Parathyroid hormone regulates the expression of the nuclear mitotic apparatus protein in the osteoblast-like cells, ROS 17/2.8

This study assessed the prevalence of tobacco and alcohol use in top-grossing American films from 1985 to 1995. The top ten money-making films for 1985 to 1995 were viewed to identify the prevalence of alcohol and tobacco use overall and by lead characters. While the use of tobacco and alcohol was stable across the study period, use of these substances was common. Most films (98%) had references that supported tobacco use and 96% had references that supported alcohol use. Discouraging the use of these substances was uncommon. Only one third of films had any references that discouraged the use of tobacco or alcohol (38% and 37%, respectively). At least one lead character used tobacco or alcohol in 46% and 79% of the films, respectively. The hazards of smoking and drinking are not reflected in the behaviors of film characters who are potential role models for youth facing the decision to smoke or drink.     

Plasma copolymer surfaces of acrylic acid/1,7 octadiene: surface characterisation and the attachment of ROS 17/2.8 osteoblast-like cells

The purpose of this study was: (a) to examine the effect of plasma-gas composition on plasma polymer oxygen/carbon (O/C) ratio, functional group composition and stability in water, and then (b) to examine cell attachment to surfaces containing different concentrations of O/C and functional groups. Oxygen-functionalised surfaces were deposited by means of the plasma copolymerisation of acrylic acid/1,7-octadiene. The use of a diluent hydrocarbon allowed the deposition of surfaces with a range of O/C concentrations. Plasma copolymer surfaces were characterised by X-ray photoelectron spectroscopy (XPS). Changes in functional group composition with % acrylic acid monomer and the non-dispersive and dispersive parts of the surface energy of these plasma copolymers were measured. The solubility of the plasma copolymers was assessed by means of XPS. The degree of attachment of ROS 17/2.8 osteoblast-like cells to plasma copolymer surfaces deemed to be 'stable' in aqueous medium was measured. Tissue culture polystyrene (TCPS) was included as a control. Attachment was found to be greatest to the plasma copolymer surface with an O/C of 0.11. This surface had a carboxylic acid concentration of ca. 3%. Attachment did not correlate with increased surface wettability (i.e. the non-dispersive component of the surface energy).     

Opposing influences of dexamethasone and retinoic acid on adenylate cyclase activity in ROS 17/2.8 cells

Exposure of ROS 17/2.8 cells to dexamethasone (DEX) or retinoic acid (RA) increases and decreases, respectively, adenylate cyclase activity (ACA) in response to isoproterenol, forskolin, guanylylimidodiphosphate, or NaFl. Despite dramatic changes in ACA, there were no significant changes in levels of cholera toxin- or pertussis toxin (PT)-dependent ADP-ribosylation of membranes prepared from cells after DEX or RA exposure as compared to controls. Similarly, immunochemical detection of alpha S, alpha i1-3, and alpha O, as well as Northern blot analysis of messenger RNA for each of the respective GTP binding proteins, also failed to demonstrate an influence of DEX or RA when contrasted with controls. In a novel use of the cyc- reconstitution assay, wherein the influence of inhibitory guanine nucleotide binding proteins in the extracts of control, DEX-, and RA-treated membranes is removed by a previous 24-h incubation with PT in the intact cell, we demonstrate that this PT treatment markedly enhances ACA in the cyc- reconstitution assay for all three preparations, but that the fold-increase due to PT-treatment is greatest in RA-treated cells. The greater magnitude of the effect of PT on RA-treated ROS 17/2.8 cells, in the absence of any obvious quantitative changes in the levels of the PT substrates, suggests that the effect of RA on ROS 17/2.8 cells appears to be an augmentation of the influence of inhibitory guanine nucleotide binding proteins, ultimately leading to reduced ACA.     

