In Situ Hybridization

To obtain a deeper insight into the genes and gene networks involved in the development of placentopathies, we have assessed global gene expression in three different models of placental hyperplasia caused by interspecies hybridization (IHPD), cloning by nuclear transfer, and mutation of the Esx1 gene, respectively. Comparison of gene expression profiles of approximately 13,000 expressed sequence tags (ESTs) identified specific subsets of genes with changed expression levels in IHPD, cloned, and Esx1 mutant placentas. Of interest, only one gene of known function and one EST of unknown function were found common to all three placentopathies; however, a significant number of ESTs were common to IHPD and cloned placentas. In contrast, only one gene was shared between IHPD and Esx1 mutant, and cloned and Esx1 mutant placentas, respectively. These genes common to different abnormal placental growth genotypes are likely to be important in the occurrence of placentopathy. Developmental Dynamics 230: 149-164, 2004.

Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to labelfree and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples.

Although the precise mechanisms explaining loss of, and failure to regain, function after spinal cord injury are unknown, there is increasing interest in the role of "secondary cell death." One prevalent theme in cell loss in other regions of the CNS involves apoptosis executed by the intracellular caspase proteases. A recent study demonstrated that spinal cord injury rapidly increased the activation of caspase-3. Our previous studies demonstrated peak apoptosis in three of four cellular compartments 3 days after controlled contusion in the rat. We have extended these analyses to include enzyme and substrate studies of caspase subfamilies both in rostral and in caudal adjacent segments compared to the lesion site. Although presumed activation of programmed proenzyme is considered the mechanism for enhanced caspases, our novel analyses were designed to detect upregulation of gene expression. We surveyed traumatically injured spinal cord for caspase family messages with a modified differential mRNA display approach and found that the caspase-3 (CASP3) message was present and upregulated severalfold after injury. Our results clearly demonstrate that cell death in the spinal cord occurs after posttranslational activation of caspases that follow, at least for caspase-3, initial upregulation of CASP3 mRNA levels.

To start determining the nature of meiotic incompetence in goat oocytes, we have examined the expression of one of the potential pre-MPF subunits: the cyclin B 1 . We have been isolating a small DNA probe encoding the goat cyclin B 1 box to analyze the expression of the cyclin B 1 gene in competent and incompetent goat oocytes. This probe was easily obtained by polymerase-chain-reaction (PCR) on reversetranscribed mRNA from granulosa cells, using cyclin B specific primers derived from a bovine cDNA. The transcript corresponding to cyclin B 1 in goat granulosa cells is 1.8 kb. In situ hybridization analysis indicated that competent and incompetent oocytes contained cyclin B 1 mRNA, but also that active cyclin B 1 mRNA synthesis occured at the end of the growth phase, e.g., when oocytes progressed in the acquisition of meoitic competence. Western blot analysis, performed with a monoclonal anticyclin B 1 antibody, revealed in competent and incompetent oocytes a polypeptide of 65kDa corresponding to the goat cyclin B 1 protein. This pattern of cyclin B 1 expression further suggested that meiotic incompetence in goat oocytes could not be primarily correlated with a lack of cyclin B 1 protein as potential pre-MPF subunit, but to a limiting amount of this protein. Mol. Reprod. Dev. 47:222-228, 1997. r 1997 Wiley-Liss, Inc.

dysplastic and malignant IP, it may have a higher detection rate than another series which contains only nondysplastic IP. We hypothesize that the higher rates of HPV detection in dysplastic and malignant IP may be related to HPV integration. The implication of this is that HPV sub-type testing may identify patients at risk for recurrence, or progression to dysplasia and malignancy, and thus may impact surveillance protocols.

The neural mechanisms underlying benzodiazepine dependence remain equivocal. The present studies tested the hypothesis that similar neural systems are recruited during diazepam tolerance and withdrawal, and that these are associated with changes in GABA A receptor properties. 2-Deoxyglucose quantitative autoradiography was employed to map the brain structures affected during chronic treatment and withdrawal from diazepam (5 mg/kg IP daily) in rats. Acute administration of diazepam evoked widespread reductions in local rates of cerebral glucose (LCGU) utilization throughout the brain. Brain structures associated with sensory processing developed tolerance to these depressant effects of diazepam after 3 days of treatment, whereas tolerance occurred in the Papez circuit of emotion after 28 days of treatment. These data suggest that adaptive changes in different neuroanatomical circuits may underlie tolerance to the various effects of diazepam. During flumazenil-precipitated withdrawal from diazepam there were marked increases in glucose use in structures of the Papez circuit, the nucleus accumbens, and the basolateral amygdala. These data suggest that the Papez circuit features strongly in diazepam tolerance and withdrawal and supports a common adaptive process being involved in these phenomena. While GABA enhancement of benzodiazepine binding was reduced in the nucleus accumbens after repeated diazepam treatment, there was little evidence to support adapative changes in GABA A receptors or GABA A subunit gene expression (␥ 2 , ␣ 1 , or ␣ 4) as underlying the functional changes in the identified circuits. Alternative neurochemical mechanisms, such as changes in glutamatergic function should be considered.

