Mouse arylamine N-acetyltransferase 2 (Nat2) expression during embryogenesis: a potential marker for the developing neuroendocrine system

Arylamine N-acetyltransferase (NAT) genes in humans and in rodents encode polymorphic drug metabolizing enzymes. Human NAT1 (and the murine equivalent mouse Nat2) is found early in embryonic development and is likely to have an endogenous role. We report the detailed expression of the murine gene (Nat2) and encoded protein in mouse embryos, using a transgenic mouse model bearing a lacZ transgene inserted into the coding region of mouse Nat2. In mouse embryos, the transgene was expressed in sensory epithelia, epithelial placodes giving rise to visceral sensory neurons, the developing pituitary gland, sympathetic chain and urogenital ridge. In Nat2 ^+/+ mice, the presence and activity of Nat2 protein was detected in these tissues and their adult counterparts. Altered expression of the human orthologue in breast tumours, in which there is endocrine signalling, suggests that human NAT1 should be considered as a potential biomarker for neuroendocrine tissues and tumours.     

Insight into the structure of Mesorhizobium loti arylamine N-acetyltransferase 2 (MLNAT2): a biochemical and computational study

The arylamine N-acetyltransferases (NAT; EC 2.3.1.5) are xenobiotic-metabolizing enzymes (XME) that catalyze the transfer of an acetyl group from acetylCoA (Ac-CoA) to arylamine, hydrazines and their N-hydroxylated metabolites. Eukaryotes may have up to three NAT isoforms, but Mesorhizobium loti is the only prokaryote with two functional NAT isoforms (MLNAT1 and MLNAT2). The three-dimensional structure of MLNAT1 has been determined (Holton, S.J., Dairou, J., Sandy, J., Rodrigues-Lima, F., Dupret, J.M., Noble, M.E.M. and Sim, E. (2005) Structure of Mesorhizobium loti arylamine N-acetyltransferase 1. Acta Cryst, F61, 14-16). No MLNAT2 crystals have yet been produced, despite the production of sufficient quantities of pure protein. Using purified recombinant MLNAT1 and MLNAT2, we showed here that MLNAT1 was intrinsically more stable than MLNAT2. To test whether different structural features could explain these differences in intrinsic stability, we constructed a high-quality homology model for MLNAT2 based on far UV-CD data. Despite low levels of sequence identity with other prokaryotic NAT enzymes ( approximately 28% identity), this model suggests that MLNAT2 adopts the characteristic three-domain NAT fold. More importantly, molecular dynamics simulations on the structures of MLNAT1 and MLNAT2 suggested that MLNAT2 was less stable than MLNAT1 due to differences in amino-acid sequence/structure features in the alpha/beta lid domain.     

Biochemical and molecular characterization of galectins from zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis

Galectins are a family of beta-galactoside-binding lectins that on synthesis are either translocated into the nucleus or released to the extracellular space. Their developmentally regulated expression, extracellular location, and affinity for extracellular components (such as laminin and fibronectin) suggest a role in embryonic development, but so far this has not been unequivocally established. Zebrafish constitute an ideal model for developmental studies because of their external fertilization, transparent embryos, rapid growth, and availability of a large collection of mutants. As a first step in addressing the biological roles in zebrafish embryogenesis, we identified and characterized members of the three galectin types: three protogalectins (Drgal1-L1, Drgal1-L2, Drgal1-L3), one chimera galectin (Drgal3), and one tandem-repeat galectin (Drgal9-L1). Like mammalian prototype galectin-1, Drgal1-L2 preferentially binds to N-acetyllactosamine. Genomic structure of Drgal1-L2 revealed four exons, with the exon-intron boundaries conserved with the mammalian galectin-1. Interestingly, this gene also encodes an alternatively spliced form of Drgal1-L2 that lacks eight amino acids near the carbohydrate-binding domain. Zebrafish galectins exhibited distinct patterns of temporal expression during embryo development. Drgal1-L2 is expressed postbud stage, and its expression is strikingly specific to the notochord. In contrast, Drgal1-L1 is expressed maternally in the oocytes. Drgal1-L3, Drgal3, and Drgal9-L1 are expressed both maternally and zygotically, ubiquitously in the adult tissues. The distinct temporal and spatial patterns of expression of members of the zebrafish galectin repertoire suggest that each may play distinct biological roles during early embryogenesis.     

