The proteomic profiling of multiple tissue damage in chickens for a selenium deficiency biomarker discovery.

Over the past decades, substantial advances have been made in both the early diagnosis and accurate prognosis of numerous cancers because of the impressive development of novel proteomic strategies. Selenium (Se) is an essential trace element in humans and animals. Se deficiency could lead to Keshan disease in humans, mulberry heart disease in pigs and damage of tissues including cardiac injury, apoptosis in the liver, reduction in the immune responses in spleen and cerebral lesions in chickens. However, it is well know that plasma biomarkers are not specific and also show alterations in various diseases including those caused by Se deficiency. Therefore, new definition biomarkers are needed to improve disease surveillance and reduce unnecessary chicken losses due to Se deficiency. To identify new biomarkers for Se deficiency, we performed exploratory heart, liver, spleen, muscle, vein, and artery proteomic screens to further validate the biomarkers using Venn analysis, GO enrichment, heatmap analysis, and IPA analysis. Based on the bioinformatics methods mentioned above, we found that differentially expressed genes and proteins are enriched to the PI3K/AKT/mTOR signal pathway and insulin pathway. We further used western blot to detect the expression of proteins related to the two pathways. Results showed that the components of the PI3K/AKT/mTOR signal pathway were definitely decreased in heart, liver, spleen, muscle, vein and artery tissues in the Se deficient group. Expression IGF and IGFBP2 of the insulin pathway were differentially increased in the heart, liver, and spleen in Se deficient group samples and decreased in muscle and artery. In conclusion, 5 proteins, namely PI3K, AKT, mTOR, IGF, and IGFBP2, were differentially expressed, which could be potentially useful Se deficient biomarkers. In the present study, proteomic profiling was used to elucidate protein biomarkers that distinguished Se deficient samples from the controls, which might provide a new direction for the diagnosis and targeted treatment induced by Se deficiency in chickens.

The Effect of Diet-induced Obesity on Toxicological Parameters in the Polygenic Sprague-Dawley Rat Model.

The obese rodent serves as an indispensable tool for proof-of-concept efficacy and mode-of-action pharmacology studies. Yet the utility of this disease model as an adjunct to the conventional healthy animal in the nonclinical safety evaluation of anti-obesity pharmacotherapies has not been elucidated. Regulatory authorities have recommended employing disease models in toxicology studies when necessary. Our study investigated standard and exploratory toxicology parameters in the high-fat diet (HFD)-induced obese, polygenic Sprague-Dawley rat model in comparison to chow diet (CD)-fed controls. We sought to establish feasibility of the model for safety testing and relevance to human obesity pathophysiology. We report that both sexes fed a 45% kcal HFD for 29 weeks developed obesity and metabolic derangements that mimics to a certain extent, common human obesity. Minor clinical pathologies were observed in both sexes and considered related to CD versus HFD differences. Histopathologically, both sexes exhibited mild obesity-associated findings in brown and subcutaneous white fat, bone, kidneys, liver, lung, pancreas, salivary parotid glands, and skeletal muscle. We conclude that chronic HFD feeding in both sexes led to the development of an obese but otherwise healthy rat. Therefore, the diet-induced obese Sprague-Dawley rat may serve as a suitable model for evaluating toxicity findings encountered with anti-obesity compounds.

Changes in expression of neuropeptides and their receptors in the hypothalamus and gastrointestinal tract of calorie restricted hens.

The list of orexigenic and anorexigenic peptides, those are known to alter feed intake, is continuously growing. However, most of them are studied in mammalian species. We aimed to investigate plasma level and mRNA expression of the pituitary adenylate cyclase-activating polypeptide (PACAP), gene expression of its receptor (PAC1), furthermore the gene expression of galanin (GAL), neuromedin U (NMU), and its two receptors (NMUR1 and NMUR2) in the hypothalamus, proventriculus, and jejunum of hens exposed to 40% calorie restriction. Feed restriction resulted in a 88% increase in mRNA and a 27% increase in peptide level of PACAP in proventriculus measured with qPCR and RIA, respectively. Increases were found in the gene expression of PAC1 (49%) and NMUR1 (63%) in the hypothalamus. Higher expressions of peptide encoding genes (76% for PACAP, 41% for NMU, 301% for NMUR1 and 308% for GAL, P < 0.05) were recorded in the jejunum of hens exposed to restricted nutrition. The results indicate that PACAP level responds to calorie restriction in the proventriculus and jejunum, but not in the hypothalamus and plasma.

