Tissue-specific Variation Research Articles

Expression of splice variants of 1α-hydroxylase in mcf-7 breast cancer cells

1,25-Dihydroxyvitamin D3 (calcitriol) is the most active natural metabolite of Vitamin D3. It has strong antiproliferative and differentiating effects on various cell types including breast cancer cells. 25-Hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase, CYP27B1) is one of the key enzymes in the formation of calcitriol. It has been found in breast cancer cells suggesting an autocrine regulation of formation of calcitriol in these cells. Alternative splicing of the encoding genes for this enzyme can possibly play a role in regulating the enzyme level and can explain tissue specific variations of 1α-hydroxylase activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. In order to obtain more information on the abundance of 1α-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The full-length sequence of 1α-hydroxylase and two different splice variants of this enzyme containing intron 1 were isolated. By Western blot technique we then confirmed the protein products of the full-length enzyme and its splice variants. We hypothesize that that the expression of splice variants can lead to a quantitatively lower expression of the mRNA of the full-length enzyme. The abundance of less active 1α-hydroxylase protein variants can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues.

Normal development and neoplasia: the imprinting connection.

The observation that a number of autosomal genes are expressed in a parent of origin-dependent monoallelic manner has fuelled a frantic research effort into the underlying mechanisms and biological functions of this phenomenon, termed genomic or parental imprinting. The level of intrigue associated with this subject has been heightened by the discovery that the "transcriptional phenotype" of some imprinted genes shows developmental and tissue-specific variation, and that some imprinted genes are expressed biallelically in tumors. Here we describe some further examples of variation in the allele-specific transcription of an imprinted gene, human IGF2. Analysis of different sub-clones of an established tumor cell line (Jeg-3) revealed examples of both a switch from monoallelic to biallelic expression, as well as monoallelic expression from the opposite parental allele. Examination of IGF2 expression in adult human liver clearly demonstrated that the functional imprinting is manifested in a promoter-specific manner. The P1 promoter produced biallelically derived transcripts, whereas the remaining three promoters were utilized in a complex pattern of mono- and biallelic expression which varied from sample to sample. These observations emphasize the need to re-examine the imprinting phenomenon and its plasticity in terms of the cis elements and trans-acting factors involved in the transcriptional regulation of these genes both in the normal and pathological contexts.

Ribosomal Protein S24 Gene Is Mutated in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare congenital red-cell aplasia characterized by anemia, bone-marrow erythroblastopenia, and congenital anomalies and is associated with heterozygous mutations in the ribosomal protein (RP) S19 gene (RPS19) in approximately 25% of probands. We report identification of de novo nonsense and splice-site mutations in another RP, RPS24 (encoded by RPS24 [10q22-q23]) in approximately 2% of RPS19 mutation-negative probands. This finding strongly suggests that DBA is a disorder of ribosome synthesis and that mutations in other RP or associated genes that lead to disrupted ribosomal biogenesis and/or function may also cause DBA.