Mouse Mesenchyme forkhead 2 (Mf2): expression, DNA binding and induction by sonic hedgehog during somitogenesis

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs revealed an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system Mf2 is transcribed in restricted regions of the mid- and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing sonic hedgehog (Shh) and in explant cultures of presomitic mesoderm Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH.     

Serum vitamin D receptor (VDR) levels as a potential diagnostic marker for colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of mortality and morbidity worldwide, and there has been a significant increase in the incidence of CRC in recent decades. Therefore, there is an urgent need to identify blood biomarkers that can be used for early diagnosis. It is not yet clear whether the level of vitamin D and its receptor, vitamin D receptor (VDR), in the blood are helpful factors in the diagnosis of CRC. Therefore, the study focuses on determining the VDR serum level’s contribution and other chemical parameters to the risk of CRC. A total of 189 Saudi participants (66 CRC patients and 123 control patients) aged 20–80 years old were enrolled in this case-control study. A serum sample was collected from each participant, and the levels of VDR and other bone profile tests were determined using ELISA or chemiluminescent assays. P values < 0.05 were considered statistically significant. The results showed a highly significant reduction in the levels of total vitamin D (P < 0.0001), VDR (P < 0.0001), vitamin D₃ (P < 0.05), and calcium (P < 0.0001) in the serum of CRC patients compared to the controls. However, the alkaline phosphatase level was higher in CRC patients compared to the controls (P < 0.0001). None of the blood markers showed a significant correlation to the progression of CRC (P > 0.05). More investigation is needed to elucidate different physiological processes that can be affected by these blood biomarkers, therefore changing the carcinogenesis of CRC.     

Chromosome mapping of the genes for murine arylamine N-acetyltransferases (NATs), enzymes involved in the metabolism of carcinogens: identification of a novel upstream noncoding exon for murine Nat2

Arylamine N-acetyltransferases (NATs) catalyse acetylation reactions which can result in either detoxification or activation of arylamine carcinogens. The human NAT loci (NAT1, NAT2, and a pseudogene, NATP) have been mapped to human chromosome 8p22, a region frequently deleted in tumours. There are three functional genes in mice (Nat1, Nat2, and Nat3) encoding for three NAT isoenzymes. Different alleles at the Nat2 locus are responsible for the acetylation polymorphism identified in different mouse strains. We show that Nat3 is close to Nat1 and Nat2, by screening of a P1 artificial chromosome (PAC) library and provide cytogenetic evidence for co-localisation of the three genes in chromosome region 8 B3.1-B3.3. The Nat region of mouse and human is homologous. We also provide sequence information and a restriction map in the vicinity of Nat1 and Nat2 and describe a noncoding exon located 6 kb upstream of the Nat2 coding region.     

The embryogenesis of the Tick Rhipicephalus (Boophilus) microplus: The establishment of a new chelicerate model system

Summary: Chelicerates, which include spiders, ticks, mites, scorpions, and horseshoe crabs, are members of the phylum Arthropoda. In recent years, several molecular experimental studies of chelicerates have examined the embryology of spiders; however, the embryology of other groups, such as ticks (Acari: Parasitiformes), has been largely neglected. Ticks and mites are believed to constitute a monophyletic group, the Acari. Due to their blood‐sucking activities, ticks are also known to be vectors of several diseases. In this study, we analyzed the embryonic development of the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). First, we developed an embryonic staging system consisting of 14 embryonic stages. Second, histological analysis and antibody staining unexpectedly revealed the presence of a population of tick cells with similar characteristics to the spider cumulus. Cumulus cell populations also exist in other chelicerates; these cells are responsible for the breaking of radial symmetry through bone morphogenetic protein signaling. Third, it was determined that the posterior (opisthosomal) embryonic region of R. microplus is segmented. Finally, we identified the presence of a transient ventral midline furrow and the formation and regression of a fourth leg pair; these features may be regarded as hallmarks of late tick embryogenesis. Importantly, most of the aforementioned features are absent from mite embryos, suggesting that mites and ticks do not constitute a monophyletic group or that mites have lost these features. Taken together, our findings provide fundamental common ground for improving knowledge regarding tick embryonic development, thereby facilitating the establishment of a new chelicerate model system. genesis 51:803–818. © 2013 Wiley Periodicals, Inc.     

