Cloning, expression and chromosomal localization of Wnt-13, a novel member of the Wnt gene family

The Wnt genes, encoding structurally-related secreted glycoproteins, are implicated in mammary carcinogenesis induced by mouse mammary tumor virus. In search of the Wnt gene(s) expressed in human gastric cancer, a WTGC1 cDNA fragment sharing 66.9% amino-acid homology with human and mouse Wnt-2 was isolated by degenerate polymerase chain reaction. The human gene corresponding to WTGC1 was designated as Wnt-13 and overlapping Wnt-13 cDNAs were cloned. Nucleotide sequence analysis indicated that the Wnt-13 gene encodes the protein of 372 amino acids, including a signal peptide, two potential N-glycosylation sites and 24 cystein residues highly conserved among members of the Wnt gene family. The Wnt-13 mRNA of 2.5 kb in size was detected in heart, brain, placenta, lung, prostate, testis, ovary, small intestine and colon of adult human and also in brain, lung and kidney of fetal human. Among various cancer cell lines, the Wnt-13 mRNA was detected in HeLa (cervical cancer), MKN28 and MKN74 (gastric cancer). The Wnt-13 gene has been mapped to human chromosome 1p13. These results suggest that the Wnt-13 gene may be involved in normal human development or differentiation as well as in human carcinogenesis.     

Region-specific expression of chicken Sox2 in the developing gut and lung epithelium: regulation by epithelial-mesenchymal interactions

A quality control system especially designed for dosimetry in scanning proton beams has been designed and tested. The system consists of a scintillating screen (Gd2O2S:Tb), mounted at the beam-exit side of a phantom, and observed by a low noise CCD camera with a long integration time. The purpose of the instrument is to make a fast and accurate two-dimensional image of the dose distribution at the screen position in the phantom. The linearity of the signal with the dose, the noise in the signal, the influence of the ionization density on the signal, and the influence of the field size on the signal have been investigated. The spatial resolution is 1.3 mm (1 s.d.), which is sufficiently smaller than typical penumbras in dose distributions. The measured yield depends linearly on the dose and agrees within 5% with the calculations. In the images a signal to noise ration (signal/1 s.d.) of 10(2) has been found, which is in the same order of magnitude as expected from the calculations. At locations in the dose distribution possessing a strong contribution of high ionization densities (i.e., in the Bragg peak), we found some quenching of the light output, which can be described well by existing models if the beam characteristics are known. For clinically used beam characteristics such as a Spread Out Bragg peak, there is at most 8% deviation from the NACP ionization chamber measurements. The conclusion is that this instrument is a useful tool for quick and reliable quality control of proton beams. The long integration-time capabilities of the system make it worthwhile to investigate its applicability in scanning proton beams and other dynamic treatment modalities.     

Inhibition of chondrogenesis by Wnt gene expression in vivo and in vitro

The Wnt family of secreted signaling proteins are implicated in regulating morphogenesis and tissue patterning in a wide variety of organ systems. Several Wnt genes are expressed in the developing limbs and head, implying roles in skeletal development. To explore these functions, we have used retroviral gene transfer to express Wnt-1 ectopically in the limb buds and craniofacial region of chick embryos. Infection of wing buds at stage 17 and tissues in the head at stage 10 resulted in skeletal abnormalities whose most consistent defects suggested a localized failure of cartilage formation. To test this hypothesis, we infected micromass cultures of prechondrogenic mesenchyme in vitro and found that expression of Wnt-1 caused a severe block in chondrogenesis. Wnt-7a, a gene endogenously expressed in the limb and facial ectoderm, had a similar inhibitory effect. Further analysis of this phenomenon in vitro showed that Wnt-1 and Wnt-7a had mitogenic effects only in early prechondrogenic mesenchyme, that cell aggregation and formation of the prechondrogenic blastema occurred normally, and that the block to differentiation was at the late-blastema/early-chondroblast stage. These results indicate that Wnt signals can have specific inhibitory effects on cytodifferentiation and suggest that one function of endogenous Wnt proteins in the limbs and face may be to influence skeletal morphology by localized inhibition of chondrogenesis.     

The mRNA-phenotype of granuloma formation: CD4+ T cell-associated cytokine gene expression during primary murine listeriosis

In murine listeriosis, elimination of bacteria and immunity to reinfection critically depend on Thy1+ CD4- cells, while cell-mediated inflammatory phenomena such as DTH and granuloma formation are mostly mediated by CD4+ T cells. In an attempt to correlate T cell phenotype and function with a particular set of cytokines produced, we examined the cytokine gene expression profile associated with the presence or absence of Thy1+, CD4+ and/or CD8+ cells in the livers of mice during a primary infection with L. monocytogenes. The presence of CD4+ cells was found to be closely associated with mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increase in expression of TNF-alpha and GM-CSF and a 25-fold increase in expression of IFN-gamma and TNF-beta mRNAs, and temporally coincided with the development of granulomatous lesions. In vivo neutralization of TNF-alpha and, to a lesser extent, IFN-gamma resulted in abrogation of granuloma formation. A similar correlation between the presence of CD8+ cells and mRNA expression for any one of the cytokines studied did not exist, pointing to a qualitatively different mechanism of CD8+ T cell mediated cure of listeriosis.     