Regulation of the transcription of parathyroid-hormone/parathyroid-hormone-related peptide receptor mRNA by dexamethasone in ROS 17/2.8 osteosarcoma cells

The normal epithelial cell-specific 1 (NES1) gene is a recently identified novel serine protease-like gene which is down-regulated during breast cancer progression. The gene product has 34-42% identity with the members of three distinct serine protease families: the trypsin-like family, activators of kringle domain-containing growth factors, and the kallikrein family (X. L. Liu et al., (1996) Cancer Res 56, 3371-3379). Although the cDNA of this gene has been cloned, its genomic structure and chromosomal position are not as yet known. Here, we report the genomic characterization and mapping of the NES1 gene. By subcloning and sequencing a PAC clone containing the complete NES1 gene, we were able to characterize the structure of this gene. The NES1 gene spans 5.5 kb and is composed of five coding exons and one untranslated exon. The positions of the introns were similar to trypsinogen, prostate specific antigen (PSA), and tissue plasminogen activator (TPA). NES1 gene was also localized with somatic cell mapping, radiation hybrid mapping, and fluorescence in situ hybridization techniques to chromosome 19q13.3-q13.4, the same region where the human kallikrein gene family resides. Taken together, our results suggest that the NES1 gene originates from the same ancestor as trypsinogen, PSA, and TPA, but remains in close proximity to PSA.     

Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates

We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues.     

Maintained coupling of oxidative phosphorylation to creatine kinase activity in sarcomeric mitochondrial creatine kinase-deficient mice

The importance of mitochondrial creatine kinase (mi-CK) in oxidative muscle was tested by studying the functional properties of in situ mitochondria in saponin-skinned muscle fibres from sarcomeric mi-CK-deficient (mutant) mice. Biochemical analyses showed that the lack of mi-CK in mutant muscle was associated with a decrease in specific activity of MM-CK in mutant ventricle, and increase in mutant soleus (oxidative) muscle. Lactate dehydrogenase activity and isoenzyme analysis showed an increased glycolytic metabolism in mutant soleus. No change was observed in ventricular muscle. In control animals, the apparent K(m) of mitochondrial respiration for ADP in ventricle and soleus (232 +/- 36 and 381 +/- 63 microM, respectively) was significantly reduced in the presence of creatine (52 +/- 8 and 45 +/- 12 microM, respectively). There was no change in the K(m) in oxidative fibres from mutant mice (258 +/- 27 and 399 +/- 66 microM, respectively) compared with control, though surprisingly, it was also significantly decreased in the presence of creatine (144 +/- 8 and 150 +/- 27 microM, respectively) despite the absence of mi-CK. It is proposed that in mutant (and perhaps normal) oxidative tissue, cytosolic MM-CK can relocate to the outer mitochondrial membrane, where it is coupled to oxidative phosphorylation by close proximity to porin, and the adenine nucleotide translocase. Such an effect can preserve the functioning of the CK shuttle and the energetic properties of mi-CK deficient tissue.     

Influence of aluminum on the regulation of PTH- and 1,25(OH)2D3-dependent pathways in the rat osteosarcoma cell line ROS 17/2.8