Release of cytokeratin-18 and -19 fragments (TPS and CYFRA 21-1) into the extracellular space during apoptosis

Serum fragments of cytokeratins-18 and -19 (measured as TPS and CYFRA 21-1, respectively) have traditionally been considered as markers of tumor proliferation, although the evidence is scarce for a causative relationship between proliferation and levels of TPS and CYFRA 21-1. We examined whether apoptosis might produce TPS and CYFRA 21-1 fragments. MCF-7 breast cancer cells were treated with mitomycin C or agonistic anti-CD95 antibody, and levels of TPS and CYFRA 21-1 in tissue culture supernatants were compared with the frequency of cells exhibiting the following markers of cell death: intracellular cytokeratin-18 cleavage, surface staining with annexin-V, propidium iodide uptake, DNA fragmentation. Twenty-four hours after inducing apoptosis, levels of TPS and CYFRA 21-1 were elevated > or = 4-fold in culture supernatants. Elevations in TPS and CYFRA 21-1 coincided with apoptosis measured by the first three cell death markers but preceded DNA fragmentation. These mitomycin C- and CD95-mediated elevations were completely inhibited by co-incubation with the caspase inhibitors Z-VAD.fmk and Z-IETD.fmk, respectively. We conclude that TPS and CYFRA 21-1 can be abundantly released into the extracellular space during the intermediate stage of epithelial cell apoptosis.     

Thermoregulation during gravidity in the children's python (Antaresia childreni): a test of the preadaptation hypothesis for maternal thermophily in snakes

Pregnant squamate reptiles (i.e. lizards and snakes) often maintain higher and more stable body temperatures than their nonpregnant conspecifics, and this maternal thermophily enhances developmental rate and can lead to increased offspring quality. However, it is unclear when this behaviour evolved relative to the evolution of viviparity. A preadaptation hypothesis suggests that maternal thermophily was a preadaptation to viviparity. Oviparous squamates are unique among oviparous reptiles for generally retaining their eggs until the embryos achieve one fourth of their development. As a result, maternal thermophily by gravid squamates may provide the same thermoregulatory benefits, at least during early development, that have been associated with viviparity. Thus, the evolution of viviparity in squamates may reflect an expanded duration of a pre-existing maternal thermoregulatory behaviour. Despite its evolutionary relevance, thermoregulation during gravidity in oviparous squamates has not yet been explored in depth. In the present study, we examined whether gravidity was associated with thermoregulatory changes in the oviparous children's python, Antaresia childreni . First, we discovered that, compared to most snakes, A. childreni is at an advanced stage of embryonic development at oviposition. Second, using surgically implanted temperature loggers, we detected a significant influence of reproductive status on thermoregulation. Reproductive females maintained higher and less variable body temperatures than nonreproductive females and this difference was most pronounced during the last 3 weeks of gravidity. Overall, these results highlight the continuum between oviparity and viviparity in squamate reptiles and emphasize the importance of thermal control of early embryonic development independent of reproductive mode.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 499–508.     

bel-2 protein in breast cancer cells obtained by fine needle aspiration (FNA): a preliminary report