Interleukin-18 and its receptor are expressed in gonadotropin-releasing hormone neurons of mouse and rat forebrain.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine and an important mediator of peripheral inflammation and host immune response. IL-18 functions through its binding with the IL-18 receptor (IL-18R), which consists of two chains, an IL-18-binding α chain (IL-18Rα) and a signaling ß chain. IL-18 and IL-18R are expressed in the brain; however, limited information is available on IL-18R expression and the role of IL-18 in neurosecretory cells. In the present study, we used immunohistochemical techniques to investigate the distribution of IL-18Rα and IL-18 in the hypothalamus of male mice and rats. IL-18Rα-positive and IL-18-positive perikarya and fibers were found scattered throughout the medial septal nucleus, the nuclei of the vertical and horizontal limbs of the diagonal band, the organum vasculosum of the laminae terminalis, the preoptic area, and the anterior hypothalamic area. It is well known that gonadotropin-releasing hormone (GnRH) neuronal somata and/or fibers are found in these regions. Therefore, we performed double-label immunofluorescence for IL-18Rα/IL-18 and GnRH. IL-18Rα was expressed in approximately 60% of GnRH-immunopositive perikarya, and IL-18 was distributed in all GnRH-immunopositive perikarya. These observations suggest that IL-18 exerts direct effects upon the GnRH neuron via IL-18Rα and acts on GnRH neurons through an autocrine or paracrine pathway.

Anatomical regional differences in selenium levels in the human brain.

The role of selenium in human brain physiology, as well as in aging and neurodegenerative processes, remains unclear. Thus, the aim of this study was to establish the "normal" (reference) levels for selenium in the human brain, as well as anatomical regional differences and age-related changes. Using inductively coupled plasma-mass spectrometry after sample microwave-assisted acid digestion, selenium levels were measured in 14 different areas of the brain of adult individuals (n = 42; 71 ± 12, range 50-101 years old) without a known history of neurodegenerative, neurological, or psychiatric disorders. In the whole data set (n = 588; 42 individuals × 14 brain areas), selenium levels ranged from 552 to 1435 ng/g, with a mean ± SD content of 959 ± 178 ng/g (dry weight basis). Selenium distribution across the different brain areas was heterogeneous, with the highest levels in the putamen, parietal inferior lobule, and occipital cortex and the lowest expression in the medulla and cerebellum. Selenium levels were unchanged with aging. Compared with the age-matched control group, significantly increased levels of selenium were found in the globus pallidus, superior temporal gyrus, and frontal cortex of Parkinson's disease (n = 1) and Alzheimer's disease (n = 2) patients. This study provides new data on the anatomical regional differences in selenium levels in the human brain, which will aid future interpretation of studies examining brain tissue affected by neurodegenerative (and other) brain diseases.

Molecular identification and functional characterization of the human colonic thiamine pyrophosphate transporter.

