Cadmium in moose kidney and liver--age and gender dependency, and standardisation for environmental monitoring.

In the northern hemisphere moose has been found to be suitable as a monitoring animal for the presence of cadmium in the environment. The metal accumulates mainly in the kidney and the liver, with the rate of accumulation dependent on age and possibly also on gender. Collection of tissue material often results in sample selections with disparate age and gender composition, which makes comparison between different regions and different studies difficult. A previous large scale investigation of metals in kidney and liver from moose in Sweden provided Cd data (n = 3,817 and 3,802, respectively) to further explore the relation between Cd accumulation and age/gender. Based on local averages, the individual deviations were analysed with respect to the factors age and gender resulting in an 'ageing function' for each gender and organ. In addition, estimates of the pure individual variations were obtained; the standard deviations correspond to a factor 1.7-1.9 for the Cd concentration, which indicates that 25-30 samples are needed to give a representative mean value (with RSD approximately 10%). In order to be able to compare results from different studies, all individual results can be transformed to represent a 'standard moose' with respect to age and gender. A comparison along these lines was undertaken between Cd levels in Alaska and Sweden. Finally, a relationship between the Cd levels in kidney and liver was derived, providing at least rough estimates for kidney from liver values (or vice versa).

Copper deficiency and neurological disorders in man and animals.

Copper metabolism in the brain is far from being completely understood and further studies are needed on the role of copper in the CNS, starting with careful measurements, metal and biological speciation of metabolites on the molecular level, and combining copper concentration in different brain areas with morphological as well as biochemical alteration after Cu-depletion/deficiency. So far a pathological role for copper has been clearly demonstrated in some human genetic diseases (e.g., Menkes' and Wilson's diseases), but other pathological features connected with metal depletion are under investigation in several laboratories. The metabolic interaction between copper and other metal ions in some neurological disorders is also discussed in this contribution.

A perivascular niche for brain tumor stem cells.

Cancers are believed to arise from cancer stem cells (CSCs), but it is not known if these cells remain dependent upon the niche microenvironments that regulate normal stem cells. We show that endothelial cells interact closely with self-renewing brain tumor cells and secrete factors that maintain these cells in a stem cell-like state. Increasing the number of endothelial cells or blood vessels in orthotopic brain tumor xenografts expanded the fraction of self-renewing cells and accelerated the initiation and growth of tumors. Conversely, depletion of blood vessels from xenografts ablated self-renewing cells from tumors and arrested tumor growth. We propose that brain CSCs are maintained within vascular niches that are important targets for therapeutic approaches.

Public health evaluation of cadmium concentrations in liver and kidney of moose (Alces alces) from four areas of Alaska.

Liver and/or kidney samples were collected from 139 hunter-killed moose from four areas of Alaska during 1986. The concentration of cadmium in organ tissue was determined by direct-current plasma atomic emission spectrometry. All results are reported as mug/g wet weight. Concentrations of cadmium in liver ranged from 0.06 microg/g to 9.0 microg/g; in the kidney cortex they ranged from 0.10 microg/g to 65.7 microg/g. Cadmium levels were significantly associated with location and age. The highest geometric mean liver (2.11 microg/g) and kidney cortex (20.2 microg/g) cadmium concentrations were detected in moose harvested near Galena, Alaska. Limited dietary information from Alaska and Canada indicates that the intake of moose liver or kidney does not exceed, in most individuals, the World Health Organization recommendations for weekly cadmium consumption of 400 microg to 500 microg. Additionally, human biomonitoring data from Canada and Alaska indicate exposure to cadmium is low except for individuals who smoke cigarettes. Given the nutritional and cultural value of subsistence foods, the Alaska Division of Public Health continues to support the consumption of moose liver and kidney as part of a well-balanced diet. Human biomonitoring studies are needed in Alaska to determine actual cadmium exposure in populations with a lifelong history of moose liver and kidney consumption.

The anti-psoriatic drug anthralin accumulates in keratinocyte mitochondria, dissipates mitochondrial membrane potential, and induces apoptosis through a pathway dependent on respiratory competent mitochondria.