The bcl-2 protein plays a role in the regulation of programmed cell death (PCD), overriding apoptosis. Its expression has been reported in breast ductal cells, where it is believed to be involved in the hormonal regulation of hyperplasia and involution. to date, bcl-2 gene product has not been investigated on breast cancer FNA. the expression of bcl-2 protein was evaluated using an immunoalkaline phosphatase technique in 54 pre-operative breast cancer aspirates and in paraffin-embedded sections from 20 matched surgical specimens. A high rate of bcl-2 protein expression was found on FNA samples (65%) and on the corresponding tissue sections (60%); there was a nearly absolute concordance in the two specimens, with 19/20 (95%) cases showing a concordant staining. These findings concur with the view that bcl-2 gene is frequently expressed in breast cancer, possibly through a hormonal-dependent pathway.     

Leucoanthocyanidin dioxygenase gene (PpLDOX): a potential functional marker for cold storage browning in peach

Enzymatic browning of the peach fruit mesocarp is a major component of the postharvest physiological disorder commonly called chilling injury or internal breakdown (IB). Previously, we detected a major quantitative trait locus (QTL; qP-Brn5.1^m) affecting browning in peach using two related progeny populations (Pop-DG and Pop-G). In this report, a gene encoding the leucoanthocanidin dioxygenase (PpLDOX) enzyme was identified as the gene potentially responsible for this QTL. PpLDOX has a high similarity with the LDOX gene of the anthocyanin biosynthesis pathway of Arabidopsis thaliana. It was co-located with qP-Brn5.1^m via the bin mapping technique with the Prunus reference T×E map. A silent SNP within the PpLDOX coding sequence was used to locate the gene more precisely on the Pop-DG map and confirm its bin assignment. These results demonstrate both the utility of comparative mapping within Prunus using the T×E reference map and the power of the bin mapping approach for easily mapping genes in the Prunus genome. An SSR polymorphism was observed in the intron of PpLDOX gene sequence. The SSR co-segregated with the SNP and was used to assess association of PpLDOX with browning in 27 peach and nectarine cultivars. Cumulative evidence obtained indicates that PpLDOX partially explains genetic variation for cold storage browning susceptibility in peach and nectarine. This functional gene has potential use in marker-assisted breeding of new cultivars with lower IB susceptibility and for genotyping current cultivars for possible differential handling during storage to reduce symptom incidence.     

pag gene-like protein (ABP-25) of the Cynops embryo: regional distribution and gene expression during early embryogenesis

A maternal protein showing a unique distribution during early Cynops embryogenesis was screened by monoclonal antibody. The antigen protein, designated as ABP-25 (animal blastomere protein, molecular weight 25,000), was distributed uniformly in the uncleaved egg and concentrated into blastomeres of the animal half during cleavage. At the blastula stage, ABP-25 was definitely localized in cells of the animal half and a polarized distribution was observed within the cytoplasm. During gastrulation, immunohistochemical analysis indicated that the reactivity of the marginal zone (presumptive mesoderm) to the monoclonal antibody ABP-25 decreased after involution. At the end of gastrulation, a polarized distribution was still clearly observed in the ventral epidermis, but not in the neuroectoderm. Both Western and Northern blots indicated that the amount of antigen protein and the intensity of gene expresion were almost constant until the neurula stage. The deduced amino acid sequence of the ABP-25 cDNA showed a strong homology (84%) with that of the pag gene associated with cell proliferation.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the accession number D37808     

A novel carbohydrate antigen expression during development of Opisthorchis viverrini- associated cholangiocarcinoma in golden hamster: a potential marker for early diagnosis