Colonic microbiota synthesize a considerable amount of thiamine in the form of thiamine pyrophosphate (TPP). Recent functional studies from our laboratory have shown the existence of a specific, high-affinity, and regulated carrier-mediated uptake system for TPP in human colonocytes. Nothing, however, is known about the molecular identity of this system. Here we report on the molecular identification of the colonic TPP uptake system as the product of the SLC44A4 gene. We cloned the cDNA of SLC44A4 from human colonic epithelial NCM460 cells, which, upon expression in ARPE19 cells, led to a significant (p < 0.01, >5-fold) induction in [(3)H]TPP uptake. Uptake by the induced system was also found to be temperature- and energy-dependent; Na(+)-independent, slightly higher at acidic buffer pH, and highly sensitive to protonophores; saturable as a function of TPP concentration, with an apparent Km of 0.17 ± 0.064 µM; and highly specific for TPP and not affected by free thiamine, thiamine monophosphate, or choline. Expression of the human TPP transporter was found to be high in the colon and negligible in the small intestine. A cell surface biotinylation assay and live cell confocal imaging studies showed the human TPP transporter protein to be expressed at the apical membrane domain of polarized epithelia. These results show, for the first time, the molecular identification and characterization of a specific and high-affinity TPP uptake system in human colonocytes. The findings further support the hypothesis that the microbiota-generated TPP is absorbable and could contribute toward host thiamine homeostasis, especially toward cellular nutrition of colonocytes.

The effect of deposition Se on the mRNA expression levels of GPxs in goats from a Se-enriched county of China.

Previous studies revealed that Se was an important regulatory factor for glutathione peroxidase (GPx) genes. However, the relationship between Se concentrations and mRNA expression levels of GPxs were unclear in goats, especially the goats living in natural Se-enriched area. Thus, the aim of this study was to determine the Se concentrations and the mRNA expression levels of GPx-1, GPx-2, GPx-3, and GPx-4 in goats from Ziyang County (ZY-H and ZY-L goats) and Baoji City (BJ-P goats), which were Se-rich region and Se-poor region in China, respectively. Atomic fluorescence spectrometry was used as an essential method to determine the Se concentrations in heart, liver, spleen, lung, kidney, longissimus, biceps femoris, and serum, and the gene expressions were quantified in mRNA samples extracted from the above tissues by real-time quantitative reverse transcription-polymerase chain reaction. We found that the Se concentrations in ZY-H and ZY-L goats were higher than that in BJ-P goats significantly (P < 0.05), and the pertinence relations of Se levels between serum and heart, liver, spleen, and kidney were significant (P < 0.05). The mRNA levels of GPx-1 in ZY-H and ZY-L goats were higher than that in BJ-P goats very significantly (P < 0.01) except for longissimus (P < 0.05). Our results indicated a significant trend for GPx-2 in the direction of increasing mRNA levels with increasing Se concentrations in goats but had no statistical significance (P > 0.05) in our experimental conditions. As to GPx-3, its mRNA expression in spleen, lung, and kidney (P < 0.05) were upregulated and were consensual to high Se contents in ZY-H goats, but no significant effects were observed in heart, liver, longissimus, and biceps femoris among our three groups (P > 0.05). The mRNA levels of GPx-4 in heart, liver, lung, and kidney of ZY-H and ZY-L goats were higher than that of BJ-P goats (P < 0.05), and the difference was very significant in lung especially (P < 0.01), but no change in spleen, longissimus, and biceps femoris (P > 0.05). In summary, these data suggested that the goats living in Ziyang County were rich in Se, and the deposition Se played important roles in the mRNA expression of GPx-1, GPx-3, and GPx-4 in certain tissues of goats differentially.

Goat liver X receptor α, molecular cloning, functional characterization and regulating fatty acid synthesis in epithelial cells of goat mammary glands.