The role of hormonal status in the development of aluminum (Al)-dependent renal osteodystrophy, which is characterized by reduced bone matrix deposition, still remains largely unknown. To address this question, we used the osteoblast-like osteosarcoma cell line ROS 17/2.8 to evaluate the role of Al on parathyroid hormone (PTH)- and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent activities in these cells. Al (1 microM) caused an inhibition of basal and 1,25(OH)2D3-induced alkaline phosphatase, but only at low doses (< 1 nM) of the steroid. Al partly inhibited basal osteocalcin (OC) secretion in ROS cells (p < 0.001), and the dose-dependent increase in 1,25(OH)2D3-induced OC release by these cells was also reduced by 1 microM Al at low concentrations of the steroid (< or = 1 nM), whereas high doses of 1,25(OH)2D3 (> or = 5 nM) totally prevented the inhibiting effects of Al. Al also had strong inhibitory actions on PTH-dependent cAMP production by ROS cells over the concentration range tested (0.5-50 nM). This inhibitory action of Al was also observed for PTH-related peptide- (PTHrp, 50 nM) but not for Isoproterenol-dependent (100 nM) cAMP formation. To evaluate more fully the mechanism of this inhibition of cAMP formation, we investigated the effect of Al on toxin-modulated, G protein-dependent regulation of cAMP formation and on the activation of adenylate cyclase by Forskolin. Cholera toxin (CT, 10 micrograms/ml), applied to cells for 4 h prior to PTH challenge, enhanced cAMP production about 2-fold above PTH alone (p < 0.001), a process that was further stimulated by Al. Pertussis toxin (PT, 1 microgram/ml, 4 h) did not modify basal PTH-dependent cAMP formation by ROS cells. However, PT treatment prevented the inhibitory effect of Al on cAMP formation by these cells (p < 0.025). The stimulation of adenylate cyclase by Forskolin (0.1 and 1 microM), which bypasses G protein regulation, was not modified by Al, indicating that Al does not affect adenylate cyclase directly. Northern blot analysis of PTH receptor mRNA levels showed that Al did not modify PTH receptor message in ROS cells. Likewise, Western blot analyses of G protein subunits showed that Al did not significantly alter Gs alpha subunit levels, in accordance with the results obtained for cAMP-dependent formation in response to CT. In contrast, Gi alpha-1 and Gi alpha-2 subunits were decreased by Al treatment, consistent with PT-restricted increases in cAMP formation in Al-treated ROS cells. Taken together, these results suggest that Al has multiple actions in osteoblast-like ROS cells. The effects of Al are modulated by hormonal control of the pathways investigated. Al affects 1,25(OH)2D3-regulated functions only when this steroid is low. Al has large inhibitory effects on PTH- and PTHrp-dependent cAMP formation. This last feature is related to the ability of Al to alter the G protein transducing pathway for PTH/PTHrp-dependent formation of cAMP since it does not affect adenylate cyclase activity directly and does not affect the PTH receptor message level. Thus, Al has stronger deleterious effects in osteoblast-like cells with an already compromised 1,25(OH)2D3 status and can modulate specifically PTH/PTHrp-mediated cAMP formation at the postreceptor level.     

Design and synthesis of analogs of vitamin E: antiproliferative activity against human breast adenocarcinoma cells

Analogs of alpha-tocopherol (vitamin E; compounds 3-9) have been synthesized and tested for their antiproliferative activity using the human breast cancer cell line, MCF7. Compounds 6-9 were synthesized from a common symthom, rac-Trolox (14) and are soluble/miscible at physiological pH. Compounds 4, 8, and 9 were found to have antiproliferative activity at micromolar concentrations.     

Estrogen stimulation of creatine kinase B specific activity in 3T3L1 adipocytes after their differentiation in culture: dependence on estrogen receptor

We have demonstrated previously that rat adipose tissue showed sex and depot-specific responses to gonadal steroids. The epididymal fat pad in males responded exclusively to androgens by increased specific activity of the brain type isozyme of creatine kinase (CK). In females, the parametrial adipose tissue responded exclusively to estrogens. The present study was undertaken to follow the responsiveness to steroid hormones, and the presence of estrogen receptors (ER), in 3T3L1 cells during their differentiation from pre-adipocytes to adipocytes. In pre-adipocytes in which the basal CK specific activity is low, there was no CK response to 17beta estradiol (E2) or dihydrotestosterone (DHT). Differentiation of the cells into adipocytes was accompanied by increased basal CK activity which was stimulated by E2, but not by DHT. Responsiveness to E2 began 5 days after switching pre-adipocytes to differentiation medium. Upon differentiation, ER became demonstrable in the cell nuclei by staining with FITC labeled anti-idiotypic antibody (clone 1D5) directed against the steroid binding domain of ER. The response to E2 was time-dependent and blocked completely by cycloheximide or actinomycin D. 1D5 itself, which has an estrogen mimetic effect, stimulated CK activity in the cells similarly to E2. The antiestrogen tamoxifen which also stimulated CK activity in the adipocytes, completely blocked E2 action. The 'pure' antagonist of E2, ICI 164,384 and the tissue-selective antiestrogens, raloxifene or tamoxifen methiodide were also complete antagonists with no agonistic effects. The response of the 3T3L1 adipocytes to E2 was upregulated by 1,25(OH)2D3. Moreover, IGF1 was also a potent stimulator of CK in these cells, and therefore may mediate partially the stimulation by E2. Transient transfection of the pre-adipocytes with ER permitted E2 induction of CK. Thus, the appearance of ER and concomitant responsiveness to E2 is another hormone-related change occurring in 3T3L1 cells during differentiation, in addition to changes such as development of insulin responsiveness. The interactions in this system provide a useful in vitro model for investigating the development of responsiveness to E2.     