Pregnancy-associated plasma protein-A and-A2 (PAPPA and PAPPA2) are proteases that cleave IGF binding proteins (IGFBPs) and thereby increase the bioavailability of growth factors. PAPPA has long been recognized as a marker of fetal genetic disorders and adverse pregnancy outcomes. In contrast, although PAPPA2 is also highly expressed in human placenta, its physiological importance is not clear. To establish whether mice will be a useful model for the study of PAPPA2, we compared the patterns of expression of PAPPA2 in the placentae of mouse and human. We show, for the first time, that Pappa2 is highly expressed in mouse placenta, as is the case in humans. Specifically, it is expressed at the interface of the maternal and fetal layers of the mouse placenta at all gestational stages studied (10. 5-16. 5 days post coitum). Similarly, PAPPA2 is expressed in the syncytiotrophoblast layer of human placental villi and is also detected in some invasive extravillous trophoblasts in the first trimester. These results are consistent with a model whereby PAPPA2 cleaves IGFBPs produced in the maternal decidua to promote fetoplacental growth, and indicate that this protein may play analogous roles in human and mouse placenta. PAPPA2 protein is detectable in the circulation of pregnant mice and humans during the first trimester and at term, raising the possibility that PAPPA2 may be a useful biomarker of placental dysfunction. Pappa2 expression also shows specific localization within the mouse embryo and therefore may play roles in fetal development, independent of its action in the placenta.

The earliest signs of the lymphatic vascular system are the lymph sacs, which develop adjacent to specific embryonic veins. It has been suggested that sprouts from the lymph sacs form the complete lymphatic vascular system. We have studied the origin of the jugular lymph sacs (JLS), the dermal lymphatics and the lymph hearts of avian embryos. In day 6.5 embryos, the JLS is an endothelial-lined sinusoidal structure. The lymphatic endothelial cells (LECs) stain (in the quail) positive for QH1 antibody and soybean agglutinin. As early as day 4, the anlagen of the JLS can be recognized by their Prox1 expression. Prox1 is found in the jugular section of the cardinal veins, and in scattered cells located in the dermatomes along the cranio-caudal axis and in the splanchnopleura. In the quail, such cells are positive for Prox1 and QH1. In the jugular region, the veins co-express the angiopoietin receptor Tie2. Quail-chick-chimera studies show that the peripheral parts of the JLS form by integration of cells from the paraxial mesoderm. Intravenous application of DiI-conjugated acetylated low-density lipoprotein into day 4 embryos suggests a venous origin of the deep parts of the JLS. Superficial lymphatics are directly derived from the dermatomes, as shown by dermatome grafting. The lymph hearts in the lumbo-sacral region develop from a plexus of Prox1-positive lymphatic capillaries. Both LECs and muscle cells of the lymph hearts are of somitic origin. In sum, avian lymphatics are of dual origin. The deep parts of the lymph sacs are derived from adjacent veins, the superficial parts of the JLS and the dermal lymphatics from local lymphangioblasts.

A sample-sequencing strategy combined with slot-blot hybridization and FISH was used to study the composition of the repetitive component of the sunflower genome. One thousand six hundred thirty-eight sequences for a total of 954,517 bp were analyzed. The fraction of sequences that can be classified as repetitive using computational and hybridization approaches amounts to 62% in total. Almost two thirds remain as yet uncharacterized in nature. Of those characterized, most belong to the gypsy superfamily of LTR-retrotransposons. Unlike in other species, where single families can account for large fractions of the genome, it appears that no transposon family has been amplified to very high levels in sunflower. All other known classes of transposable elements were also found. One family of unknown nature (contig 61) was the most repeated in the sunflower genome. The evolution of the repetitive component in the Helianthus genus and in other Asteraceae was studied by comparative analysis of the hybridization of total genomic DNAs from these species to the sunflower smallinsert library and compared to gene-based phylogeny. Very little similarity is observed between Helianthus species and two related Asteraceae species outside of the genus. Most repetitive elements are similar in annual and perennial Helianthus species indicating that sequence amplification largely predates such divergence. Gypsy-like elements are more represented in the annuals than in the perennials, while copia-like elements are similarly represented, attesting a different amplification history of the two superfamilies of LTR-retrotransposons in the Helianthus genus. Sequences from Helianthus annuus randomly sheared genomic DNA library and sequences of genes used for phylogenetic analyses are available at the URL: https://services.appliedgenomics.org/sequencesexport/26-Helianthus/.

Marek's disease virus (MDV) isolated from poultry flocks in three states of India was monitored for the virus occurrence in the field. The MDV genome was isolated from feather follicles, spleen, and liver of the chicken (173 samples). Twenty two samples were positive for MDV genome in PCR and belonged to the serotype 1. The sequencing of MEQ gene of 11 samples revealed that nucleotide sequences of the isolate Ind-KA-01-06 was similar to the very virulent MDV, strain RB-1B. In situ hybridization studies also confirmed a presence of MDV serotype 1 in the infected liver tissues. Furthermore, the ability of the virus to induce apoptosis detected by flow cytometry showed that the virulent MDV induced apoptosis more efficiently than Turkey herpesvirus (HVT) vaccine virus. The present study showed the presence of virulent/very virulent MDV strains in the Indian poultry flocks.