Structure, chromosomal localisation and expression of the murine dominant negative helix-loop-helix Id4 gene

In the elderly patients a greater loss of teeth is frequently seen in combination with periodontal diseases. This article presents an overview of the changes of oral structures during ageing as well as immunology, physical and psychological aspects. Also summarised are complications by systemic illnesses, drug medications and periodontal alterations. If possible illnesses and medications are taken into consideration, there should be no special problem for the elderly patients.     

A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion

Mucus hypersecretion and plugging of lower respiratory tract airways contributes to the morbidity and mortality associated with asthma. Interleukin (IL)-4 plays a putative role in some forms of asthma. Thus, transgenic mice that overexpress murine IL-4 selectively within the lung were used to study the effect of IL-4 on mucus glycoprotein gene expression and mucin release. Histologic examination of lung sections from IL-4 mice revealed that nonciliated epithelial cells from conducting airways were hypertrophic, due at least in part to the accumulation of mucus glycoprotein. The cytoplasm of these cells stained positively for glycoproteins using mucicarmine, alcian blue (AB), and periodic acid-Schiff (PAS). Ciliated cells were also enlarged but did not show any mucin-specific staining. Inclusion granules typically found in nonciliated (Clara) cells of control mice were absent in the IL-4 transgenic mice. Northern blot analysis of total RNA from lung tissue revealed that the expression of the MUC5AC, but not MUC2, mucin gene was distinctly upgraded in IL-4 transgenic mice compared to transgene-negative controls. In addition, a 5- to 10-fold increase in AB- and PAS-positive material was found in lavage fluid from IL-4 overexpressing mice compared to transgene-negative controls. Thus, the overexpression of IL-4 locally within the lung enhances mucus glycoprotein synthesis by altering gene expression, results in the accumulation of mucus glycoprotein in nonciliated epithelial cells, and induces the release of mucus into the airway lumen. We therefore hypothesize that the overproduction of mucus seen in some patients with asthma may be a direct result of the action of IL-4 within the inflamed lung.     

The control of IL-4 gene expression in activated murine T lymphocytes: a novel role for neu-1 sialidase

IL-4 is important in controlling the development of immune responses. Following activation with anti-CD3epsilon under serum-free conditions, splenocytes from most normal (neu-1b) mouse strains directly produced IL-4 and other T cell cytokines. However, splenic T cells from SM/J and B10.SM (H-2v, neu-1a) strain mice, deficient in neu-1 sialidase activity, failed to produce IL-4 but produced normal levels of IL-2 following activation. Moreover, sialidase-deficient mice produced markedly less IgE and IgG1 Abs following immunization with protein Ags than did mouse strains with normal neu-1 sialidase activity. Enriched T cells from neu-1a mice failed to be effectively primed with exogenous murine IL-4 to become IL-4-producing cells. Treatment of splenocytes or enriched T cells from neu-1a mice with bacterial sialidase prior to activation or IL-4 priming promoted their subsequent capacity to produce IL-4. In contrast, activation of T cells from neu-1b mice in the presence of a sialidase inhibitor almost completely blocked subsequent IL-4 production. The presence of IL-4 during priming enhanced T cell expression of neu-1-specific sialidase activity and increased the membrane expression of asialo-G(M1) compared with T cells activated without IL-4. These results suggest that T cell-associated neu-1 sialidase is required for early IL-4 production by splenic T cells and is involved in the IL-4 priming process of conventional T cells to become active IL-4 producers.     

Activation of DNA synthesis and expression of the JE gene by catalase in mouse osteoblastic cells: possible involvement of hydrogen peroxide in negative growth regulation

Type I diabetes susceptibility genes have been identified within the major histocompatibility complex (MHC) on chromosome 6p21.3 and near the VNTR/insulin region on chromosome 11p15.5. We have used polymorphic dinucleotide repeat markers to search the human genome for additional susceptibility genes in 162 type I diabetic families with an affected sibling pair. We report that an additional susceptibility gene is located on chromosome 2q31 near HOXD8 (P < 10(-5), maximum logarithm of odds score = 4.8) in an analysis of affected sibling pairs having specific human leukocyte antigen (HLA) and hypervariable nucleotide tandem repeat (VNTR)/insulin gene haplotypes (absence of high-risk HLA-DR3/4 haplotypes and presence of homozygous high-risk class I VNTR alleles). These results suggest the interaction of a minimum of three genes in the pathogenesis of type I diabetes in humans.     