Anthralin is a potent topical drug, inducing clearance of psoriatic plaques. Anthralin disrupts mitochondrial function and structure, but its mechanism of action remains undefined. This study aimed to determine whether anthralin induced keratinocyte apoptosis as well as to investigate molecular mechanisms and the role of mitochondria. We studied human keratinocytes and human 143B rho(0) cells, which lack mitochondrial DNA and a functional respiratory chain. We show that anthralin disrupts mitochondrial membrane potential (DeltaPsim) and causes endogenous cytochrome c release, resulting in the activation of caspase-3 and characteristic morphological changes of apoptosis. Disruption of DeltaPsim and cytochrome c release were independent of mitochondrial permeability transition or caspase activation. Human 143B rho(0) cells were resistant to anthralin-induced cell death, disruption of DeltaPsim, and cytochrome c release compared with the isogenic 143B rho+ cell line. Using the intrinsic fluorescence of anthralin, rapid accumulation within mitochondria was observed independent of DeltaPsim. Using assays that measure individual respiratory chain complexes, we show that anthralin specifically interacts with ubiquinone pool. These data indicate that anthralin induces apoptosis through a novel mitochondrial pathway dependent on oxidative respiration and involving electron transfer with the ubiquinone pool. These studies identify keratinocyte apoptosis as a potentially important mechanism involved in the clearance of psoriasis.

The structure of (S)-(-)-4-(2-benzimidazolyl)-4-hydroxybutyric acid formed during mammalian metabolism of the fungicide 2-(2-furyl)benzimidazole.

During metabolism of the fungicide 2-(2-furyl)benzimidazole (FB) in mammals, an optically active compound, metabolite A, was isolated. This compound, a principal metabolite in the biological degradation of FB, was isolated from the urine of horse and dog. When studied by IR, NMR, MS, CD and electrophoretic techniques, the metabolite proved to be the zwitterionic form of (S)-(-)-4-(2-benzimidazolyl)-4-hydroxybutyric acid. The synthesized optical active compound was in every respect identical with metabolite A. The suggested structure has been completely verified by the current investigation of its crystal structure, based on single crystal diffraction data collected at the synchrotron MAX II in Lund, Sweden. In an earlier determination, a related compound, 3-benzimidazolylpropionic acid (Ercan et al. 1996) was suggested to be non-zwitterionic. The present studies have shown that the latter compound too is zwitterionic and that the conclusions presented were apparently based on some erroneously proposed hydrogen positions.

A review of the "mysterious" wasting disease in Swedish moose (Alces alces L.) related to molybdenosis and disturbances in copper metabolism.

The main purpose of this article is to review the previously published data on so-called "moose sickness" in the light of two case studies presented here. Molybdenosis and Mo-induced disturbances of Cu metabolism in moose are characterized by numerous severe lesions caused by reduced activity of Cu-containing enzymes such as ceruloplasmin, superoxide dismutase in blood, and myocardial cytochrome c oxidase. Consequences of such metabolic disturbances (e.g. glucose intolerance, insulin resistance, and non-insulin-dependent diabetes mellitus) were first reported in moose in 2000. This was corroborated by the detection of furosine, pentosidine, and Nepsilon-(carboxymethyl)-lysine in blood plasma and the kidney, indicating long-term hyperglycemia. Increased concentrations of insulin, glucose, and urea and reduced levels of phosphate, T4, and Mg in blood were also seen. Recently, a similar toxic endocrinopathy was reported in sheep treated therapeutically with thiomolybdates because of chronic Cu toxicosis. Two case reports illustrate the difficulty of diagnosing Mo-related disturbances of Cu metabolism in moose, as analyses of Cu and Mo have not proved entirely reliable because of interaction, accumulation, and the short biological half-life of Mo. The increased bioavailability of Mo is most probably the result of increased pH in the soil, caused, for example, by liming, making Mo accessible in forage plants consumed by moose. The etiology underlying the Swedish moose disease has been difficult to determine because of the complex clinical signs and unspecific pathological findings. However, a combination of clinical chemistry, trace element analysis, and biochemistry correlated with the pathological findings has corroborated molybdenosis and Mo-induced disturbances of Cu metabolism as the probable etiological factor. Alternative etiologies suggested for the moose disease, such as viral infection, starvation because of overpopulation, and/or shortage of forage as well as senescence and phytotoxic substances, are discussed.