The liver X receptor α (LXRα) is a nuclear receptor of the transcription factor and is known to play a crucial role in lipid metabolism processes such as bile acid and fatty acid synthesis in humans and rodents. However, very little information is available on the role of LXRα in the regulation of fatty acid synthesis in the goat mammary gland. In this investigation, a cDNA was isolated from the mammary gland of Xinong Saanen dairy goats and designated as goat LXRα. RT-PCR and RACE gave rise to the full-length cDNA of LXRα, which was comprised of 1654 bp and characterized by an ORF of 1344 bp and 5'- and 3'-UTR regions of 150 and 160 bp, respectively. The deduced amino acid sequence encodes 477 amino acids with a predicted molecular weight (MW) of 50.4kDa and a theoretical isoelectric point (pI) of 6.3. Additionally, homology search and sequence multi-alignment indicated that the putative goat LXRα amino acid sequence is very similar to those of cattle, mice, rats, swine, and humans. Bioinformatic predictions demonstrated that the LXRα protein is located in the nucleus, containing characteristic signatures of a nuclear receptor with DNA-binding domain (DBD) and ligand-binding domain (LBD). Real-time quantitative PCR suggested that LXRα was predominantly expressed in the small intestine, liver, spleen and mammary gland. Treatment of goat mammary gland epithelial cells (GMEC) with different concentrations (i.e., 0.01, 0.1, 1 µM) of T0901317, a synthetic agonist of LXRα, resulted in elevated sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN) mRNA levels in response to LXRα activation. The association between different T0901317 concentrations and fatty acid composition in GMEC also was examined using gas chromatography (GC). The results showed that activation of LXRα significantly increased GMEC C18:1 and C18:2 contents, but did not affect levels of saturated fatty acids (SFA). These discoveries are consistent with the notion that LXRα plays a key role in controlling lipogenesis and regulating synthesis of unsaturated fatty acids (UFA) in the mammary gland of goats, which may prove useful in regulation of milk fat production.

Fatty acid composition of membrane bilayers: importance of diet polyunsaturated fat balance.

In one of the most extensive analyses to date we show that the balance of diet n-3 and n-6 polyunsaturated fatty acids (PUFA) is the most important determinant of membrane composition in the rat under 'normal' conditions. Young adult male Sprague-Dawley rats were fed one of twelve moderate-fat diets (25% of total energy) for 8weeks. Diets differed only in fatty acid (FA) profiles, with saturate (SFA) content ranging 8-88% of total FAs, monounsaturate (MUFA) 6-65%, total PUFA 4-81%, n-6 PUFA 3-70% and n-3 PUFA 1-70%. Diet PUFA included only essential FAs 18:2n-6 and 18:3n-3. Balance between n-3 and n-6 PUFA is defined as the PUFA balance (n-3 PUFA as % of total PUFA) and ranged 1-86% in the diets. FA composition was measured for brain, heart, liver, skeletal muscle, erythrocytes and plasma phospholipids, as well as adipose tissue and plasma triglycerides. The conformer-regulator model was used (slope=1 indicates membrane composition completely conforming to diet). Extensive changes in diet SFA, MUFA and PUFA had minimal effect on membranes (average slopes 0.01, 0.07, 0.07 respectively), but considerable influence on adipose tissue and plasma triglycerides (average slopes 0.27, 0.53, 0.47 respectively). Diet balance between n-3 and n-6 PUFA had a biphasic influence on membrane composition. When n-3 PUFA<10% of total PUFA, membrane composition completely conformed to diet (average slope 0.95), while diet PUFA balance>10% had little influence (average slope 0.19). The modern human diet has an average PUFA balance ~10% and this will likely have significant health implications.

[The influence of chromium chloride consumption on peroxidation processes and activity of antioxidant defence in rat tissues].

The supplementation of a standart vivarium food with 200 mg/kg CrCl3 x 6H2O caused an increase in chromium content and a decrease in hydroperoxide and TBARS in most tissues examined. Also in all organs and tissues of rats the activity of glutathione peroxidase, glutathione reductase and catalase incresed at action of chromium increased. In brain and kidneys the level of reduced glutathione increased. Superoxide dismutase activity was significantly higher in heart and skeletal muscles of animals and equal in lungs and liver, and in other organs--brain, kidneys and spleen in animals of the studied group the enzyme activity was lower compared to animals of control group. These results demonstrate the regulatory influence of chromium on free radical process in rat tissues.

Cloning and expression of a retinoic acid receptor ß2 subtype from the adult newt: evidence for an early role in tail and caudal spinal cord regeneration.