Energy balance in muscle activity: simulations of ATPase coupled to oxidative phosphorylation and to creatine kinase

Considering the novel functions for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the developing nervous system (reviewed in Layer and Willbold, Prog. Histochem. Cytochem., 1995) a quantitative survey of the spatiotemporal developmental profiles of both AChE and BuChE activity in the neonatal rat brain would be extremely useful. To that end, we collected six brain regions at seven developmental time points, (postnatal day 1, 4, 7, 12, 17, 21, adult; n>/=3) and measured AChE and BuChE activity using both biochemical and histological methods. These results indicated that the developmental pattern of AChE and BuChE activity varied with respect to brain region and age: (1) the ontogeny of either AChE or BuChE specific activity in one region was not necessarily indicative of the developmental pattern of the same cholinesterase in other regions; (2) the AChE developmental profile in a given region did not necessarily predict the BuChE developmental pattern for that same region. The data were also analyzed from a different perspective, i.e., the ratio of BuChE-AChE activity, in order to determine if BuChE activity preceded AChE activity during development as has been proposed for the chick nervous system (Layer, Proc. Natl. Acad. Sci. USA, 1983). Our analysis showed that, in general, the BuChE-AChE ratio decreased as the region matured, data which parallel the pattern of development of these esterases in the chick nervous system.     

Sequence and activity of parathyroid hormone/parathyroid hormone-related protein receptor promoter region in human osteoblast-like cells

Aspartic proteinase cathepsin D (CD) is believed to be associated with proteolytic processes leading to local invasion and seeding of tumour cells. To estimate a potential prognostic value of cathepsin D in squamous cell carcinoma of the head and neck, its total concentration was measured immunoradiometrically (ELSA-CATH-D kit, CIS bio international) in cytosols of tumour and adjacent normal tissue samples from 111 patients; in 42/111 patients, the CD concentration was determined in serum samples obtained at diagnosis (serum no. 1) and after the therapy (serum no. 2) from each of these patients. Sera of 15 healthy volunteers served as controls. A significantly elevated concentration of CD was measured in tumour cytosols as compared to normal tissue cytosols (31.1 versus 12.6 pmol/mgp, P < 0.0001) and in cytosols of normal laryngeal tissue than of the oral cavity or pharynx (13.3 versus 11.2 pmol/mgp, P = 0.03). The higher CD tumour concentration correlated with the age of the patients (< or =60 versus >60 years, 28.8 versus 32.8 pmol/mgp, P = 0.045) and histopathological tumour grade (G1+2 versus G3, 32.6 versus 24.4 pmol/mgp, P = 0.02). In serum samples, a lower concentration of CD was measured in the control group than in the patients (3.6 versus 4.1 pmol/mls, P = 0.045) and in serum no. 1 than in serum no. 2 (4.1 versus 5.1 pmol/ mls. P = 0.05). The CD concentration in sera obtained at diagnosis was stage-dependent (S(I-III) versus S(IV), 3.9 versus 4.7 pmol/ mls. P = 0.09); there was a trend towards lower CD concentrations with an increasing time delay in serum no. 2 sampling (Rs = -0.20, P = 0.21). No correlation was observed between cytosolic and serum concentrations of CD. We conclude that our results confirm a specific role of CD in the process of invasion and metastasis of squamous cell carcinoma of the head and neck, which might also be of prognostic value in this particular cancer type.     

Dopamine fails to stimulate protein kinase C activity in renal proximal tubules of spontaneously hypertensive rats