Comparison of two monitoring systems for Cu and Mo in the Swedish environment.

Bio-geochemical samples (BGS) are roots of certain aquatic plants and mosses suitable for monitoring elements dissolved in stream water. The moose, a wild ruminant living in most parts of Sweden, represents higher trophic level and another manifestation of bioavailability. By analyzing BGS (n approximately 33600) and moose liver (n approximately 2400), a systematic survey has been performed in the terrestrial environment of Sweden. Cu and Mo are essential elements for life, and their presence is especially important for the Cu-dependent processes in ruminants. The availability of Cu and Mo as monitored in BGS and moose was visualized in the form of maps and subjected to further statistical analysis. The medians, with lower and upper quartiles indicated as intervals, for the country as a whole were: moose liver, Cu = 34 (20-59), Mo = 0.82 (0.58-1.06) mg kg(-1) wet weight; BGS, Cu = 50 (35-77), Mo = 9.0 (5.3-18.0) mg kg(-1) dry weight. The ranges of medians for the 22 Swedish counties were: moose liver, Cu = (20-62), Mo = (0.54-1.18) mg kg(-1) wet weight; BGS, Cu = (28-115), Mo = (5-47) mg kg(-1) dry weight. The relationships between the counties and the connections between the monitoring variables were elucidated by principal component analysis (PCA). It was demonstrated that two monitoring systems could give divergent results. An unexpectedly strong negative correlation was found between the county medians for Cu in BGS and moose liver. A possible explanation, based on the interaction between Cu and Mo in moose, could not be verified.

The TP53-ARF tumor suppressor pathway is frequently disrupted in large/cell anaplastic medulloblastoma.

We analyzed the TP53 and INK4A/ARF loci in 29 pediatric medulloblastomas. Mutually exclusive mutation in TP53, methylation of P14(ARF) or deletion of INK4A/ARF were identified in 21% (6/29) of tumors. Five of these alterations were detected in large cell/anaplastic medulloblastomas or tumors with significant anaplasia. Our data provide the first evidence that alterations within the TP53-ARF tumor suppressor pathway contribute to development of aggressive forms of medulloblastoma.

Nova Scotia moose mystery--a moose sickness related to cobalt- and vitamin B12 deficiency.

A wasting, debilitating disease with uncertain aetiology affecting moose (Alces alces americana) in Eastern North America has been reported repeatedly ever since the 1910s. Despite the intensive studies during 1930-1960s the cause of the sickness could not be established. In the 1960s a parasitic nematode (Parelaphostrongylus tenuis) was reported as constituting a probable explanation for the sickness, although several clinical and pathological signs remained unexplained. In Sweden, a moose disease with similar signs, has been shown to be caused by molybdenosis resulting from a nutritional imbalance. The findings of this investigation were applied in Nova Scotia to determine trace element concentrations in tissues from indigenous moose. Co deficiency was found in about half of the cases and the investigation was complemented by determining the vitamin B12 level, which proved nutritional Co/vitamin B12 deficiency, further verified by an increased MMA (methylmalonic acid) level in plasma. Deficiencies were found mainly in the Tobeatic and Cape Breton Highland regions. No indications of molybdenosis or other trace element disturbances were found in Nova Scotia. Otherwise, extremely high Cd levels (148 mg Cd/kg kidney wet wt., maximum) were found, though probably not contributing to the moose sickness. The Cd burden of moose on mainland Nova Scotia was more than 50% higher than that of moose in Huntville and Alonquin (Ontario, Canada) and five- to six-fold is higher than the highest Cd levels found in Sweden. To counteract the bio-geochemical effects of Co deficiency in the moose environment, provision of Co-containing salt licks is suggested.

Medulloblastoma sensitivity to 17-allylamino-17-demethoxygeldanamycin requires MEK/ERKM.

