HRAS1-selected, chromosome-mediated transformants vary in phenotype in vitro and tumorigenic potential in vivo

Transfection of mouse C127 cells with mitotic chromosomes isolated from a human EJ bladder carcinoma cell line gave rise, at high frequency, to foci of transformed cells. Independent, HRAS1-selected chromosome-mediated transformants displayed distinctive cellular morphologies in monolayer culture and colony-forming abilities in low-melting-point agarose. Subcutaneous inoculation of neonatally thymectomized, Ara-C-protected, total-body-irradiated CBA mice was used to compare the tumorigenic potential of each transformant. Significant quantitative and qualitative differences in tumorigenicity were found between transformants which correlated with differences in malignant phenotype observed in vitro. The sensitivity of the tumorigenicity assay is such that rare transformation events can be selected directly in vivo.     

Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption.

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. INTRODUCTION: Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. MATERIALS AND METHODS: Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1. RESULTS: In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. CONCLUSIONS: We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.     

Are nonresorbing osteoclasts sources of bone anabolic activity?

Some osteopetrotic mutations lead to low resorption, increased numbers of osteoclasts, and increased bone formation, whereas other osteopetrotic mutations lead to low resorption, low numbers of osteoclasts, and decreased bone formation. Elaborating on these findings, we discuss the possibility that osteoclasts are the source of anabolic signals for osteoblasts. In normal healthy individuals, bone formation is coupled to bone resorption in a tight equilibrium. When this delicate balance is disturbed, the net result is pathological situations, such as osteopetrosis or osteoporosis. Human osteopetrosis, caused by mutations in proteins involved in the acidification of the resorption lacuna (ClC-7 or the a3-V-ATPase), is characterized by decreased resorption in face of normal or even increased bone formation. Mouse mutations leading to ablation of osteoclasts (e.g., loss of macrophage-colony stimulating factor [M-CSF] or c-fos) lead to secondary negative effects on bone formation, in contrast to mutations where bone resorption is abrogated with sustained osteoclast numbers, such as the c-src mice. These data indicate a central role for osteoclasts, and not necessarily their resorptive activity, in the control of bone formation. In this review, we consider the balance between bone resorption and bone formation, reviewing novel data that have shown that this principle is more complex than originally thought. We highlight the distinct possibility that osteoclast function can be divided into two more or less separate functions, namely bone resorption and stimulation of bone formation. Finally, we describe the likely possibility that bone resorption can be attenuated pharmacologically without the undesirable reduction in bone formation.     

Occupational exposure of deck crews to carcinogenic agents on crude oil tankers

Occupational exposure to carcinogenic agents on the decks on six Norwegian crude oil tankers was examined in five harbors. The purpose of the study was to evaluate the need for improving the working environment on deck on these tankers. Technical arrangments and the work itself on the deck were observed during loading or unloading. Occupational monitoring was performed by active sampling of benzene, polyaromatic hydrocarbons, and some aldehydes. The crew answered a questionnaire concerning their work, use of protective equipment, and occurrence of acute symptoms. The levels of air-borne carcinogenic agents were low, probably due to closed loading systems on all tankers. However, the seamen reported discomfort during the work that may be related to other chemical agents in the cargo. The seamen were frequently painting with lead chromate paint without using personal protective equipment. This type of chemical exposure should be evaluated.     

Turbulent stress measurements downstream of three bileaflet heart valve designs in pigs

Objective: Mechanical heart valves can cause thromboembolic complications, possibly due to abnormal flow patterns that produce turbulence downstream of the valve. The objective of this study was to investigate whether three different bileaflet valve designs would exhibit clinically relevant differences in downstream turbulent stresses. Methods: Three bileaflet mechanical heart valves (Medtronic Advantage^®, CarboMedics© Orbis™ Universal and St. Jude Medical^® Standard) were implanted into 19 female 90 kg pigs. Blood velocity was measured during open chest conditions in the cross sectional area downstream of the valves with 10 MHz ultrasonic probes connected to a modified Alfred^® Pulsed Doppler equipment. As a measure of turbulence, Reynolds normal stress (RNS) was calculated at three different cardiac output ranges (3–4, 4.5–5.5, 6–7 L/min). Results: Data from 12 animals were obtained. RNS correlated with increasing cardiac outputs. The highest instantaneous RNS observed in these experiments was 47 N/m², and the mean RNS taken spatially over the cross sectional area of the aorta during systole was between 3 N/m² and 15 N/m². In none of the cardiac output ranges RNS values exceeded the lower critical limit for erythrocyte or thrombocyte damage for any of the valve designs. Conclusions: Reynolds normal stress values were below 100 N/m² for all three valve designs and the difference in design was not reflected in generation of turbulence. Hence, it is unlikely that any of the valve designs causes flow induced damage to platelets or erythrocytes.     