Background Mucus hyper-secretion is a feature of several airways diseases such as chronic rhinosinusitis, asthma, and cystic fibrosis (CF). Since mucins are major components of mucus, the knowledge of their distribution and regulation in nasal tissues is likely to improve mucus hyper-secretion therapy. Objective The aim of this study was to evaluate and compare mucin gene expression at epithelial and glandular levels, and to identify potential mucin expression patterns for specific upper airways pathologies. Methods Immunohistochemistry for MUC1, MUC2, and MUC4-MUC8 mucins was performed on healthy nasal mucosa (NM; n = 12), bilateral nasal polyps (NP; n = 38), NP from CF patients (n = 10), and antrochoanal (AC) polyps (n = 11). MUC2, MUC4, MUC5AC, and MUC6 mRNA expression were also analysed by in situ hybridization. Results MUC1, MUC4, and MUC5AC mucins were highly expressed in the epithelium and their expression pattern was similar in all NP types, MUC1 and MUC4 being increased and MUC5AC decreased compared with NM. MUC8 was highly detected at both epithelial and glandular levels with marked variability between groups. MUC5B was mainly detected in glands and the expression in all polyp types was higher than in NM. Moreover, MUC5B expression was higher in NP epithelia from CF patients than in bilateral NP and healthy NM. Although MUC2 expression was low, especially in AC polyps, it was detected in most samples. In NM, MUC6 and MUC7 were scarcely detected and MUC7 expression was restricted to glands. Conclusions These results suggest that NP have a different pattern of mucin expression than healthy NM and that CF polyps (increased MUC5B) and AC polyps (decreased MUC2) have a different mucin expression pattern than bilateral NP.

Receptor proteins for photoreception have been studied for several decades. More recently, putative receptors for olfaction have been isolated and characterized. In contrast, no receptors for taste have been identified yet by molecular cloning. This report describes experiments aimed at identifying a receptor responsible for the taste of monosodium glutamate (MSG). Using reverse transcriptase (IV)-PCR, we found that several ionotropic glutamate receptors are present in rat lingual tissues. However, these receptors also could be detected in lingual tissue devoid of taste buds. On the other hand, RT-PCR and RNase protection assays indicated that a G-protein-coupled metabotropic glutamate receptor, mGluR4, also is expressed in lingual tissues and is limited only to taste buds. In situ hybrid-ization demonstrated that mGluR4 is detectable in 40-70% of vallate and foliate taste buds but not in surrounding nonsensory epithelium, confirming the localization of this metabotropic receptor to gustatory cells. Expression of mGluR4 in taste buds is higher in preweaning rats compared with adult rats. This may correspond to the known higher sensitivity to the taste of MSG in juvenile rodents. Finally, behavioral studies have indicated that MSG and L-2-amino-4-phosphonobutyrate (L-AP4), a ligand for mGluR4, elicit similar tastes in rats. We conclude that mGluR4 may be a chemosensory receptor responsible, in part, for the taste of MSG.

This study reports the spatio-temporal pattern of BEN expression (a molecule of the immunoglobulin superfamily) during early stages of the first axonal tract formation, in the fore-and midbrain of chick embryos [Hamburger and Hamilton (HH) stages 12-22]. The expression of BEN has been analysed using immunohistochemistry and non-radioactive in situ hybridization. Furthermore, double labelling experiments (combining anti-class Ill P-tubulin, a pan-neuronal marker, and anti-BEN antibodies) have been carried out to determine whether BEN is expressed by all first axonal tracts. The first neurons expressing BEN appear around stage HH13-14, in the caudal diencephalon. They belong to the interstitial nucleus of Cajal, and their axons are the first components of the medial longitudinal fasciculus. By HH14, two other early axonal tracts appear: the tract of the postoptic commissure and the descending root of the mesencephalic nucleus of the trigeminal nerve. Only the latter expresses BEN. At later stages of development numerous new axonal tracts appear in the telencephalic, diencephalic and mesencephalic domains. Only a few of them (the fourth nerve, the lemniscus lateralis, the tectobulbar and habenulopeduncular tracts) express BEN. In all BEN positive systems, the cell bodies, axons and growth cones are uniformly labelled by the antibody. We have found that none of the early axonal tracts grows preferentially at interneuromeric boundaries. Moreover, each tract is formed by several thin fascicles rather than a single one. The expression of BEN is transient and disappears shortly before hatching. These results suggest that BEN may serve to promote axonal outgrowth of precise neuronal systems involved in 'axonal scaffolding'.