Tyrosine-533 of rat dopamine transporter: involvement in interactions with 1-methyl-4-phenylpyridinium and cocaine

To improve our understanding of structure-function relationships for neurotransmitter transporters, we performed site-directed mutagenesis of the rat dopamine transporter (DAT) and assessed the functions of the mutants in transiently-expressing COS cells. Tyrosine-533 of rat DAT lies in the 11th transmembrane region, where the corresponding amino acid of human DAT is phenylalanine. Alanine substitution of tyrosine-533 (Y533A) conferred an increased affinity for 1-methyl-4-phenylpyridinium (MPP+). Phenylalanine substitution of tyrosine-533 (Y533F) increased the velocity of MPP+ uptake but decreased DAT's affinity for MPP+. Cocaine's potency in inhibiting dopamine uptake was unchanged with Y533A, but increased with Y533F. Differences in the uptake kinetics and inhibitory potency of cocaine between rat and human DATs were similar to the differences observed between the wild-type and Y533F mutants DATs. Tyrosine-533 may be important for the DAT function and for species differences in transporter functions, including differential sensitivities to cocaine and 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in humans and rats.     

Controlling gene expression in living cells through small molecule-RNA interactions

Short RNA aptamers that specifically bind to a wide variety of ligands in vitro can be isolated from randomized pools of RNA. Here it is shown that small molecule aptamers also bound their ligand in vivo, enabling development of a method for controlling gene expression in living cells. Insertion of a small molecule aptamer into the 5' untranslated region of a messenger RNA allowed its translation to be repressible by ligand addition in vitro as well as in mammalian cells. The ability of small molecules to control expression of specific genes could facilitate studies in many areas of biology and medicine.     

Gamma delta T lymphocytes in oriental cutaneous leishmaniasis: occurrence and variable delta gene expression

It has been suggested that T lymphocytes expressing gamma delta T-cell receptors could play an important role in defence against some intracellular infectious pathogens. The present study was undertaken to characterize the occurrence and variable delta gene expression of T lymphocytes expressing the gamma delta T-cell receptor in oriental cutaneous leishmaniasis. Eleven cases of oriental cutaneous leishmaniasis were investigated by immunohistological analysis using an alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. In three cases, we observed an increased percentage of gamma delta T cells (about 20% of CD3+ cells). In these cases gamma delta T cells generally expressed the V delta 2 segment, and only rarely the V delta 1 gene product. V delta 2+ cells were predominantly localized in the dermis, and were virtually absent in the epidermal compartment. The rare gamma delta T cells observed in the epidermis were almost exclusively V delta 1+. This study demonstrates that an increase of gamma delta T cells may be found in oriental cutaneous leishmaniasis, although it is not a constant feature of the disease. The finding of a preferential expansion of the V delta 2 subset suggests that this subpopulation of gamma delta T cells might be selectively involved in the recognition of Leishmania antigens. The distinct compartmentalization of gamma delta T-cell subpopulations indicates that these subsets may recognize distinct sets of antigens.     

Aspirin inhibits tumor necrosis factoralpha gene expression in murine tissue macrophages

Postnatal development of the gerbil ventral cochlear nucleus (VCN) was studied quantitatively under the light microscope in Nissl-stained serial sections at postnatal day 0 (P0), P5, P7, P10, P12, P15, and P140. VCN boundaries were unambiguous at all ages, and nucleus volume was calculated planimetrically for all groups. Measurements of neuron soma cross-sectional area and number were made in all groups except P0. Both VCN volume and soma area doubled between P5 and P10. Although somatic growth did not continue beyond P10, VCN volume increased a further 57% between P15 and P140. Neuron number did not change significantly between P5 and P10, averaging approximately 36,000 neurons. Between P10 and P12, neuron number decreased significantly by 22%, with no further change thereafter. Our data show that, following significant postnatal growth in the gerbil VCN, a brief period of naturally occurring neuron death begins at the onset of hearing.     

Characterization of the transport of a cationic octapeptide, octreotide, in rat bile canalicular membrane: possible involvement of P-glycoprotein

Several approaches were successfully performed to directly assign and characterize auxin binding of ABP44 in gel. The 44 kDa high affinity auxin binding protein ABP44 from pea was tested for its ability to bind 5-azido-[7-3H]-IAA in photoaffinity labeling experiments. Competition experiments with several auxin analogues confirm data published previously (Reinard and Jacobsen 1995). Critical reflections of the limitations of the method are also discussed. Immunostaining using the antibody D16 (Napier and Venis 1992), which is directed against the putative binding site of ABP1, revealed that ABP44's auxin binding site is at least partially related to the corresponding site of ABP1. Nevertheless, both proteins do not share any further immunological relationships. Our results with D16 recommend a careful reconsideration of data published by other authors. Furthermore, a 80 kDa, dimeric glutathione dependent formaldehyde dehydrogenase (FDH) from mung bean, described recently, was found to be different from ABP44. In contrast to the described FDH, ABP44 exhibited no FDH activity.