western analyses on aliquots of each of the fractions obtained with the use of a specific polyclonal antiserum against 3 beta-hydroxysteroid dehydrogenase-isomerase, the characterization of which is described. In control experiments, the degree of contamination of the fractions was determined by assessing the presence of known subcellular fraction markers with Western analysis. In the bovine and mouse adrenal glands, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to be localized solely in the microsomal fraction, while in the rat, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to have dual subcellular distribution: the microsomes and the inner mitochondrial membrane. We conclude that there is a species difference in the subcellular distribution of this important steroidogenic enzyme and that this species difference may be related to the steroidogenic pathway preferred in that species.

The use of running shoes to reduce plantar pressures in patients who have diabetes.

We compared the plantar pressures generated by walking in leather-soled Oxford-style shoes and by walking in inexpensive running shoes with those generated by walking in thin socks on a hard surface for thirty-nine individuals (thirteen who had diabetes and neuropathy, and thirteen who had diabetes without neuropathy, and thirteen who had neither diabetes nor neuropathy [controls]). Except for two anatomical regions, the plantar pressure associated with the Oxford-style shoes were not different from those associated with walking without shoes. In comparison, the inexpensive running shoes relieved plantar pressure in the forefoot and heel by a mean (and standard deviation) of 31 +/- 9.1 per cent, with the most relief occurring in the feet that had the highest pressures when they were unshod. There were significant reductions in pressure in all regions of the foot except for the midfoot (p < 0.01), and there were no significant differences between the groups. Individuals who have insensate feet should be discouraged from wearing leather-soled Oxford-style shoes because of the risk of ulceration due to elevated plantar pressures. Inexpensive running shoes should be viewed as the very minimally acceptable choice for footwear for these individuals if the feet are free of deformity.

Frequency content of normal and diabetic plantar pressure profiles: implications for the selection of transducer sizes.

How small do pressure transducers need to be in order to faithfully measure the plantar pressure profiles (PPPs) under normal and diabetic feet? In this study, pressures were collected from five diabetic and six non-diabetic subjects using a commercial measurement system with 25 mm2 transducers. Discrete Fourier Transform techniques were then used to determine (i) the spatial frequency content of diabetic and non-diabetic PPPs, and (ii) the effects of quadrupling the transducer area (from 5 mm x 5 mm to 10 mm x 10 mm). When the data were filtered to represent the effects of using 10 mm x 10 mm transducers, it was found that the ensuing reductions in peak pressure in the toe region (50 kPa) were significantly greater than in all other regions of the foot (p < 0.05). There was a significant correlation between pressure underestimations and measured peak pressures in the metatarsal regions. Based on data collected with 25 mm2 transducers it was concluded that transducer sizes greater than 6.36 mm x 6.18 mm (medio-lateral and antero-posterior directions) would result in sub-optimal sampling of PPPs.

Locomotion in a rotating space station: a synthesis of new data with established concepts.

Despite technical problems associated with designing a rotating space station it is still thought that such a device may provide a more tolerable work environment and prevent some of the physiological changes that currently pose a threat to long-duration space missions. In the present analysis four case studies are presented and the results show that centrifugal and Coriolis effects could hinder one's ability to walk or run in a natural way in such an environment. In a rotating station that has a nominal 'G-level' equal to that on earth it can be shown that a person running at 3.8 m s -1 could experience foot 'heaviness' effects that range from 1 to 3 g and fore-aft foot 'forces' that range fom -0.5 to +0.5 g. In contrast the hip region could sense a relatively constant 'force' equal to 2 g. With regard to the body as a whole there would be 'weight changes' that depended on the direction of gait. While these conditions imply that locomotion in a rotating space station would be different from normal gait, it is likely that given sufficient training, astronauts could learn optimal strategies to account for centrifugal and Coriolis effects on individual body segments. The learning process would also entail developing strategies on which route to take when moving from one location to another, since in many cases the shortest route would not be the least energy consuming. Such training would be justified if it were shown that artificial gravity was an effective countermeasure to the problems of muscle atrophy and bone loss.

A biomechanical evaluation of one-stage vs two-stage bilateral knee arthroplasty patients.

In this study the functional abilities of eight one-stage bilateral total knee replacement (TKR) patients were compared to five two-stage bilateral TKR and nine control subjects. The TKR individuals were an average of 62 months post-operative. Based on gait analysis, ground reaction force profiles during walking and isometric knee strength assessment, the one-stage individuals did not differ significantly from the control subjects. The two-stage individuals had significantly less knee range of motion during gait and smaller vertical ground reaction forces during the braking phase than the control and one-stage individuals. To compare left and right sides, a symmetry index was computed and there were no significant differences among the three groups. Based on the variables tested in this biomechanical evaluation it can be concluded that for individuals facing bilateral knee replacement a one-stage procedure can result in functional capabilities at least comparable to a two-stage procedure.

Quantifying muscle activity in non-ambulatory children with spastic cerebral palsy before and after selective dorsal rhizotomy.

Cerebral palsy is a condition that results in varying degrees of functional deficits. The goal of this study was to develop an objective measure of muscle activity during a prescribed voluntary motor task in non-ambulatory children with spastic cerebral palsy. While performing a simultaneous hip/knee flexion task from the supine position, followed by return to the starting position, electromyographic and kinematic data were obtained from the right leg of eight children before and after selective dorsal rhizotomy and compared with eight age-matched controls. The electromyographic and kinematic data were combined to determine for each muscle of interest (tibialis anterior, soleus, vastus lateralis, biceps femoris) the percentage of the movement cycle for which the muscle was acting concentrically, eccentrically, isometrically or was considered inactive. Averaged over the four muscles, isometric activity decreased by 38% post-op and the time the muscles were inactive increased by 37% following surgery. The percentages of concentric and eccentric activity did not differ significantly between pre- and post-op conditions. Post-operatively, the percentage muscle activity patterns of the children with cerebral palsy more closely resembled that of the control children: averaged across all muscles and contraction types, the difference between the control children and the children with cerebral palsy was reduced by 50% following surgery. This measurement technique indicates promise as a method for quantifying muscle activity during voluntary motor tasks in non-ambulatory children with cerebral palsy.

Role-taking, moral development, and mental retardation.

Role-taking ability and level of moral development were measured in three groups of boys and girls including 16 moderately retarded adolescents (mean chronological age 15; mean mental age 9), 16 nonretarded adolescents matched for chronological age, and 16 nonretarded children matched for mental age. The mentally retarded adolescents scored significantly lower on role-taking ability and moral development than the adolescents matched for chronological age. They did not differ from children of their same mental age in role-taking ability; and they tended to score lower in moral development. The results supplied qualified support for Kohlberg's theory of moral development.

Rapid and extensive surface changes near Titan's equator: evidence of April showers.

Although there is evidence that liquids have flowed on the surface at Titan's equator in the past, to date, liquids have only been confirmed on the surface at polar latitudes, and the vast expanses of dunes that dominate Titan's equatorial regions require a predominantly arid climate. We report the detection by Cassini's Imaging Science Subsystem of a large low-latitude cloud system early in Titan's northern spring and extensive surface changes (spanning more than 500,000 square kilometers) in the wake of this storm. The changes are most consistent with widespread methane rainfall reaching the surface, which suggests that the dry channels observed at Titan's low latitudes are carved by seasonal precipitation.