Background. Transforming growth factor-b 1 (TGF-b 1) is the major fibrogenic growth factor implicated in the pathogenesis of renal scarring. Proteinuria is a poor prognostic feature for various types of glomerular disease and its toxic action may be related to the activation of tubular epithelial cells towards increased production of cytokines and chemoattractant peptides. In this work we studied the site of synthesis and expression profile of TGF-b 1 in the renal tissue of patients with heavy proteinuria and examined the relation of this expression with the urinary excretion of TGF-b 1. Methods. Twenty-five patients with heavy proteinuria (8.4"3.0 gu24 h) were included in the study. All patients underwent a diagnostic kidney biopsy and were commenced on immunosuppressive therapy with corticosteroids and cyclosporin. The sites of synthesis and expression profile of TGF-b 1 mRNA and protein in the kidney were examined by in situ hybridization and immunohistochemistry. Urinary and plasma TGF-b 1 levels were determined by ELISA before the initiation of treatment and 6 months later and compared with those of normal subjects and of patients with IgA nephropathy and normal urinary protein excretion. Results. The site of synthesis and expression of TGF-b 1 in the renal tissue of patients with heavy proteinuria was mainly localized within the cytoplasm of tubular epithelial cells. Interstitial expression was also present but glomerular TGF-b 1 expression was found only in patients with mesangial proliferation. Urinary TGF-b 1 excretion was significantly higher in nephrotic patients compared with normal subjects and with patients with IgA nephropathy and normal urinary protein excretion (783"280 vs 310"140 and 375"90 ngu24 h, respectively; P-0.01). In patients with remission of proteinuria after immunosuppressive therapy, urinary TGF-b 1 excretion was significantly reduced (from 749"290 to 495"130 ngu24 h; P-0.01), while in patients with persistent nephrotic syndrome, it remained elevated. Conclusions. The localization of TGF-b 1 mRNA and protein within tubular epithelial cells, along with its increased urinary excretion in patients with nephrotic syndrome, suggest the activation of these cells by filtered protein towards increased TGF-b 1 production.

factor expressed in several regions of the developing mammalian central nervous system. First found in the midbrain/hindbrain region, Pax-2 expression is later found in the cerebellum, hindbrain, and spinal cord. We have examined the expression pattern of Pax-2 from embryonic day 12 (E12) through postnatal day 35 (P35) using immunohistochemistry and in situ hybridization. Expression of Pax-2 is found in scattered cells of the cerebellar ventricular zone at E13. Pax-2-expressing cells migrate away from this germinative center to positions in the deep cerebellar nuclei (DCN), internal granule cell layer, molecular layer, and folial whitematter tracts of the cerebellum. Immunocytochemistry of both tissue sections and primary dissociated cultures demonstrates that Pax-2 is expressed by cells of a neuronal lineage, but not by cells of either an astrocytic or oligodendrocytic lineage. Specifically, the presence of Pax-2 identifies the entire population of ␥-aminobutyric acid (GABA)ergic interneurons in the cerebellar cortex

Cocaine-and amphetamine-regulated transcript (CART) is expressed in the hypothalamus, and putative peptides encoded by CART potently inhibit feeding when administered centrally. CART is strongly down-regulated in the lateral hypothalamic area and the arcuate nucleus in animal models of obesity with disrupted leptin signaling. Here we have used in situ hybridization and immunohistochemistry to study CART expression in mice homozygous for the anorexia (anx) mutation which are characterized by a much reduced food intake and premature death. anx /anx mice had significantly decreased levels of CART mRNA label and peptide-immunoreactive cell bodies and fibers in the arcuate nucleus and a lower number of detectable CART-expressing cells in the dorsomedial hypothalamic nucleus / lateral hypothalamic area. Moreover, serum leptin levels were significantly lower in anx /anx mice compared to normal littermates, most likely due to the prominent depletion of body fat in these animals. The decrease in the anorexigenic agents leptin and CART, may reflect a compensatory down-regulation in response to the energy-deprived state of anx /anx mice. Alternatively, the reduced arcuate CART expression may be a consequence of a molecular defect in the arcuate nucleus of these animals.

The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during embryogenesis. A loss of function mutation, heph(03429), results in varied defects in embryonic developmental processes, leading to embryonic lethality. However, the suite of molecular functions that are disrupted in the mutant remains unknown. We have used an unbiased high throughput sequencing approach to identify transcripts that are misregulated in this mutant. Misregulated transcripts show evidence of significantly altered patterns of splicing (exon skipping, 5' and 3' splice site switching), alternative 5' ends, and mRNA level changes (up and down regulation). These findings are independently supported by reverse-transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridization. We show that a group of genes, such as Zerknüllt, z600 and screw are among the most upregulated in the mutant and have been functionally linked to dorso-ventral pattern...