Ribosomal Protein S24 Gene Is Mutated in Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia with marked clinical heterogeneity, an increased risk of malignancy and mutations in ribosomal protein (RP) S19 in 25% of probands. To identify other gene(s) mutated in DBA and investigate their expression and function, we performed a genome-wide screen using a 10,000 single nucleotide polymorphism mapping set (Affymetrix) on a large family comprising 10 informative meioses. We found linkage of the DBA phenotype to regions on chromosome 8q, 10 and 6. The RP gene RPS24, is located in the linked region on chromosome 10; we sequenced exons, intron-exon boundaries and the promoter regions in this family, and found a nonsense mutation (316C>T) in exon 4 of RPS24 in five affected individuals, and a wild type sequence in five unaffected family members. This mutation causes the change of Gln106STOP and is predicted to result in formation of a truncated RPS24 protein. Subsequently, we sequenced DNA from 215 unrelated DBA probands, 30 with RPS19 mutations and 185 without. We found another nonsense mutation in exon 2 in a sporadic case, and a splice site deletion resulting in skipped exon 2 in another proband and in his father; over 200 control individuals did not have any of the above sequence changes, indicating that they are pathogenic mutations. To explore the normal role of RPS24 and consider how its dysfunction might result in DBA we performed real time RT-PCR (rt-PCR) and western blotting experiments on 20 normal human tissues and on lymphoblastoid cell lines from diseased and control individuals. Interestingly, rt-PCR of total human RPS24 and RPS19 mRNA revealed a tissue-specific variation in expression level. We found co-ordinate expression of both genes in the majority of studied tissues. Lymphoblastoid cell lines from both probands with nonsense mutations showed a reduced level of RPS24 mRNA, suggesting degradation of mutated transcripts due to nonsense mediated decay, while the RPS19 mRNA level in these patients was normal or elevated. Western blot experiments revealed a reduction of RPS24 protein in lymphoblastoid cell lines from all three mutated probands compared to control samples. Interestingly, co-ordinate expression of RPS24 and RPS19 protein was found in these patients as well as in other patients with RPS19 mutations or without any mutations, suggesting co-regulation of RP expression. To determine whether recruitment of mRNA to polysomes was impaired in DBA patients, we separated lymphoblast cell line lysates from nine diseased and four control individuals on sucrose gradients. We did not detect any significant difference in the RNA ratio of polysome-bound/free ribosomal subunits between diseased and control samples (p<0.3). It is likely that lymphoblasts, which are not defective in DBA, have sufficient ribosomes to mask an abnormality in translation.In summary, our results suggest that in addition to RPS24 and RPS19, which are mutated in ~ 27% of index cases, other DBA genes are also RP genes or genes involved in ribosome biogenesis or translation, and reinforce the notion that DBA is a ribosomal disease. This study opens new avenues for studying and understanding the pathogenesis of DBA.

Convergence, constraint and the role of gene expression during adaptive radiation: floral anthocyanins in Aquilegia

Convergent phenotypes are testament to the role of natural selection in evolution. However, little is known about whether convergence in phenotype extends to convergence at the molecular level. We use the independent losses of floral anthocyanins in columbines (Aquilegia) to determine the degree of molecular convergence in gene expression across the anthocyanin biosynthetic pathway (ABP). Using a phylogeny of the North American Aquilegia clade, we inferred six independent losses of floral anthocyanins. Via semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we monitored developmental and tissue-specific variation in expression of the six major structural ABP loci in three Aquilegia species, two that produce anthocyanins (A+) and one that does not (A-). We then compared ABP expression in petals of old-bud and pre-anthesis flowers of 13 Aquilegia species, eight wild species and two horticultural lines representing seven independent A- lineages as well as three wild A+ species. We only found evidence of down-regulation of ABP loci in A- lineages and losses of expression were significantly more prevalent for genes late in the pathway. Independent contrast analysis indicates that changes in expression of dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) are strongly phylogenetically correlated consistent with the multilocus targets of trans-regulatory elements in the ABP of other systems. Our findings strongly suggest that pleiotropy constrains the evolution of loss of floral anthocyanins to mutations affecting genes late in the ABP mostly through convergent changes in regulatory genes. These patterns support the hypothesis that rapid evolutionary change occurs largely through regulatory rather than structural mutations.

Presentation of splice variants of 1-alpha-hydroxylase in MCF-7 breast cancer cells

20097 Background: Calcitriol (1,25-dihydroxyvitamin D3) is the most active metabolite of vitamin D. It is known for its antiproliferative and differentiating effects on many cell populations including MCF-7 and other breast cancer cells. 1α-hydroxylase (CYP27B1) one of the key enzymes has been found in breast cancer cells suggesting an autocrine regulation of producing calcitriol. An alternative splicing of the encoding genes for the1α-hydroxylase might play a role in regulating enzyme levels and can explain tissue specific variations of the enzyme activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. Methods: In order to obtain more information on the abundance of 1α-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The protein products of the full length enzyme and its slice variants were confirmed by Western Blot. Results: The full length sequence of 1α-hydroxylase and two different splice variants including intron 1 were isolated. Conclusion: We hypothesize that the expression of the splice variants can lead to a quantitatively lower expression the mRNA of the full length enzyme. The abundance of less active 1α-hydroxylase protein products can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues. No significant financial relationships to disclose.