Contractile properties of in situ perfused skeletal muscles from rats with congestive heart failure

We hypothesized that in congestive heart failure (CHF) slow-twitch but not fast-twitch muscles exhibit decreased fatigue resistance in the sense of accelerated reduction of muscle force during activity. Experiments were carried out on anaesthetized rats 6 weeks after induction of myocardial infarction or a sham operation (Sham). Animals with left ventricular end-diastolic pressure (LVEDP) > 15 mmHg under anaesthesia were selected for the CHF group. There was no muscle atrophy in CHF. Force generation by in situ perfused soleus (Sol) or extensor digitorum longus (EDL) muscles was recorded during stimulation (trains at 5 Hz for 6 s (Sol) or 10 Hz for 1.5 s (EDL) at 10 or 2.5 s intervals, respectively) for 1 h in Sol and 10 min in EDL at 37 °C. Initial force was almost the same in Sol from CHF and Sham rats, but relaxation was slower in CHF. Relaxation times (95–5 % of peak force) were 177 ± 55 and 131 ± 44 ms in CHF and Sham, respectively, following the first stimulation train. After 2 min of stimulation the muscles transiently became slower and maximum relaxation times were 264 ± 71 and 220 ± 45 ms in CHF and Sham, respectively (   P < 0.05  ). After 60 min they recovered to 204 ± 60 and 122 ± 55 ms in CHF and Sham, respectively (   P < 0.05  ). In CHF but not in Sham rats the force of contraction of Sol declined from the second to the sixtieth minute to 70 % of peak force. The EDL of both CHF and Sham fatigued to 24–28 % of initial force, but no differences in contractility pattern were detected. Thus, slow-twitch muscle is severely affected in CHF by slower than normal relaxation and significantly reduced fatigue resistance, which may explain the sensation of both muscle stiffness and fatigue in CHF patients.     

Liver pathology associated with the use of anabolic-androgenic steroids

Abstract: This review examines the liver-damaging side effects of anabolic-androgenic steroids (AAS). It seems that AAS can cause development of peliosis hepatis, subcellular changes of hepatocytes, hepatocellular hyperplasia and hepatocellular adenomas. On the other hand, it has not been convincingly proved that AAS can cause development of hepatocellular carcinomas when used in the usual therapeutic doses. Tumours reported as hepatocellular carcinomas caused by AAS seem to be hyperplastic lesions of a benign nature able to regress with withdrawal of the putative agent. The effects of untraditional combinations and high-dose AAS are not yet known, leaving the possibility of a carcinogenic effect in those cases.     

Effects of polymerization heat and monomers from acrylic cement on canine bone

We investigated the effects of polymerization heat and toxicity of polymethyimetacrylate bone cement in the canine tibial diaphysis. Heat was studied by filling the tibias with either bone cement or bone wax contained in a monomer tight membrane pouch. Toxicity was studied by filling both tibias with cement, with the control side contained in the membrane pouch. Bone blood perfusion was measured by microsphere technic, and bone remodeling by ^99mTc-methylene diphosphonate uptake and by histologic technique. In bone exposed to the combination of polymerization heat and monomer, both perfusion and remodeling were impaired. We did not find any effects of polymerization heat alone.

We conclude that hot toxic chemicals from bone cement during polymerization may inhibit bone blood perfusion and remodeling, whereas heat alone seems to be of minor importance for the regenerative processes in cemented diaphyseal bone.     

The lack of effects of zinc and nitric oxide in initial state of pilocarpine-induced seizures.

In this study we investigated whether intracerebroventricular (i.c.v.) injection of L-NAME (a nitric oxide synthase inhibitor) or CaEDTA (an extracellular zinc chelator) or the combination of the two could affect the initial phase of pilocarpine induced (2 h) seizures. Two groups of rats were used. Animals from both groups were given with i.c.v. injections of either saline (10 microl), L-NAME (150 microg/10 microl), CaEDTA (100 mM/10 microl) or L-NAME and CaEDTA. One group received pilocarpine HCl (380 mg/kg i.p.) the other served as control. Pilocarpine HCl was injected intraperitoneally 10 min later. The behavior of the animals was observed for 2h and the intensity of their seizures was scored. The rats were then sacrificed and their brains were removed and analyzed for zinc ions by using the immersion autometallography and the TSQ fluorescence staining. All the animals which received pilocarpine HCl developed seizures. Despite treatment with L-NAME and/or CaEDTA we found that the latency and the intensity of seizures were similar in both groups investigated. The distribution of stainable zinc ions and the intensity of staining in hippocampus were not affected by pilocarpine and found unchanged after L-NAME and/or CaEDTA injections in both the control animals and the pilocarpine treated animals. The data suggest that the nitric oxide system and zinc ions do not affect pilocarpine-induced seizures in their initial state.     

Neural classifier construction using regularization, pruning and test error estimation

In this paper we propose a method for construction of feed-forward neural classifiers based on regularization and adaptive architectures. Using a penalized maximum likelihood scheme, we derive a modified form of the entropic error measure and an algebraic estimate of the test error. In conjunction with optimal brain damage pruning, a test error estimate is used to select the network architecture. The scheme is evaluated on four classification problems.