Poor prognosis of cholangiocarcinoma (CCA) is primarily due to delayed diagnosis because of the lack of appropriate tumor marker(s) to detect cancer development at an early stage. We have recently established a S121 monoclonal antibody (mAb) which recognizes an unidentified glycan epitope on MUC5AC, designated as CCA-associated carbohydrate antigen (CCA-CA). This antigen is expressed in human CCA cells but not in normal biliary epithelia. Detection of CCA-CA effectively distinguished CCA patients' sera from normal control sera with high specificity and sensitivity. In the present study, we examined a time profile of the expression of CCA-CA by immunohistochemical methods in the liver tissues of Opisthorchis viverrini (Ov)-associated CCA in a hamster model. Hamsters were divided into four groups; non-treated, Ov infected, NDMA (N-nitrosodimethamine) treated and Ov + NDMA treated groups, and animals from each group were euthanized at 1, 3 and 6 months post-treatment. CCA-CA was not detected in normal biliary cells of non-treated hamsters throughout the course of experiment. CCA-CA became detectable in the cytoplasm and apical surface of biliary cells of the NDMA and Ov + NDMA groups at early stage (1 month) of tumor development and increased with tumor progression. In contrast, CCA-CA was detected as nuclear staining at the 1 month post Ov infection and declined thereafter. These results suggest the possibility of CCA-CA as an early marker for CCA.     

MCF-7/VD(R): a new vitamin D resistant cell line

Several in vitro and in vivo experiments have demonstrated potent cell regulatory effects of vitamin D compounds in cancer cells. Moreover, a promising phase I study with the vitamin D analogue Seocalcitol (EB 1089) in patients with advanced breast and colon cancer has already been carried out and more clinical trials evaluating the clinical effectiveness of EB 1089 in other cancer types are in progress (M?rk Hansen et al. [2000a]). However, only little is known about the mechanisms underlying the actions of vitamin D or about the possible development of drug resistance in the patients. Therefore, in an attempt to gain more insight into these aspects, we have developed the MCF-7/VD(R) cell line, a stable subclone of the human MCF-7 breast cancer cell line, which is resistant to the growth inhibitory and apoptosis inducing effects of 1alpha,25(OH)(2)D(3). Despite this characteristic, receptor studies on the VDR have clearly demonstrated that the MCF-7/VD(R) cells contain fully functional VDRs, although in a lower number than seen with the parental MCF-7 cells. The regulation of the 24-hydroxylase enzyme appeared to be intact in the MCF-7/VD(R) cells and no differences with regard to growth rate and morphological appearance between the MCF-7/VD(R) cells and the parental MCF-7 cells were observed. Interestingly, however, the sensitivity of the MCF-7/VD(R) cells to the pure anti-estrogen ICI 182,780 was found to be increased. The MCF-7/VD(R) cell line shows characteristics different from those of previously described vitamin D resistant breast cancer cell lines but also some similarities. Together such vitamin D resistant cell lines therefore serve as a useful tool for studying the exact mechanism of action of vitamin D and the development of vitamin D resistance.     

Identifying potential regulators of infantile hemangioma progression through large-scale expression analysis: a possible role for the immune system and indoleamine 2,3 dioxygenase (IDO) during involution

Hemangiomas are benign endothelial tumors. Often referred to as hemangiomas of infancy (HOI), these tumors are the most common tumor of infancy. Most of these lesions proliferate rapidly in the first months of life, and subsequently slowly involute during early childhood without significant complications. However, they often develop on the head or neck, and may pose a significant cosmetic concern for families. In addition, a fraction of these tumors can grow explosively and ulcerate, bleed, or obstruct vision or airway structures. Current treatments for these tumors are associated with significant side effects, and our knowledge of the biology of hemangiomas is limited. The natural evolution of these lesions creates a unique opportunity to study the changes in gene expression that occur as the endothelium of these tumors proliferates and then subsequently regresses. Such information may also increase our understanding of the basic principals of angiogenesis in normal and abnormal tissue. We have performed large-scale genomic analysis of hemangioma gene expression using DNA microarrays. We recently identified insulin-like growth factor 2 as a potentially important regulator of hemangioma growth using this approach. However, little is known about the mechanisms involved in hemangioma involution. Here we explore the idea that hemangioma involution might be an immune-mediated process and present data to support this concept. We also demonstrate that proliferating hemangiomas express indoleamine 2,3 dioxygenase (IDO) and discuss a possible mechanism that accounts for the often slow regression of these lesions.     

A novel marker for assessment of liver matrix remodeling: An enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M)

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.