Tissue-specific effect of ascorbic acid supplementation on the expression of cytochrome P450 2E1 and oxidative stress in streptozotocin-induced diabetic rats

Here, we investigated the effect of l-ascorbic acid (AA) supplementation on the CYP2E1 expression level and oxidative stress in various tissues such as the liver, kidney, pancreas, and brain of streptozotocin (STZ)-induced diabetic rats. An increased cytochrome P450 2E1 (CYP2E1) expression level with a concomitant increase in the production of reactive oxygen species were found in all the tissues of STZ-induced diabetic rats tested compared with an untreated control, suggesting the possible diabetes-induced tissue injury. In contrast, the AA supplementation to the diabetic rats alleviated these experimental parameters in a tissue-specific manner. AA affected the liver most severely followed by the kidney. There was little or no effect of AA supplementation on the brain and pancreas. The circulation level of the ketone bodies, inducers of CYP2E1, was also decreased by AA supplementation compared with those of the diabetic rats. Therefore, the suppression of ketone production by AA can be one of the mechanisms of a reduction in CYP2E1. These results suggest that AA plays an important role in reducing elevated CYP2E1 expression level and the oxidative stress mediated by type 1 diabetes with a tissue-specific variation.

Preparative laser capture microdissection and single-pot cell wall material preparation: a novel method for tissue-specific analysis

In adaptation to their function the walls of plant cell display tissue-specific variations of composition according to their developmental stage, cell type and stress of various origin. It is therefore important to obtain a precise analytical data describing the cell wall composition with respect to these different factors. In the present work, laser capture microdissection (LCM) was used for isolating different tissues from the stem of Urtica dioica L. at a semi-preparative scale. The technique was associated for the first time to a one-pot sequential cell wall preparation and hydrolysis for the carbohydrate analysis of each cell type. The results demonstrate that the combination of LCM and micro-analytical methods can provide individual cell type composition and should improve our knowledge of the biochemical diversity of cell walls in plants. This approach will be of potential interest for the understanding of the effects of stress or genetic engineering on the composition of the cell walls.

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.

New quantitative Methylation‐Sensitive Arbitrarily Primed PCR (qMS AP‐PCR) method to determine DNA methylation level in cancer

The aim of the study was to develop a sensitive method to identify and quantify methylated DNA fragment in cancer. The genomic DNA of three different tissues (primary cancer of the liver (T-Lv), lung (T-Lg) and kidney (T-Kd)) was analyzed and compared to adjacent normal tissues from the same individuals. After DNA digestion with RsaI, then with a methylation-sensitive and non-sensitive restriction enzymes (HpaII and MspI), digested products were submitted to the AP-PCR amplification. Amplicons were subsequently separated on a Labchip of the Agilent bioanalyzer and the methylated fragments were sized and quantified. We have identified a set of methylated DNA fragments. Although results shown tissue specific variation of methylation level some methylated fragments were common in cancer tissues. There was 100% methylation for a 333 bp fragment in T-Lg and T-Kd and in normal tissues. This same fragment was hypomethylated in T-Lv. Partial hypermethylation of a 357 pb fragment was observed in T-Kd, T-Lv and normal tissues. In T-Lg, methylation was 100 %. A 398 bp fragment was 100 % methylated in T-Kd, but not methylated in T-Lv and T-Lg. The fragment was hypomethylated in normal kidney and liver tissue, 62 % methylated in normal lung tissue. The method is a helpful tool to identify and quantify specific DNA fragments sensitive to methylation.

Expression and regulation of 1-acyl-sn-glycerol- 3-phosphate acyltransferases in the epidermis

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred. Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.

Antiglioma activity of 2,2′:6′,2ʺ-terpyridineplatinum(II) complexes in a rat model—Effects on cellular redox metabolism