ERBB2 increases the sensitivity of breast cancer cells to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). This has been attributed to the disruption of ERBB3/ERBB2 heterodimers that maintain a crucial cell survival signal via phosphatidylinositol 3-kinase/AKT. ERBB2 confers a poor clinical outcome in medulloblastoma, the most common malignant pediatric brain tumor. Here, we show that medulloblastoma cell sensitivity to 17-AAG is directly related to ERBB2 expression level. Furthermore, overexpression of exogenous ERBB2 in these cells induces spontaneous homodimerization, further enhancing cell sensitivity to 17-AAG. In contrast to breast cancer cells, this increased sensitivity to 17-AAG does not result from cell dependence on AKT1 activity. Rather, we show that 17-AAG generates a dose- and time-dependent increase in MEK/ERK signaling that is required for the drug to inhibit the proliferation of medulloblastoma cells and that ERBB2 sensitizes medulloblastoma cells to 17-AAG by up-regulating basal MEK/ERK signaling. We further show that down-regulation of MEK1 activity markedly reduces the sensitivity of medulloblastoma, breast, and ovarian cancer cells to 17-AAG, whereas expression of a constitutively active MEK1 potentiates the activity of 17-AAG against these cells. Therefore, intact MEK/ERK signaling may be required for optimal 17AAG activity against a variety of tumor cell types. These data identify a new mechanism by which 17-AAG inhibits the proliferation of cancer cells. Defining the precise mode of action of these agents within specific tumor cell types will be crucial if this class of drugs is to be efficiently developed in the clinic.

Quantification of DNA adducts in individual cells by immunofluorescence: effects of variation in DNA conformation.

Previously reported detection of melphalan-DNA adducts by immunofluorescent staining indicated considerable intercell variation in fluorescence levels. Investigations were undertaken to determine whether this variation reflected actual intercell differences in adduct levels. Melphalan-treated CCRF-CEM leukaemia cells were analysed by the trapped-in-agarose DNA immunostaining (TARDIS) method using fluorescein immunofluorescence and Hoechst dye-DNA fluorescence. Increasing the time of DNA denaturation in alkali affected the staining intensity, in agreement with known adduct properties, but failed to reduce intercell heterogeneity. To test the hypothesis that heterogeneity resulted from variation in levels of DNA strand breaks, drug-treated cells were exposed to ionising radiation. An increase in level and reduction in heterogeneity of immunofluorescence were observed, optimal at 10 Gy. When samples were irradiated after lysis, 1 Gy was optimal. At the optimal doses, irradiation before or after lysis resulted in similar levels of DNA strand breaks. Our conclusions are as follows: (a) There was no major intercell variation in the number of adducts other than from variation in DNA content. (b) Detection of melphalan, and possibly other adducts, by immunofluorescence can be markedly influenced by the level of strand breaks present in the DNA. (c) Samples analysed for melphalan adducts by immunofluorescence should be irradiated to minimise errors due to this factor.

ERBB2 up-regulates S100A4 and several other prometastatic genes in medulloblastoma.

Medulloblastoma is frequently disseminated throughout the central nervous system by the time of diagnosis. Conventional therapeutic approaches have not reduced the high mortality associated with metastatic medulloblastoma and little is known regarding the molecular mechanisms that promote tumor invasion. Previously, we reported that overexpression of ERBB2 in medulloblastoma is associated with poor prognosis and metastasis. Here, we demonstrate that ERBB2 overexpression increases the migration of medulloblastoma cells across basement membranes in vitro. Furthermore, using microarray expression profiling, we show that ERBB2 up-regulates the expression of prometastatic genes in medulloblastoma cells. These include S100A4, which was previously shown to promote metastasis of breast cancer. We demonstrate that S100A4 is a direct target of ERBB2 signaling in medulloblastoma cells via a pathway involving phosphatidylinositol 3-kinase, AKT1, and extracellular signal-regulated kinase 1/2 and that levels of ERBB2 and S100A4 are tightly correlated in samples of primary medulloblastoma. Finally, we show that ERBB2-dependent medulloblastoma cell invasion in vitro and prometastatic gene expression in vivo can be blocked using the ERBB tyrosine kinase inhibitor OSI-774. These data identify an ERBB2 driven prometastatic pathway that may provide a novel target for therapeutic intervention in metastatic medulloblastoma.

ERBB receptor signaling promotes ependymoma cell proliferation and represents a potential novel therapeutic target for this disease.