Retinoic acid receptor beta 2 (RARß2) has been proposed as an important receptor mediating retinoid-induced axonal growth and regeneration in developing mammalian spinal cord and brain. In urodele amphibians, organisms capable of extensive central nervous system (CNS) regeneration as adults, this receptor had not been isolated, nor had its function been characterized. We have cloned a full-length RARß2 cDNA from adult newt CNS. This receptor, NvRARß2, is expressed in various adult organs capable of regeneration, including the spinal cord. Interestingly, both the NvRARß2 mRNA and protein are up-regulated during the first 2 weeks after amputation of the tail, primarily in the ependymoglial and meningeal tissues near the rostral cut surface of the cord. Treatment with LE135, a RARß-selective antagonist, caused a significant inhibition of ependymal outgrowth and a decrease in tail regenerate length. These data support an early role for this receptor in caudal spinal cord and tail regeneration in this amphibian.

Selenium, selenoproteins and the thyroid gland: interactions in health and disease.

The trace element selenium is an essential micronutrient that is required for the biosynthesis of selenocysteine-containing selenoproteins. Most of the known selenoproteins are expressed in the thyroid gland, including some with still unknown functions. Among the well-characterized selenoproteins are the iodothyronine deiodinases, glutathione peroxidases and thioredoxin reductases, enzymes involved in thyroid hormone metabolism, regulation of redox state and protection from oxidative damage. Selenium content in selenium-sensitive tissues such as the liver, kidney or muscle and expression of nonessential selenoproteins, such as the glutathione peroxidases GPx1 and GPx3, is controlled by nutritional supply. The thyroid gland is, however, largely independent from dietary selenium intake and thyroid selenoproteins are preferentially expressed. As a consequence, no explicit effects on thyroid hormone profiles are observed in healthy individuals undergoing selenium supplementation. However, low selenium status correlates with risk of goiter and multiple nodules in European women. Some clinical studies have demonstrated that selenium-deficient patients with autoimmune thyroid disease benefit from selenium supplementation, although the data are conflicting and many parameters must still be defined. The baseline selenium status of an individual could constitute the most important parameter modifying the outcome of selenium supplementation, which might primarily disrupt self-amplifying cycles of the endocrine-immune system interface rectifying the interaction of lymphocytes with thyroid autoantigens. Selenium deficiency is likely to constitute a risk factor for a feedforward derangement of the immune system-thyroid interaction, while selenium supplementation appears to dampen the self-amplifying nature of this derailed interaction.

Organ-specificity of placebo effects on blood pressure.

There is increasing evidence that verbal suggestions accompanying placebo interventions can alter autonomic functions. The underlying mechanisms of these changes are not well understood. However, previous studies point at the specificity of such effects. The aim of the experiment was to lower blood pressure by a placebo intervention and to investigate the specificity of autonomic changes. Forty-five healthy participants received a single administration of an active drug (a homeopathic remedy), an identically-looking placebo drug, or no drug. Active drugs and placebo drugs were administered in a double-blind design and were accompanied by verbal suggestions of a blood-pressure lowering effect. Systolic and diastolic blood pressure, the electrocardiogram, electrodermal activity, and the electrogastrogram were recorded during 30min before and after the intervention, and changes in situational anxiety were assessed. Results indicated a decrease of systolic blood pressure in the placebo group, as compared to the control group. Diastolic blood pressure levels, heart rate, respiratory sinus arrhythmia, skin conductance, gastric slow-wave frequency and situational anxiety did not change differentially between groups. In conclusion, the reduction in systolic blood pressure following the placebo intervention could not be attributed to stress relief or anxiety reduction. Rather, results suggest that the placebo intervention specifically reduced systolic blood pressure.

Evaluation of deltamethrin kinetics and dosimetry in the maturing rat using a PBPK model.