The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors, although the range of responding genes is still incompletely defined. Here we show that the tumor-associated carbonic anhydrases (CAs) are tightly regulated by this system. Both CA9 and CA12 were strongly induced by hypoxia in a range of tumor cell lines. In renal carcinoma cells that are defective for the von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is associated with loss of regulation by hypoxia, consistent with the critical function of pVHL in the regulation of HIF-1. Further studies of CA9 defined a HIF-1-dependent hypoxia response element in the minimal promoter and demonstrated that tight regulation by the HIF/ pVHL system was reflected in the pattern of CA IX expression within tumors. Generalized up-regulation of CA IX in VHL-associated renal cell carcinoma contrasted with focal perinecrotic expression in a variety of non-VHL-associated tumors. In comparison with vascular endothelial growth factor mRNA, expression of CA IX demonstrated a similar, although more tightly circumscribed, pattern of expression around regions of necrosis and showed substantial although incomplete overlap with activation of the hypoxia marker pimonidazole. These studies define a new class of HIF-1-responsive gene, the activation of which has implications for the understanding of hypoxic tumor metabolism and which may provide endogenous markers for tumor hypoxia.

Three potent broad-spectrum antimicrobial peptides (HFIAP-1, -2, and -3) isolated from intestinal tissues of Myxine glutinosa (Atlantic hagfish) are identified as ancient members of the cathelicidin family of antimicrobial peptides, hitherto known only from mammals. In situ hybridization reveals that HFIAPs are produced in nests of myeloid cells within the loose connective tissue of the gut wall, a tissue reminiscent of both gut-associated lymphoid tissue (GALT) and vertebrate spleen. We suggest that this tissue organization provides local defense of the hagfish gastrointestinal tract via innate immunity and possibly served as the architectural plan upon which the adaptive immune system evolved.

We show with transgenic mice that targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) in undifferentiated spermatogonia promotes malignant testicular tumors, which express germ-cell markers. The tumors are invasive and contain aneuploid cells, but no distant metastases have been found. By several histological, molecular, and histochemical characteristics, the GDNF-induced tumors mimic classic seminomas in men, representing a useful experimental model for testicular germ-cell tumors. The data also show that a deregulated stimulation of a normal proto-oncogene by its ligand can be an initiative event in carcinogenesis.

Regucalcin is involved in maintenance of calcium homeostasis due to the activation of Ca 2+ pumping enzymes in the plasma membrane. It has a suppressive effect in cell proliferation, DNA and RNA synthesis, and may be associated with the abnormal cell division on tumor tissues. On the other hand both estrogens and Ca 2+ are implicated in breast and prostate cancer but there are no studies focused on the expression of regucalcin in rat mammary gland or prostate. Furthermore, it is known that the expression of regucalcin in rat liver and kidney is regulated by 17b-estradiol (E 2). The aim of this study is to analyze if regucalcin is expressed in rat mammary gland and prostate and if it is regulated by E 2 in these tissues. We demonstrated for the first time that regucalcin mRNA and protein are present in rat mammary gland and prostate by in situ hybridization and immunohistochemistry, respectively. Furthermore, we show by Real-time PCR that E 2 down-regulates regucalcin expression in rat mammary gland and prostate.

We reported previously that human geneMAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with aMAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12MAGE genes are located in the q terminal region of chromosome X. LikeMAGE-1, the 11 additionalMAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that ofMAGE-1. The coding sequences of theMAGE genes predict the same main structural features for allMAGE proteins. In contrast, the promoters and first exons of the12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of eachMAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of theMAGE family includingMAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

by in situ X-ray observations based on the same pressure scale in the previous study . The overpressure was calculated using these boundaries. We observed the pressure to drop during the transformation by 1-2 GPa, but this would not affect the transformation rate significantly because the overpressure is very large in the present study. This has been confirmed in the post-spinel transformation 7 .

Gametophytic apomictic plants form nonreduced embryo sacs that generate clonal embryos by parthenogenesis, in the absence of both meiosis and eggcell fertilization. Here we report the sequence and expression analysis of a lorelei-like Paspalum notatum gene, n20gap-1, which encodes a GPI-anchored protein previously associated with apomixis in this species. Phylogeny trees showed that n20gap-1 was evolutionary related to the Arabidopsis thaliana lorelei genes At4g26466 and At5g56170. The lorelei At4g26466 disruption was shown to be detrimental to sperm cell release in arabidopsis. RFLP (Restriction Fragment Length Polymorphism) analysis revealed the occurrence of several homologous sequences in the Paspalum notatum genome, exhibiting polymorphisms genetically linked to apomixis. Real-time PCR showed that lorelei-family genes present a minor activity peak at pre-meiosis and a major one at anthesis. The apomictic genotype analyzed showed a significantly increased activity at pre-meiosis, post-meiosis and anthesis with respect to a sexual genotype. In situ hybridization assays revealed expression in integuments, nucellus and the egg-cell apparatus. Several n20gap-1 alleles differing mainly at the 3 0 UTR sequence were identified. Allelespecific real-time PCR experiments showed that allele 28 was significantly induced in reproductive tissues of the apomictic genotype with respect to the sexual genotype at anthesis. Our results indicate that P. notatum lorelei-like genes are differentially expressed in representative sexual (Q4188) and apomictic (Q4117) genotypes, and might play a role in the final stages of the apomixis developmental cascade. However, the association of n20gap-1 expression with the trait should be confirmed in significant number of sexual and apomictic genotypes.