The mammalian thioredoxin system, comprising the selenoenzyme thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin (Trx), is implicated in thiol-mediated antioxidant defense and redox regulatory processes including transcriptional control, DNA synthesis, and apoptosis. Cell proliferation supported by the thioredoxin system can be suppressed by TrxR inhibition. In this study, we assessed the effects of the potent hTrxR inhibitors 4-mercaptopyridine (4′-chloro-2,2′:6′,2ʺ-terpyridine)platinum nitrate (I 232N) and 2-mercaptopyridine (4′-chloro-2,2′:6′,2ʺ-terpyridine)platinum nitrate (I 252N) on glioblastoma in a rat model. These compounds show no or little cross-resistance with cisplatin and are thus of great clinical interest. Triple intravenous application of 25–35 mg/kg of the compounds led to a significant decrease of tumor growth as determined by magnetic resonance imaging. Metabolic as well as redox parameters in the blood of the animals were not altered. However, TrxR activity was significantly decreased in the tumor tissue, and redox parameters—including glutathione concentrations, total antioxidant status, and the activities of different antioxidant enzymes—showed tissue-specific variations. As indicated by different apoptotic markers, the antitumor activity of I 232N is not mediated by the induction of programmed cell death but rather by hTrxR inhibition and DNA intercalation leading to cell cycle arrest.

Copy Number of mtDNA Common Deletion (δmtDNA 4977) Showing Tissue-Specific and Age-Related in the Same Individual Determined by Real-Time PCR

There have been controversial arguments on both the case for and against on the accumulation of mtDNA deletion in association to tissue and age. The debate continues as to whether this mutation is a major contributor to the phenotypic expression of aging and common degenerative diseases or simply a clinical in significant epiphenomenona. The objective of this study was to determine whether δmtDNA4977 copy number is correlated with age-related and tissue-specific variation. Prospective pilot study One hundred and forty tissues from blood, ovary, uterine muscle, and abdominal muscle were obtained from patients ranging in age from 20-59 years. After reviewing the clinical reports, patients with mitochondrial disorder were excluded from this study. Total DNA isolated from all samples. Sample previously identified as δmtDNA4977 deleted DNA served as control based on real-time PCR. The δmtDNA4977 plasmid DNA standard was constructed for the area around deleted mtDNA into pGEM-T Easy Vector System II (thereafter called pGEM-δmtDNA4977) and identified by bidirectional sequencing. Real-time PCR amplification was carried out in Opticon 2 (MJ Research) with 50 ng of DNA and QuantiTect SYBR Green PCR master mix. Tabled 1 In the present study, we found that copy number of δmtDNA4977 was rarely detectable in the lymphocytes whereas the deletion was frequently observed in ovary, uterine muscle and abdominal muscle. This study confirms that absence of the mtDNA deletion in rapidly dividing cells, such as lymphocytes, is understandable if we accept that such tissues may lose the defect by selective pressure. On the other hand, high copy number of δmtDNA4977 was found in ovary, uterine muscle and abdominal muscle during the ageing process. The mechanisms for the accumulation of mtDNA deletions in the ageing process are still unknown. Slip-misparing has been proposed to be the most common mechanism associated with the genesis of mtDNA rearrangements. Oxidative damage to mtDNA also increases with age, suggesting that these two mechanisms may be associated to the generation of age-related deletions. One of he main sources of free radicals play a role in the genesis of mtDNA rearrangements and oxidative damage to mtDNA is the respiratory chain. Respiratory chain defects can also show marked within-tissue variation; classic examples are mtDNA deletions in skeletal muscle. Within-tissue variation can be particularly pronounced in the ovaries; a striking difference in the mutant loads of individual oocytes has been reported from a carrier of the mtDNA T8993G mutation. This is presumably a germ cell segregation effect related to the mitochondria bottleneck phenomenon. In conclusion, copy number of δmtDNA4977 showing age-related are tissue-specificity.

Tissue-specific variation of δ13C in mature canopy trees in a temperate forest in central Europe

Stable carbon isotope analysis has become a key toot in functional ecology, yet considerable natural variability often limits the interpretations. In this study we document the spatial, taxonomic, temporal and tissue-specific delta(13)C variability in 10 tree species of a temperate European forest. The Swiss Canopy Crane provided access to the three dimensional space within 55 trees 30-35 m high representing the genera Acer, Carpinus, Fagus, Prunus, Quercus, Tilia, Abies, Larix, Picea and Pinus. The results from six broad-leaved and four conifer species (seven deciduous, three evergreen) documented that the species effect was not significant in contrast to tissue-specific and spatial differences in the canopy. Year-to-year differences were not large but still significant. Our analysis confirmed a significant difference between delta(13)C of foliage collected in the upper and lower canopy, but revealed no systematic differences with respect to azimuthal directions in tree crowns of the broad-leaved trees, as opposed to the conifers, which show clear differences between the sun-exposed and the shaded side. Tissue-specific differences were significant, despite surprisingly similar mean values for most tissue types. Such tissue effect was largely due to young branch xylem, which exhibited a systematic less negative deviation from the other tissue types. These findings were consistent across the species tested and provided some guidelines towards a representative sampling strategy for 13 C analysis.