Immature rats are more susceptible than adults to the acute neurotoxicity of pyrethroid insecticides like deltamethrin (DLM). A companion kinetics study (Kim et al., in press) revealed that blood and brain levels of the neuroactive parent compound were inversely related to age in rats 10, 21, 40 and 90 days old. The objective of the current study was to modify a physiologically based pharmacokinetic (PBPK) model of DLM disposition in the adult male Sprague-Dawley rat (Mirfazaelian et al., 2006), so blood and target organ dosimetry could be accurately predicted during maturation. Age-specific organ weights and age-dependent changes in the oxidative and hydrolytic clearance of DLM were modeled with a generalized Michaelis-Menten model for growth and the summary equations incorporated into the PBPK model. The model's simulations compared favorably with empirical DLM time-courses in plasma, blood, brain and fat for the four age-groups evaluated (10, 21, 40 and 90 days old). PND 10 pups' area under the 24-h brain concentration time curve (AUC(0-24h)) was 3.8-fold higher than that of the PND 90 adults. Our maturing rat PBPK model allows for updating with age- and chemical-dependent parameters, so pyrethroid dosimetry can be forecast in young and aged individuals. Hence, this model provides a methodology for risk assessors to consider age-specific adjustments to oral Reference Doses on the basis of PK differences.

The yin and yang of vitamin D receptor (VDR) signaling in neoplastic progression: operational networks and tissue-specific growth control.

Substantive evidence implicates vitamin D receptor (VDR) or its natural ligand 1alpha,25-(OH)2 D3 in modulation of tumor growth. However, both human and animal studies indicate tissue-specificity of effect. Epidemiological studies show both inverse and direct relationships between serum 25(OH)D levels and common solid cancers. VDR ablation affects carcinogen-induced tumorigenesis in a tissue-specific manner in model systems. Better understanding of the tissue-specificity of vitamin D-dependent molecular networks may provide insight into selective growth control by the seco-steroid, 1alpha,25-(OH)2 D3. This commentary considers complex factors that may influence the cell- or tissue-specificity of 1alpha,25-(OH)2 D3/VDR growth effects, including local synthesis, metabolism and transport of vitamin D and its metabolites, vitamin D receptor (VDR) expression and ligand-interactions, 1alpha,25-(OH)2 D3 genomic and non-genomic actions, Ca2+ flux, kinase activation, VDR interactions with activating and inhibitory vitamin D responsive elements (VDREs) within target gene promoters, VDR coregulator recruitment and differential effects on key downstream growth regulatory genes. We highlight some differences of VDR growth control relevant to colonic, esophageal, prostate, pancreatic and other cancers and assess the potential for development of selective prevention or treatment strategies.

Cloning and expression of Na+/K+ -ATPase and osmotic stress transcription factor 1 mRNA in black porgy, Acanthopagrus schlegeli during osmotic stress.

We cloned complementary DNA (cDNA) encoding the Na(+)/K(+)-ATPase (NKA) and the osmotic stress transcription factor 1 (OSTF1) from the kidney and gill, respectively, of the black porgy, Acanthopagrus schlegeli. Black porgy NKA full-length cDNA consists of 3078 base pairs (bp) and encodes a protein of 1025 amino acids; OSTF1 partial cDNA consists of 201 bp. To investigate the osmoregulatory ability of black porgy when black porgy were transferred to freshwater (FW), we examined the expression of NKA and OSTF1 mRNA in osmoregulatory organs, i.e., gill, kidney and intestine, using quantitative polymerase chain reaction (QPCR). To determine the hypoosmotic stressor specificity of the induction of NKA and OSTF1, black porgy were exposed to 30 degrees C water temperature for 24 h. In the gill, NKA mRNA was 4.2 times higher in FW, its expression in the kidney was 5.7 times higher in 10 per thousand seawater (10 per thousand SW) than in SW. In contrast, OSTF1 mRNA in the gill was 3.7 times higher in FW than in SW. The expression of heat shock protein 90 (HSP90) mRNA occurred not only during transfer to FW, but also in high-temperature water in all tested tissues, although the mRNA levels were not significantly different. Plasma osmolality level was decreased and cortisol level was increased when the fish were transferred from SW to FW. These results suggest that NKA and OSTF1 genes play important roles in hormonal regulation in osmoregulatory organs and that these genes are specific to hypoosmotic stress, improving the hyperosmoregulatory ability of black porgy in hypoosmotic environments.

TM14 is a new member of the fibulin family (fibulin-7) that interacts with extracellular matrix molecules and is active for cell binding.

We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin beta1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7.