PURPOSE: This study was designed to investigate the biological and therapeutic significance of ERBB1, ERBB2, ERBB3, and ERBB4 in childhood ependymoma. EXPERIMENTAL DESIGN: The expression frequency and clinical significance of ERBB1-4 was analyzed in a large cohort of pediatric ependymoma (n = 121) using immunohistochemistry, Western blotting, and reverse transcription-PCR analysis. Histological markers of anaplasia (necrosis, microvascular proliferation, and Ki-67 proliferative index) were also determined. Functional assessment of ERBB-dependent cell signaling and proliferation, in addition to novel therapeutic inhibition of these processes, was conducted using short-term cultures of human ependymoma cells. RESULTS: Coexpression of ERBB2 and ERBB4 was identified in over 75% of tumors. High-level coexpression of these receptors was significantly related to tumor proliferative activity [P < 0.05; Ki-67 labeling index (LI)] and, in combined survival analysis of clinical (degree of surgical resection) and molecular (ERBB2/ERBB4 expression status and Ki-67 LI) factors, enabled a greater resolution of patient prognosis than any individual variable alone. Ligand-dependent activation of ERBB receptor-signaling in cultured ependymoma cells resulted in AKT phosphorylation and cellular proliferation that was significantly blocked in a dose-dependent manner using WAY-177820, a novel inhibitor of ERBB2 tyrosine kinase activity. CONCLUSIONS: This study suggests that ERBB receptor signaling results in aggressive disease behavior in ependymoma by promoting tumor cell proliferation. An analysis of ERBB2 and ERBB4 expression, in association with Ki-67 LI and the degree of surgical resection, may provide an accurate tool for assessing disease risk in children with this disease. In addition, these receptors may serve as a target for novel therapeutic approaches in ependymoma.

Myocardial cytochrome c oxidase activity in Swedish moose (Alces alces L.) affected by molybdenosis.

Since the mid-1980s, a 'mysterious' wasting disease has been afflicting the moose (Alces alces L.) population of south-western Sweden. In 1994, molybdenosis combined with copper deficiency was suggested as the cause of this complex syndrome of clinical signs, diversity of necropsy findings and changes in trace element concentrations. These findings were corroborated by scientists in many countries by similar observations in other ruminants, particularly cattle and sheep, and also by changes in trace element concentrations and clinical chemical findings in our model experiments with goats. The biochemistry of copper is dependent on a number of copper-dependent enzymes in the animal organism. An important example is cytochrome c oxidase (COX), responsible for oxidative phosphorylation and energy production within the cell. In the present study, COX activity and trace element concentrations were determined in myocardium from affected and healthy moose. Citrate synthase (CS) activity was also measured for use as a mitochondrial marker. COX activity had decreased by 45% and the COX/CS ratio by 37%, while Mo and Na were found to have increased by 140% and 25%, respectively. The increase in Na was indicative of the frequently reported oedematous changes in 'flabby' moose heart. The concentrations of the elements Cu, Mg, Mn, P and Zn had decreased by 20%, 20%, 35%, 7% and 19%, respectively. The simultaneous decrease in COX activity and Cu concentration and the increase in Mo further support the hypothesis that molybdenosis is the cause of the moose disease.

A new urolith in four cats and a dog: composition and crystal structure.

A previously unreported urolith is presented. The concrement was found in the bladder of four Persian cats and one collie dog. It contained potassium, magnesium and phosphorus, all determined by chemical analysis. The infrared spectrum of the X-ray-dense urolith displayed absorption bands in the phosphate region. A single absorption maximum at 930 cm(-1) indicated the presence of inorganic pyrophosphate. A single crystal structure determination of the urolith has been performed. The results obtained are consistent with the formula [K(1.0)Mg(1.5)](4+)[P(2)O(7)](4-.)(H(2)O)(5), which corresponds to 11.5% K, 10.7% Mg and 18.2% P. The structure contains negatively charged slabs of composition MgP(2)O(7) small middle dot(H(2)O)(2)(2-)alternating with positively charged, hydrated Mg(2+) and K(+) ions. The observed P(2)O(7) is that of a distorted eclipsed syn conformation, which is the most common geometry in the solid state according to a statistical analysis of known structural data. A simple numerical descriptor has been derived, classifying observed X(2)O(7) conformations. It is suggested that some genetic enzymatic dysfunction of pyrophosphate-hydrolysing alkaline phosphatase might cause the formation of this unusual urolith.