In situ hybridization histochemistry was performed to analyse the distribution of the messenger RNA (mRNA) of three putative somatostatin (SRIF) receptors in rat brain, using oligonucleotide probes derived from the cDNA coding for SSTR-1, SSTR-2, and SSTR-3 receptors.

The selective antagonist for the 5-HT 2A serotonin receptor MDL 100,907, recently characterized autoradiographically in rat brain, has been characterized as a radioligand for the visualization of this receptor in human and monkey brain. In both species [ 3 H]MDL 100,907 binding to brain sections was saturable, had sub-nanomolar affinity (K d = 0.14 -0.19 nM in human brain; K d = 0.16-0.19 nM in monkey brain) and presented a pharmacological profile consistent with its binding to 5-HT 2A receptors (rank order of affinity for [ 3 H]MDL 100,907-labeled receptors: MDL 100,907 \spiperone\ketanserin\ mesulergine). The autoradiographical signal obtained with [ 3 H]MDL 100,907 was compared to the signal obtained with [ 3 H]ketanserin, [ 3 H]RP62203 and [ 3 H]mesulergine in both species, and to the distribution of 5-HT 2A receptor mRNA as determined by in situ hybridization in monkey brain. At variance with the other radioligands, [ 3 H]MDL 100,907 showed a single population of binding sites with extremely low levels of non-specific binding. As expected, mesulergine showed low affinity for [ 3 H]MDL 100,907-labeled receptors and the autoradiographic pattern shown by [ 3 H]mesulergine confirmed the lack of labeling of the 5-HT 2A receptor by this radioligand in primate brain. The similarity of the distribution of [ 3 H]MDL 100,907-labeled receptors and 5-HT 2A mRNA in monkey brain, supports the selectivity of this radioligand for 5-HT 2A receptors and suggests a somatodendritic localization of these receptors. The present results confirm [ 3 H]MDL 100,907 as the radioligand of choice at present for the autoradiographic visualization of 5-HT 2A receptors in mammalian brain including post-mortem human brain.

BACKGROUND: Previously, we showed that prenatal exposure to boric acid (BA), an industrial agent with large production, causes alterations of the axial skeleton in rat embryos, reminiscent of homeotic transformations. Indeed, Sprague-Dawley rats exposed in utero to BA on gestation day 9 (GD 9) had only six, rather than the normal seven, cervical vertebrae. This finding, observed in 91% of GD 21 fetuses, suggests posterior transformations of vertebrae. The present study attempts to determine if these skeletal alterations could be explained by modifications of the hox code, involved in the establishment of positional information along the craniocaudal axis of the embryo. METHODS: Pregnant rats were treated by gavage with BA (500 mg/kg, twice) on GD 9. Embryos were collected on GD 11 or GD 13.5 and processed for in situ hybridization. Several hox genes were selected according to the position of their cranial limit of expression in the cervical and thoracic region. RESULTS: At GD 13.5, we detected a cranial shift of the anterior limit of expression of hoxc6 and hoxa6. We observed no difference between control and treated embryos in the location of the cranial limit of expression of the other genes: hoxd4, hoxa4, hoxc5, and hoxa5. CONCLUSIONS: Our results demonstrate that following in utero exposure to BA on GD 9, a disturbance of the expression of hox genes involved in the specification of most anterior vertebrae is observed at GD 13.5. Based on their expression domain and on their implication in the definition of the cervicothoracic vertebral boundary, it is likely that the anteriorization of hoxc6 and hoxa6 reported here is correlated to the morphological phenotype observed in

Ornithine aminotransferase (OAT) is a reversible enzyme expressed mainly in the liver, kidney and intestine. OAT controls the interconversion of ornithine into glutamate semi-aldehyde, and is therefore involved in the metabolism of arginine and glutamine which play a major role in N homeostasis. We hypothesised that OAT could be a limiting step in glutamine -arginine interconversion. To study the contribution of the OAT enzyme in amino acid metabolism, transgenic mice that specifically overexpress human OAT in the liver, kidneys and intestine were generated. The transgene expression was analysed by in situ hybridisation and real-time PCR. Tissue (liver, jejunum and kidney) OAT activity, and plasma and tissue (liver and jejunum) amino acid concentrations were measured. Transgenic male mice exhibited higher OAT activity in the liver (25 (SEM 4) v. 11 (SEM 1) nmol/min per mg protein for wild-type (WT) mice; P, 0·05) but there were no differences in kinetic parameters (i.e. K m and maximum rate of reaction (V max )) between WT and transgenic animals. OAT overexpression decreased plasma and liver ornithine concentrations but did not affect glutamine or arginine homeostasis. There was an inverse relationship between ornithine levels and OAT activity. We conclude that OAT overexpression has only limited metabolic effects, probably due to the reversible nature of the enzyme. Moreover, these metabolic modifications had no effect on phenotype.