Dentin matrix protein 4, a novel secretory calcium-binding protein that modulates odontoblast differentiation.

Formation of calcified tissues is a well regulated process. In dentin, the odontoblasts synthesize several biomolecules that function as nucleators or inhibitors of mineralization. To identify genes that are odontoblast-specific, a subtractive hybridization technique was employed that resulted in the identification of a previously undescribed novel gene synthesized by the odontoblasts. Based on the nomenclature in our laboratory, this gene has been named dentin matrix protein 4 (DMP4). The protein encoded by mouse DMP4 cDNA contained 579 amino acids, including a 26-amino acid signal peptide. Analysis of the protein sequence demonstrated the presence of a Greek key calcium-binding domain and one conserved domain of unknown function in all the species examined thus far. Calcium binding property was confirmed by (45)Ca binding assays and the corresponding change in conformation by far-ultraviolet circular dichroism. Northern analysis demonstrated high expression levels of a single 3-kb mRNA transcript in tooth, whereas low expression levels were detected in other tissues. In situ hybridization analysis showed high expression levels of DMP4 in odontoblasts and low levels in osteoblasts and ameloblasts during tooth development. Gain and loss of function experiments demonstrated that DMP4 had the potential to differentiate mesenchymal precursor cells into functional odontoblast-like cells.

Expression of glucose-6-phosphatase system genes in murine cortex and hypothalamus.

The glucose-6-phosphatase (G6Pase) system participates in the regulation of glucose homeostasis by converting glucose-6-phosphate (G6P) into glucose and inorganic phosphates. We have used an RT-PCR-based cloning and sequencing approach to study the expression of components of the G6Pase system in the hypothalamus and cortex tissues of the ob/ob mouse. We observed the expression of hepatic G6Pase catalytic subunit, G6PC, in both tissues, although increased template inputs were required for its detection. Conversely, expression of both the mouse homologue of the previously-described brain-specific G6P translocase T1 (G6PT1) variant and of the hepatic G6PT1 isoform was easily detectable in hypothalamus and cortex tissues. Of the proposed G6Pase catalytic subunit homologues, the expression of murine ubiquitous G6Pase catalytic subunit-related protein (UGRP, G6PC3) was also easily detectable in both tissues. However, islet-specific G6Pase catalytic subunit-related protein (IGRP, G6PC2) was expressed in a tissue-specific manner, and was detectable only in hypothalamus tissue at increased template inputs. We conclude that cells within ob/ob mouse hypothalamus and cortex tissues express genes with either established or proposed roles in G6P hydrolysis.

Selective transcriptional down-regulation of anther invertases precedes the failure of pollen development in water-stressed wheat.

Water deficit during male meiosis in wheat (Triticum aestivum L.) causes pollen sterility. With a view to identifying the internal trigger for this failure, it was found that water stress specifically impairs the activities of vacuolar and cell-wall invertases in anthers prior to the arrest of pollen development. The enzymes are affected only when water deficit occurs around meiosis. Three invertase cDNAs, two encoding the cell-wall (Ivr1, Ivr3) and one the vacuolar (Ivr5) isoform, were isolated from an anther cDNA library. RNA gel-blot analysis using floral organs of well-watered plants revealed that these genes were expressed preferentially, though not exclusively, in anthers. Semi-quantitative RT-PCR demonstrated that transitory water deficit during meiosis selectively down-regulated the transcription of two of the three genes, one encoding the vacuolar (Ivr5) and the other a cell-wall (Ivr1) isoform, without affecting the Ivr3 message. Their expression did not recover upon resumption of watering. Another homologue of Ivr1 was also down-regulated, but only during the post-stress period. The stress effects on invertase transcripts were consistent with those on the developmental profiles of the corresponding enzyme activities. In situ hybridization revealed that the stress-sensitive invertase genes, unlike an insensitive one, were expressed within the microspores. No evidence for an invertase inhibitor under stress was found. Together the results show that the decline in invertase activity is probably regulated primarily at the transcriptional level in a gene- and cell-specific manner.