Case definitions for human poisonings postulated to palytoxins exposure.

A series of case reports and anecdotal references describe the adverse effects on human health ascribed to the marine toxin palytoxin (PLTX) after different exposure routes. They include poisonings after oral intake of contaminated seafood, but also inhalation and cutaneous/systemic exposures after direct contact with aerosolized seawater during Ostreopsis blooms and/or through maintaining aquaria containing cnidarian zoanthids. The symptoms commonly recorded during PLTX intoxication are general malaise and weakness, associated with myalgia, respiratory effects, impairment of the neuromuscular apparatus and abnormalities in cardiac function. Systemic symptoms are often recorded together with local damages whose intensity varies according to the route and length of exposure. Gastrointestinal malaise or respiratory distress is common for oral and inhalational exposure, respectively. In addition, irritant properties of PLTX probably account for the inflammatory reactions typical of cutaneous and inhalational contact. Unfortunately, the toxin identification and/or quantification are often incomplete or missing and cases of poisoning are indirectly ascribed to PLTXs, according only to symptoms, anamnesis and environmental/epidemiological investigations (i.e. zoanthid handling or ingestion of particular seafood). Based on the available literature, we suggest a "case definition of PLTX poisonings" according to the main exposure routes, and, we propose the main symptoms to be checked, as well as, hemato-clinical analysis to be carried out. We also suggest the performance of specific analyses both on biological specimens of patients, as well as, on the contaminated materials responsible for the poisoning. A standardized protocol for data collection could provide a more rapid and reliable diagnosis of palytoxin-poisoning, but also the collection of necessary data for the risk assessment for this family of toxins.

Melastatin expression and prognosis in cutaneous malignant melanoma.

PURPOSE: Melastatin (MLSN-1), a novel melanocyte-specific gene recently identified using a genomic approach, is expressed in murine and human melanoma cells at levels inversely proportional to metastatic rates in vivo. We studied the relationship between expression of melastatin mRNA in the primary cutaneous tumor and prognosis in patients with localized malignant melanoma. PATIENTS AND METHODS: Melastatin mRNA was evaluated by in situ hybridization in primary cutaneous melanoma from 150 patients with localized disease (American Joint Committee on Cancer [AJCC] stage I and II). Multivariate Cox proportional hazards regression analysis was performed to assess the prognostic utility of melastatin mRNA expression while adjusting for other prognostic indicators. RESULTS: Uniform melastatin mRNA expression in the primary tumor was correlated with prolonged disease-free survival (P < .0001). Multivariate analysis revealed that melastatin status, mitotic rate, and tumor thickness influence disease-free survival independently. The 8-year disease-free survival rate in AJCC stage I patients whose tumors diffusely expressed melastatin mRNA was 100%, whereas in stage I patients with melastatin loss, the disease-free survival rate was 77% +/- 15% (median +/- SE). In patients with stage II disease whose tumors diffusely expressed melastatin mRNA, the 8-year disease-free survival rate was 90% +/- 7%, whereas in patients with melastatin loss, the disease-free survival rate was 51% +/- 8%. CONCLUSION: Downregulation of melastatin mRNA in the primary cutaneous tumor is a prognostic marker for metastasis in patients with localized malignant melanoma and is independent of tumor thickness and other variables. Used in combination, melastatin status and tumor thickness allow for the identification of subgroups of patients at high and low risk of developing metastatic disease.

Patterns of melastatin mRNA expression in melanocytic tumors.