Genome size and endonuclear DNA replication in spiders

Although genome sizes (C-values) are now available for 115 arachnid species (Gregory and Shorthouse [2003] J Hered 94:285-290), the extent of genome amplification (endonuclear DNA replication or polyploidization) accompanying tissue differentiation in this diverse and abundant class of invertebrates remains unknown. To explore this aspect of arachnid development, samples of hemolymph and other tissues were taken from wild-caught specimens as air-dried smears, stained with the Feulgen reaction for DNA, and assayed using both scanning and image analysis densitometry. Cells from midgut diverticula and Malpighian tubules of Argiope and Lycosa (=Pardosa) often showed giant nuclei with 50-100 pg of DNA per nucleus, reflecting at least four cycles of endonuclear DNA replication when compared to the DNA content of hemocytes or sperm from the same specimen. Nuclei with markedly elevated DNA levels also appeared, but far less frequently, in tissue samples from several other arachnid species (Antrodiaetus, Hypochilus, Latrodectus, Liphistus and Loxosceles), but revealed no correlation with differences in somatic cell (2C) genome sizes. Our data show that several DNA classes of polysomatic nuclei regularly arise during tissue differentiation in some species of spiders and may provide an interesting model system for further study of patterns of tissue-specific variation in DNA endoreduplication during development.

Sex-related Differences in Gene Expression in Salivary Glands of BALB/c Mice

Sex-related differences exist in the structure and function of the major glands in a variety of species. Moreover, many of these variations appear to be unique to each tissue. We hypothesized that this sexual dimorphism is due, at least in part, to gland-specific differences in gene expression between males and females. Glands were collected from male and female BALB/c mice (n = 5/sex/experiment), and total RNA was isolated. Samples were analyzed for differentially expressed mRNAs with CodeLink microarrays, and data were evaluated by GeneSifter. Our results demonstrate that significant (P < 0.05) sex-related differences exist in the expression of numerous genes in the major salivary glands, and many of these differences were tissue-specific. These findings support our hypothesis that sex-related differences in the salivary glands are due, at least in part, to tissue-specific variations in gene expression.

Molecular and functional analysis of Caenorhabditis elegans CHIP, a homologue of Mammalian CHIP

A recently identified molecule C-terminus of Hsc70 interacting protein (CHIP) has been reported to be an E3 ubiquitin ligase collaborating with molecular chaperones for the degradation of misfolded or unfolded proteins. The physiological roles of CHIP in animal and plant development remain largely unknown. Here, we show that the knockdown of CeCHIP by RNAi and knockout by a deletion mutation arrests the development of the animal at the larval stage. CeCHIP expresses ubiquitously in all tissues but there are tissue specific variations of expression level. CeCHIP produces dose dependent phenotypes in vivo. Over expression of CHIP causes embryonic lethality, while a comparatively lower level of over expression causes locomotion and egg laying defects, and the CHIP over expressed animals form dauers at a higher temperature.

Chemokines, chemokine receptors and adhesion molecules on different human endothelia: discriminating the tissue-specific functions that affect leucocyte migration