The levels and cellular localization of mRNA for complement C1q and C3 were examined by RNA gel blot and nonradioactive in situ hybridization in the frontal cortex of patients with Alzheimer's disease (AD) and age-matched controls. We found that the hybridization signal for C1q mRNA was markedly increased (approx. 3.5-fold) in the frontal cortex of AD patients compared to that in age-matched controls. In contrast to previous reports we also found that the levels of C3 mRNA, although well expressed, did not differ significantly between AD cases and age-matched controls. Nonradioactive in situ hybridization using digoxigenin-labeled riboprobes revealed that transcripts coding for both C1q and C3 were closely associated with neurons. These results support the hypothesis that complement could play a role in neuronal degeneration which has been observed in the brain of AD patients.

A 35-year-old black man with end-stage renal disease from biopsy-proven focal segmental glomerulosclerosis developed worsening function of his renal allograft 160 days after living related donor renal transplantation. Renal biopsy showed necrotizing and crescentic glomerulonephritis (NCGN) and presence of intraglomerular viral inclusions confirmed by immunocytochemical stain and in situ hybridization techniques to be cytomegaloviral in origin. Electron microscopy showed no immune complexes, and workup for other causes of NCGN was negative. The patient was treated with ganciclovir without other changes in his immunosuppressive regimen. After 8 weeks of ganciclovir therapy, a second renal transplant biopsy showed resolution of the glomerular process and disappearance of the cytomegalovirus (CMV) inclusions. The resolution of the glomerular process with treatment for CMV infection, and without other change in therapy, strongly supports a causative link between CMV and NCGN in this patient. This case represents the first report of CMV-associated NCGN in a renal transplant patient. 1998 by the National Kidney Foundation, Inc.

Multiple alternatively spliced 5' untranslated regions (S'UTRs) have been identified in growth hormone (GH) receptor mRNA isolated from hepatic and adipocyte tissue. In the present study, the preferential utilisation of a GC-rich S'UTR, designated exon lB, was observed following the isolation of ovine (0) GH receptor cDNA clones from a skeletal muscle cDNA library. Although exon lB-oGH receptor mRNA was expressed in all tissues examined, marked differences in the level of expression relative to the whole GH receptor transcript pool were observed between tissues. A single genomic clone (19) was isolated that encompassed exon lB, together with 6 kilobase pairs of 5' and 12 kilobase pairs of 3' flanking sequence. Multiple transcription initiation sites were identified using RNase protection analysis on skeletal muscle poly(A)+ RNA, a result consistent with the absence of a proximal TATA box element. A CAAT box (-37 to -33) and a putative binding site for SPl (a GC box -68 to -63) were found in the sense orientation immediately upstream of major transcription initiation site. Transfection of a series of overlapping promoter fragments linked to the luciferase reporter gene into HuH7, CHO and HeLa cells defined a core promoter element of 134 base pairs that was sufficient for maximum promoter activity. The emerging complexity of the 5' regulatory region of the GH receptor gene was emphasised by the observation that probes derived from exon 1B and the distal 3' intron boundary do not hybridise with previously cloned genomic sequences that span the liver-specific PI promoter and exon 2.

How divergent genetic systems regulate a common pathway during the development of two serial structures, forelimbs and hindlimbs, is not well understood. Specifically, HAND2 has been shown to regulate Shh directly to initiate its expression in the posterior margin of the limb mesenchyme. Although the Hand2-Shh morphoregulatory system operates in both the forelimb and hindlimb bud, a recent analysis suggested that its upstream regulation is different in the forelimb and hindlimb bud. A combination of all four Hox9 genes is required for Hand2 expression in the forelimb-forming region; however, it remains elusive what genetic system regulates the Hand2-Shh pathway in the hindlimb-forming region. By conditional inactivation of Islet1 in the hindlimb-forming region using the Hoxb6Cre transgene, we show that Islet1 is required for establishing the posterior hindlimb field, but not the forelimb field, upstream of the Hand2-Shh pathway. Inactivation of Islet1 caused the loss of posterior structures in the distal and proximal regions, specifically in the hindlimb. We found that Hand2 expression was downregulated in the hindlimb field and that Shh expression was severely impaired in the hindlimb bud. In the Hoxb6Cre; Islet1 mutant pelvis, the proximal element that is formed in a Shh-independent manner, displayed complementary defects in comparison with Pitx1 -/hindlimbs. This suggests that Islet1 and Pitx1 function in parallel during girdle development in hindlimbs, which is in contrast with the known requirement for Tbx5 in girdle development in forelimbs. Our studies have identified a role for Islet1 in hindlimb-specific development and have revealed Islet1 functions in two distinct processes: regulation upstream of the Hand2-Shh pathway and contributions to girdle development.

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-b-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect rootto-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.