The melanocyte-specific gene Melastatin (MLSN1) shows an inverse correlation of mRNA expression with metastatic potential in human and murine cell lines in vitro. Melastatin mRNA expression in primary cutaneous melanoma also has been found to correlate with disease-free survival. The histologic patterns of Melastatin mRNA expression in nevi, primary melanoma, and melanoma metastases have not been described previously. Using in situ hybridization with (35)S-labeled probes, we examined Melastatin mRNA expression in 64 cases of normal skin, benign melanocytic nevi, primary cutaneous melanomas, and melanoma metastases. Ubiquitous melanocytic expression of Melastatin mRNA was observed in all benign melanocytic proliferations (14 of 14), although some nevi showed a gradient of reduced Melastatin expression with increased dermal depth (3 of 14). Uniform expression of Melastatin mRNA was observed in 49% of primary cutaneous melanomas (18 of 37 cases, including 1 case of in situ melanoma). Melastatin mRNA loss by a portion of the melanoma was identified in 53% of the invasive melanoma samples (19 of 36) and 100% of the melanoma metastases (11 of 11). Primary melanomas without mRNA loss ranged in thickness from 0.17 to 2.75 mm (median, 0.5 mm; mean, 0.73 mm), whereas tumors that showed Melastatin mRNA down-regulation ranged in thickness from 0.28 to 5.75 mm (median, 1.7 mm; mean, 2.13 mm). A focal aggregate or nodule of melanoma cells without detectable signal was the most commonly observed pattern of Melastatin loss (13 of 19 cases), whereas complete loss of Melastatin mRNA expression by all of the dermal melanoma cells was observed in only 4 of the 19 cases. Two invasive melanomas displayed a scattered, nonfocal pattern of Melastatin mRNA loss. Of the 11 melanoma metastases examined, 64% displayed focal Melastatin mRNA loss, and 36% had complete loss of Melastatin mRNA expression. We observed several patterns of Melastatin mRNA expression in primary melanoma that may be distinguished from expression in benign melanocytic nevi. Melastatin mRNA expression appears to correlate with melanocytic tumor progression, melanoma tumor thickness, and the potential for melanoma metastasis. HUM PATHOL 31:1346:1356.

Targeted deletion of the tub mouse obesity gene reveals that tubby is a loss-of-function mutation.

The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and cochlear degeneration. A positional cloning effort to find the gene responsible for this phenotype led to the identification of tub, a member of a novel gene family of unknown function. A splice defect mutation in the 3' end of the tub gene, predicted to disrupt the C terminus of the Tub protein, has been implicated in the genesis of the tubby phenotype. It is not clear, however, whether the Tub mutant protein retains any biological activity, or perhaps has some dominant function, nor is it established that the tubby mutation is itself responsible for all of the observed tubby phenotypes. To address these questions, we generated tub-deficient mice and compared their phenotype to that of tubby mice. Our results demonstrate that tubby is a loss-of-function mutation of the tub gene and that loss of the tub gene is sufficient to give rise to the full spectrum of tubby phenotypes. We also demonstrate that loss of photoreceptors in the retina of tubby and tub-deficient mice occurs by apoptosis. In addition, we show that Tub protein expression is not significantly altered in the ob, db, or melanocortin 4 receptor-deficient mouse model of obesity.

Metallothionein-like proteins in the freshwater oligochaete Limnodrilus udekemianus and their role as a homeostatic mechanism against cadmium toxicity.

The purpose of this study was to determine if metallothioneins are present in the aquatic oligochaete Limnodrilus udekemianus and to determine the interplay between the presence of these proteins, cadmium (Cd) exposure, and Cd toxicity. The latter was geared specifically towards evaluating the role of metallothionein as a homeostatic mechanism against Cd toxicity. These issues are important for evaluating the usefulness of the quantification of metallothioneins as a biomonitoring tool. Worms in sediment were exposed to Cd under static conditions, with Cd initially added to the aqueous phase. Survival was monitored while respiration (as a measure of sublethal Cd effects) was determined immediately following exposure. Metallothioneins were separated from the cytosol by gel permeation high performance liquid chromatography (HPLC) while Cd levels were quantified in whole worms, cytosol and cytosolic fractions. Also, a Cd-saturation assay was used to determine the amounts of Cd bound to metallothionein and the total Cd-binding capacity of the metallothionein. Limnodrilus udekemianus has a metallothionein-like protein (an inducible cytosolic protein with an apparent molecular weight of approximately 15 kD that binds high levels of Cd and shows a red shift upon Cd binding). Sediment Cd levels above 60 microg/g were lethal to the worms (in 8-day exposures). Respiration rates at 13 and 41 microg/g Cd were not significantly different from controls, though cytosolic Cd levels were substantially increased in the 41 microg/g exposure. In this latter cytosol, Cd levels were significantly elevated in the low molecular weight pool (which includes metallothioneins) but not in the other pools, while the Cd-saturation assay also showed that worms in this group had significantly elevated levels of metallothionein-bound Cd. However, in all treatments the metallothionein was far from saturated by Cd. These observations indicate that no 'spill-over' of Cd was evident as lethal levels of Cd were approached. The overall cytosolic Cd distribution, and the degree of metallothionein saturation in Limnodrilus udekemianus thus do not appear to be good predictors of Cd toxicity in this species.