The selective accumulation of different leucocyte populations during inflammation is regulated by adhesion molecules and chemokines expressed by vascular endothelium. This study examined how chemokine production and the expression of adhesion molecules and chemokine receptors vary between endothelia from different vascular beds. Human saphenous vein endothelium was compared with lung and dermal microvascular endothelia and with umbilical vein endothelium and a bone-marrow endothelial cell line. All endothelia produced CCL2 and CXCL8 constitutively, whereas CXCL10 and CCL5 were only secreted after tumour necrosis factor (TNF)-alpha or interferon (IFN)-gamma stimulation. In combination with TNF-alpha, IFN-gamma suppressed CXCL8 but enhanced CCL5 and CXCL10, whereas transforming growth factor (TGF)-beta reduced secretion of all chemokines. Basal chemokine secretion was higher from umbilical vein than other endothelial cells. Chemokine receptors, CXCR1, CXCR3 and CCR3, were present on all endothelia but highest on saphenous vein. CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 were also detected at variable levels on different endothelia. The variation between endothelia in chemokine secretion was much greater than the variations in adhesion molecules, both on resting cells and following cytokine stimulation. These results indicate that it is the tissue-specific variations in endothelial chemokine secretion rather than variations in adhesion molecules that can explain the different patterns of inflammation and leucocyte traffic seen in non-lymphoid tissues.

Identification and characterization of mouse Rab32 by mRNA and protein expression analysis

Rab proteins, a subfamily of the ras superfamily, are low molecular weight GTPases involved in the regulation of intracellular vesicular transport. Cloning of human RAB32 was recently described. Presently, we report the cloning and characterization of the mouse homologue of Rab32. We show that murine Rab32 exhibits a ubiquitous expression pattern, with tissue-specific variation in expression level. Three cell types with highly specialized organelles, melanocytes, platelets and mast cells, exhibit relatively high level of Rab32. We show that in murine amelanotic in vitro transformed melanocytes as well as in human amelanotic metastatic melanoma cell lines, the expression of Rab32 is markedly reduced or absent, in parallel with the loss of expression of two key enzymes for the production of melanin, tyrosinase and Tyrp1. Therefore, in both mouse and human systems, the expression of Rab32 correlates with the expression of genes involved in pigment production. However, in melanoma samples, amelanotic due to a mutation in the tyrosinase gene, the expression of Rab32 remains at levels comparable to those observed in pigmented melanoma samples. Finally, we observed co-localization of Rab32 and the melanosomal proteins, Tyrp1 and Dct, indicating an association of Rab32 with melanosomes. Based on these data, we propose the inclusion of Rab32 to the so-called melanocyte/platelet family of Rab proteins.

Tissue-specific HSP70 levels and reproductive physiological responses in fishes inhabiting a metal-contaminated creek.

The 70-kDa stress protein family (HSP70) plays important roles in a variety of physiological processes, including protein chaperoning, protection against apoptosis, steroidogenesis, and general cellular stress responses in vertebrate organisms, and has also been proposed as a biochemical marker of environmental stress, such as toxicant exposure. The objectives of this study were to determine HSP70 protein expression in head kidney, liver, gill, and ovarian tissues and to examine reproductive physiological responses in female fishes exposed chronically to sublethal metal concentrations. Female black bullhead (Ameiurus melas) and bluegill sunfish (Lepomis macrochirus) were collected from Tar Creek, Oklahoma (flowing through the Tri-State mining district) and from a nearby reference creek (Lytle Creek) during spring (prespawning; 26.5 +/- 0.95 degrees C water temperature) and winter (ovarian recrudescence; 4.8 +/- 0.80 degrees C water temperature). Aqueous (dissolved and suspended) concentrations of Cd and Zn and liver concentrations of Cd and Zn in both fish species were significantly greater at Tar Creek compared to Lytle Creek. HSP70 expression was consistently elevated in the head kidney of both fish species collected at Tar Creek in comparison to fish collected from the reference creek. In contrast, no consistent differences were observed in HSP70 expression in liver, gill, or ovarian tissues between sites. Significant seasonal differences were observed in expression of HSP70 in gill tissue of both species, in ovarian and liver tissue of bluegill sunfish and in head kidney of black bullhead. Serum testosterone concentration was significantly reduced in sunfish collected from Tar Creek during winter. Gonadosomatic and hepatosomatic indices were significantly lower in black bullhead collected from Tar Creek during spring, and condition factors were lower in black bullhead collected from Tar Creek during both spring and winter. There was no significant difference in the extent of ovarian follicular cell apoptosis in either species collected during spring. In conclusion, we observed significant tissue specific differences and seasonal variation in expression of HSP70, as well as alterations in circulating testosterone levels in female fish chronically exposed to metals.