We have previously reported that dopamine-1 receptor-mediated activation of phospholipase C is diminished in renal cortical slices of spontaneously hypertensive rats. The present study was carried out to examine the effect of dopamine on protein kinase C (PKC), which is one of the enzymes involved in the signal-transduction pathway leading to dopamine-induced inhibition of Na+/K(+)-ATPase in the renal proximal tubule. Renal proximal tubule suspensions were obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats of 10-12 weeks old. The tubules were incubated with dopamine in the presence or absence of DA-1 receptor antagonist SCH 23390. The PKC activity was measured by using a specific fluorescent peptide substrate (sequence, PKSRTLSVAAK). We found that dopamine produced a concentration-dependent increase in protein kinase C activity in the WKY rats, however, it failed to stimulate PKC activity in the SHR. Peak stimulation of 3.828 +/- 0.35 (ng/micrograms) protein in the WKY rats was observed at dopamine concentration of 1 microM, which was blocked in a concentration-dependent manner by SCH 23390 (0.25 microM). These results provide evidence that dopamine directly stimulates PKC activity via activation of DA-1 receptors in WKY rats. Furthermore, we discovered that dopamine fails to stimulate PKC activity in the SHR. This phenomenon may be responsible for the failure of dopamine to inhibit Na+/K(+)-ATPase activity in the hypertensive animals.     

Effect of vitamin D metabolites and analogs on renal and intestinal calbindin-D in the rat

The effects of endotoxin (20 mg kg-1 i.p.) on the mesenteric vascular responses to acetylcholine, bradykinin, sodium nitroprusside, and to transient occlusion of the superior mesenteric artery were examined in rats anesthetized with pentobarbitone. Mesenteric vasodilator responses to close arterial injections of acetylcholine and bradykinin were reduced at 1.5 h after endotoxin and almost abolished by 4 h; responses to sodium nitroprusside were unaffected. Occlusion of the superior mesenteric artery for 30, 60, or 120 s produced, on release of the occlusion, a time-dependent vasodilator response in the mesenteric circulation (post-occlusion hyperemia). This hyperemia was markedly reduced by nitro-L-arginine methyl ester (L-NAME); L-NAME did not modify acetylcholine-induced vasodilation. Endotoxin-pretreatment did not modify mesenteric post-occlusion hyperemia 1.5 h after administration but markedly reduced the response by 2.5 h. The administration of L-NAME to endotoxin-treated rats did not further attenuate the hyperemic responses. Mesenteric vasoconstrictor responses to phenylephrine were not modified by endotoxin, although systemic pressor responses to this agent were impaired. We concluded that endotoxin impairs endothelium and nitric oxide-dependent vasodilator responses in the mesenteric circulation.     

Cardiac markers in the early hours of acute myocardial infarction: clinical performance of creatine kinase, creatine kinase MB isoenzyme (activity and mass concentration), creatine kinase MM and MB subform ratios, myoglobin and cardiac troponin T

We compared early markers of acute myocardial infarction (AMI) in the first 6 h from the onset of symptoms in 133 non-traumatized patients arriving at the emergency department with chest pain suggestive of AMI. Clinical performance parameters were calculated on the basis of 45 patients with AMI and 88 patients with a non-AMI diagnosis. At admission and in the first 0-3 h after the onset of chest pain the creatine kinase-MB (CK-MB) subform ratio was the most sensitive test at a comparable specificity level of 0.95. In the time interval of 3-5 h, myoglobin, the CK-MB mass concentration and the CK-MB subform ratio were associated with the greatest areas under receiver operating characteristic (ROC) curves, but differences between these tests were small and non-significant. At 6 h from the onset of pain, differences in clinical performance between the same three tests were even smaller whether or not samples drawn after the start of thrombolytic treatment were included in the test comparison. For confirmation of AMI at 6 h after onset of pain, CK-MB (activity and mass concentration) demonstrated the highest positive likelihood ratio, and for exclusion of AMI at 6 h the CK-MB subform ratio was associated with the highest negative likelihood ratio. However, differences between the CK-MB subform ratio, CK-MB mass concentration and myoglobin were not significant as estimated by the substantial overlap between the confidence intervals of the likelihood ratios and the ROC areas at 6 h. Cardiac troponin T (cTnT) demonstrated an ROC area equal to the CK-MB isoform ratio and myoglobin at 6 h. However, the likelihood ratio for ruling out AMI was lower, mostly due to the elevated cTnT in unstable coronary disease not defined as AMI. We conclude that the CK-MB subform ratio, CK-MB mass concentration and myoglobin do not demonstrate any significant differences in clinical performance for ruling in or ruling out acute myocardial infarction at 6 h after the onset of chest pain.