Targeted mutation reveals a central role for SR-BI in hepatic selective uptake of high density lipoprotein cholesterol.

Scavenger receptor BI (SR-BI) is a cell surface receptor that binds high density lipoproteins (HDL) and mediates selective uptake of HDL cholesteryl esters (CE) in transfected cells. To address the physiological role of SR-BI in HDL cholesterol homeostasis, mice were generated bearing an SR-BI promoter mutation that resulted in decreased expression of the receptor in homozygous mutant (designated SR-BI att) mice. Hepatic expression of the receptor was reduced by 53% with a corresponding increase in total plasma cholesterol levels of 50-70% in SR-BI att mice, attributable almost exclusively to elevated plasma HDL. In addition to increased HDL-CE, HDL phospholipids and apo A-1 levels were elevated, and there was an increase in HDL particle size in mutant mice. Metabolic studies using HDL bearing nondegradable radiolabels in both the protein and lipid components demonstrated that reducing hepatic SR-BI expression by half was associated with a decrease of 47% in selective uptake of CE by the liver, and a corresponding reduction of 53% in selective removal of HDL-CE from plasma. Taken together, these findings strongly support a pivotal role for hepatic SR-BI expression in regulating plasma HDL levels and indicate that SR-BI is the major molecule mediating selective CE uptake by the liver. The inverse correlation between plasma HDL levels and atherosclerosis further suggests that SR-BI may influence the development of coronary artery disease.

Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis.

We have used differential cDNA display to search for genes whose expression correlates with an aggressive phenotype in variants of the B16 murine melanoma line, B16-F1 and B16-F10. This analysis identified a novel gene, termed melastatin, that is expressed at high levels in poorly metastatic variants of B16 melanoma and at much reduced levels in highly metastatic B16 variants. Melastatin was also found to be differentially expressed in tissue sections of human melanocytic neoplasms. Benign nevi express high levels of melastatin, whereas primary melanomas showed variable melastatin expression. Melastatin transcripts were not detected in melanoma metastases. Within the set of human primary cutaneous melanomas examined, melastatin expression appeared to correlate inversely with tumor thickness. The expression pattern observed suggests that loss of melastatin expression is an indicator of melanoma aggressiveness.

Vascular MADs: two novel MAD-related genes selectively inducible by flow in human vascular endothelium.

Vascular endothelium is an important transducer and integrator of both humoral and biomechanical stimuli within the cardiovascular system. Utilizing a differential display approach, we have identified two genes, Smad6 and Smad7, encoding members of the MAD-related family of molecules, selectively induced in cultured human vascular endothelial cells by steady laminar shear stress, a physiologic fluid mechanical stimulus. MAD-related proteins are a recently identified family of intracellular proteins that are thought to be essential components in the signaling pathways of the serine/threonine kinase receptors of the transforming growth factor beta superfamily. Smad6 and Smad7 possess unique structural features (compared with previously described MADs), and they can physically interact with each other, and, in the case of Smad6, with other known human MAD species, in endothelial cells. Transient expression of Smad6 or Smad7 in vascular endothelial cells inhibits the activation of a transfected reporter gene in response to both TGF-beta and fluid mechanical stimulation. Both Smad6 and Smad7 exhibit a selective pattern of expression in human vascular endothelium in vivo as detected by immunohistochemistry and in situ hybridization. Thus, Smad6 and Smad7 constitute a novel class of MAD-related proteins, termed vascular MADs, that are induced by fluid mechanical forces and can modulate gene expression in response to both humoral and biomechanical stimulation in vascular endothelium.

Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation.

Chemokines are small secreted proteins that stimulate the directional migration of leukocytes and mediate inflammation. During screening of a murine choroid plexus complementary DNA library, we identified a new chemokine, designated neurotactin. Unlike other chemokines, neurotactin has a unique cysteine pattern, Cys-X-X-X-Cys, and is predicted to be a type 1 membrane protein. Full-length recombinant neurotactin is localized on the surface of transfected 293 cells. Recombinant neurotactin containing the chemokine domain is chemotactic for neutrophils both in vitro and in vivo. Neurotactin messenger RNA is predominantly expressed in normal murine brain and its protein expression in activated brain microglia is upregulated in mice with experimental autoimmune encephalomyelitis, as well as in mice treated with lipopolysaccharide. Distinct from all other chemokine genes, the neurotactin gene is localized to human chromosome 16q. Consequently we propose that neurotactin represents a new delta-chemokine family and that it may play a role in brain inflammation processes.

Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development.

To test the hypothesis that PTH-related peptide (PTHrP) is a paracrine regulator of endochondral bone development, we localized PTHrP and its cognate receptor during normal skeletal development at both messenger RNA (mRNA) and protein levels and compared the growth plate phenotypes of PTHrP-deficient [(PTHrP(-/-)] mice to those of normal littermates [PTHrP(+/+]. PTHrP mRNA was expressed adjacent to uncavitated joints, in the perichondrium of long bones and to a lower level in proliferating chondrocytes. In contrast, PTHrP protein was most evident at the interface of proliferating and hypertrophic zones, where it colocalized with PTH/PTHrP receptor mRNA and protein. Most strikingly, the proliferating zone was dramatically shorter in PTHrP(-/-) cartilage, although the percentage of cells in S-phase of the cell cycle in the proliferating zone was indistinguishable between PTHrP(+/+) and PTHrP(-/-) mice. Terminal differentiation of chondrocytes, which was characterized by cell hypertrophy, apoptosis (DNA fragmentation and decreased bcl-2 mRNA expression), and matrix mineralization, was more advanced in growth cartilage of PTHrP(-/-), compared with PTHrP(+/+) animals. These data demonstrate that PTHrP acts principally as a paracrine factor, which promotes elongation of endochondral bone by restraining or delaying the pace of chondrocytic development and terminal differentiation of growth-plate chondrocytes.

Identification and characterization of the mouse obesity gene tubby: a member of a novel gene family.

The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.

Localization of parathyroid hormone/parathyroid hormone-related peptide receptor mRNA in kidney.

In kidney, parathyroid hormone (PTH) exerts differential distal effects along the nephron. To define cells expressing receptors for PTH in kidney, we localized PTH/ PTH-related peptide (PTH/PTHrP) receptor mRNA in rat kidney by in situ hybridization. PTH/PTHrP receptor mRNA is localized to glomerular podocytes, convoluted and straight proximal tubules, the cortical portion of thick ascending limbs, and distal convoluted tubules, but was not detected in the thin limb of Henle's loop or in collecting ducts. Northern blot analysis showed that cultured human glomerular podocytes express a unique 4.0-kb PTH/PTHrP receptor transcript but do not express detectable levels of the common approximately 2.4-kb transcript found in whole kidney and in many other tissues. Whereas the tubular localization of PTH/PTHrP receptor mRNA coincides well with previously known sites of PTH action, the intense expression and the unique size of the PTH/PTHrP receptor transcript in glomerular podocytes suggest that PTH and/or PTHrP may play a role(s) in glomerular function.

Identification and expression cloning of a leptin receptor, OB-R.

The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.

Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acids during fetal development of rats.

PTH-related peptide (PTHrP) is a causative agent of hypercalcemia associated with malignancy. PTHrP binds to and activates the same receptor as does PTH. Because PTHrP has been suggested to regulate the growth and differentiation of cells as a paracrine/autocrine factor, we examined the expression of both PTHrP and its receptor genes during fetal development of rats (15-20 days gestation) by in situ hybridization with riboprobes. Both PTHrP and its receptor messenger RNAs (mRNAs) were expressed not only in the skeleton, but also in many fetal extraskeletal tissues, such as choroid plexus, ears, lungs, tooth buds, heart, and skin. In these extraskeletal tissues PTHrP mRNA was expressed mainly in surface-lining cells, whereas its receptor mRNA was expressed mainly in adjacent mesenchyma cells. In endochondral bones, these two genes were expressed in largely discrete, but mostly neighboring, areas, although the localizations of these two mRNAs changed over developmental stages. The expression patterns of PTHrP and its receptor mRNAs during fetal development suggest PTHrP's roles as a paracrine factor and its involvement in epithelial-mesenchymal(-like) interactions.

Localization of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor mRNAs in rat brain.