Background In recent years, planaria have emerged as an important model system for research into stem cells and regeneration. Attention is focused on their unique stem cells, the neoblasts, which can differentiate into any cell type present in the adult organism. Sequencing of the Schmidtea mediterranea genome and some expressed sequence tag projects have generated extensive data on the genetic profile of these cells. However, little information is available on their protein dynamics. Results We developed a proteomic strategy to identify neoblast-specific proteins. Here we describe the method and discuss the results in comparison to the genomic high-throughput analyses carried out in planaria and to proteomic studies using other stem cell systems. We also show functional data for some of the candidate genes selected in our proteomic approach. Conclusions We have developed an accurate and reliable mass-spectra-based proteomics approach to complement previous genomic studies and to further achieve a more accurate understanding and description of the molecular and cellular processes related to the neoblasts.

The expression of proliferating cell nuclear antigen (PCNA) was studied in human papillomavirus (HPV)-infected, benign and malignant lesions of the genital tract and larynx using immunocytochemical staining of formalin-fixed clinical specimens. We observed the induction of PCNA in squamous carcinomas and adenocarcinomas, as has been demonstrated with other malignancies. In addition, the differentiated keratinocytes of the upper spinous cells and granulocytes in condylomata acuminata and low-grade intraepithelial neoplasias showed a consistent induction of PCNA compared with the normal squamous epithelium, in which only some of the parabasal and basal cells were positive. This reactivation of PCNA synthesis correlated with the presence of high copy numbers of HPV DNA and was independent of the oncogenic risk potential of the infecting HPV genotype. We postulate that HPV gene products induce the expression of PCNA and other components of the host DNA replication machinery in different...

The types of sodium channels that are expressed by neurons shape the rising phase of action potentials and influence patterns of action potential discharge. With regard to the enteric nervous system (ENS), there is uncertainty about which channels are expressed, and in particular it is unknown whether Na v 1.7 is present. We designed specific probes for the guinea pig Na v 1.7 ␣ subunit as well as for the other tetrodotoxin (TTX)-sensitive ␣ subunits (Na v 1.1, Na v 1.2, Na v 1.3, and Na v 1.6) in order to perform in situ hybridization (ISH) histochemistry on guinea pig myenteric ganglia. We established that only Na v 1.7 mRNA and Na v 1.3 mRNA are expressed in these ganglia. The ISH signal for Na v 1.7 transcripts was found in seemingly all the myenteric neurons. The expression of the Na v 1.3 ␣ subunit was confirmed by immunohistochemistry in a large proportion (62%) of the myenteric neuron population. This population included enteric sensory neurons. Na v 1.6 immunoreactivity, absent from myenteric neurons, was detected in glial cells only when a high anti-Na v 1.6 antibody concentration was used. This suggests that the Na v 1.6 ␣ subunit and mRNA are present only at low levels, which is consistent with the fact that no Na v 1.6 mRNA could be detected in the ENS by ISH. The fact that adult myenteric neurons are endowed with only two TTX-sensitive ␣ subunits, namely, Na v 1.3 and Na v 1.7, emphasizes the singularity of the ENS. Both these subunits, known to have slow-inactivation kinetics, are well adapted for generating action potentials from slow excitatory postsynaptic potentials, a mode of synaptic transmission that applies to all ENS neuron types. 1 The oligoprobes for ISH (column 2) were designed from the oligonucleotide sequences coding for the amino acid sequences included between the numbers indicated in column 3. These numbers resulted from alignment with orthologue rat amino acid sequences. The accession Nos. for these guinea pig sequences are indicated in column 4. The asterisks indicate the accession Nos. of the sequences determinated in the present study. In the case of Na v 1.1 and Na v 1.3, the guinea pig amino acid sequences included short sequences that were identical to the rat amino acid sequences used for raising the antibodies in the present study. The numbers that delimit these rat sequences are indicated in the column 5.

Although the precise mechanisms of the conversion of predentin to dentin are not well understood, several lines of evidence implicate the noncollagenous proteins (NCPs) as important regulators of dentin biomineralization. Here we compared the in vivo temporospatial expression patterns of two dentin NCP genes, dentin matrix protein 1 (Dmp1), and dentin sialophosphoprotein (DSPP) in developing molars. Reverse transcription-polymerase chain reaction was performed on embryonic day 13 to 1-day-old first molars using Dmp1- and DSPP-specific primer sets. Dmp1 transcripts appeared at the late bud stage, while DSPP mRNA was seen at the cap stage. Expression of both genes was sustained throughout odontogenesis. In situ hybridization analysis revealed interesting differences in the expression patterns of these genes. While Dmp1 and DSPP showed coexpression in young odontoblasts before the start of mineralization, the expression of these genes was notably distinct at later stages. Dmp1 expression decreased in secretory odontoblasts after the appearance of mineral, while high levels of DSPP were sustained in odontoblasts. In early secretory ameloblasts, DSPP expression was transient and down-regulated with the appearance of dentin matrix. Interestingly, Dmp1 expression became evident in ameloblasts during the maturative phase of amelogenesis. In contrast to Dspp expression that was tooth-specific, Dmp1 was expressed by osteoblasts throughout ossification in the skeleton. Probes directed to the “DSP” and “DPP” regions of the DSPP gene showed identical patterns of mRNA expression. These data show that the developmental expression patterns of Dmp1 and DSPP are distinct, implying that these molecules serve different biological functions in vivo.