Parathyroid hormone (PTH)-related peptide (PTHrP) has been identified in human tumors associated with the syndrome of humoral hypercalcemia of malignancy. PTHrP mRNA is also expressed in a variety of non-malignant tissues, suggesting that PTHrP is an endogenous peptide with as-yet unidentified autocrine or paracrine functions in normal tissues, including brain (Weir et al., Proc. Natl. Acad. Sci., 87 (1990) 108-112). In the present study, we used in situ hybridization to examine the expression of PTHrP and the common receptor for PTH and PTHrP in adult rat brain. Widespread yet anatomically discrete patterns of hybridization were observed using 35S-labeled antisense cRNA probes. PTHrP gene expression was highest in the supramamillary nucleus of the hypothalamus, medial superior olivary nucleus, and in subpopulations of cells in the neostriatum, hippocampus, and cerebral cortex. Other major sites of PTHrP gene expression included the amygdala, midline thalamic nuclei, pontine nuclei, choroid plexus, and the anterior pituitary gland. Highest levels of PTH/PTHrP receptor mRNA were in the mesencephalic portion of the trigeminal nucleus and the trigeminal ganglion, the lateral reticular, pontine and reticulotegmental nuclei, the hypoglossal nucleus and area postrema. Other major sites of PTH/PTHrP receptor expression included the anterodorsal nucleus of the thalamus, basolateral amygdala, entorhinal cortex, parasubiculum, cells in the Purkinje cell layer of the cerebellum, vestibular nuclei, ventral cochlear nucleus, the motor nucleus of the trigeminal, and the facial and external cuneate nuclei. The expression of genes encoding PTHrP and its receptor in discrete areas of the brain suggests that PTHrP may function as a neurotransmitter in the central nervous system.

Parathyroid hormone induces sequential c-fos expression in bone cells in vivo: in situ localization of its receptor and c-fos messenger ribonucleic acids.

Localization of PTH/PTH-related peptide (PTH/PTHrP) receptor mRNA and PTH induction of c-fos expression were examined in bones of 4-week-old rats by in situ hybridization. Receptor transcripts were most highly expressed by growth plate chondrocytes from lower proliferating to upper hypertrophic cell layers. PTH/PTHrP receptor mRNA also was expressed in osteoblasts as well as in some bone marrow stromal cells. Subcutaneous administration of human PTH-(1-84) (225 micrograms/kg) induced rapid, transient, and sequential expression of the protooncogene c-fos mRNA in cells in bone. Osteoblasts and chondrocytes expressing PTH/PTHrP receptor transcripts as well as some stromal cells expressed c-fos mRNA first (15-60 min), followed by transient expression in the majority of stromal cells and in osteoclasts (1-2 h). Delayed and transient induction of c-fos in cells with few or no detectable receptor transcripts suggests that PTH acts indirectly on stromal cells and osteoclasts by either stimulating osteoblasts to secrete a substance(s) that acts locally and/or inducing changes in cell-cell contacts between osteoblasts and adjacent cells.

In situ localization of PTH/PTHrP receptor mRNA in the bone of fetal and young rats.

We characterized cells that express parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor mRNA in bones of fetal and postnatal rats by in situ hybridization. During endochondral development of fetal bones, PTH/PTHrP receptor transcripts were highly expressed both in maturing chondrocytes and in osteoblasts in the periosteum and ossification center, but not in fully hypertrophic chondrocytes. Similar to the localization in the fetal bones, PTH/PTHrP receptor mRNA expression was highly localized to maturing chondrocytes in the articular cartilage and growth plate, and to osteoblasts in the femur of young rats. In both young and fetal rats, transcripts for Type X collagen were localized to hypertrophic chondrocytes, mostly between chondrocytes and bone cells both of which express PTH/PTHrP receptor mRNA. Transcripts for PTH/PTHrP receptors and alkaline phosphatase co-localized in the bone of young rats, but they did not co-localize in fetal bones at the early stages of endochondral ossification. These results show that PTH/PTHrP receptor mRNA is expressed in a cell-type and stage-specific manner during skeletal development.

Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype.

A novel adenosine receptor subtype has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA, designated RFL9, encodes a protein of 332 amino acids. The structure of RFL9 is most similar to that of the recently cloned rat A2-adenosine receptor, with a sequence identity of 73% within the presumed seven transmembrane domains. Expression of RFL9 in COS-6M cells resulted in ligand binding and functional activity characteristics of an adenosine receptor that is coupled positively to adenylyl cyclase. Examination of the tissue distribution of RFL9 mRNA by Northern blot analysis showed a restricted distribution with highest levels expressed in large intestine, cecum, and urinary bladder; this pattern was distinct from that of either the A1- or A2-adenosine receptor mRNAs. In situ hybridization studies of RFL9 mRNA showed no specific hybridization pattern in brain, but a hybridization signal was readily observed in the hypophyseal pars tuberalis. Thus, RFL9 encodes a novel A2-adenosine receptor subtype.