ABSTRACT
Mosquitoes inject saliva into a vertebrate host during blood feeding. The analysis of mosquito saliva in host skin is important for the elucidation of the inflammatory responses to mosquito bites, the development of antithrombotic drugs, and the transmission-blocking of vector-borne diseases. We produced transgenic Anopheles stephensi mosquitoes expressing the secretory luciferase protein (MetLuc) fused to a saliva protein (AAPP) in the salivary glands. The transgene product (AAPP-MetLuc) of transgenic mosquitoes exhibited both luciferase activity as a MetLuc and binding activity to collagen as an AAPP. The detection of luminescence in the skin of mice bitten by transgenic mosquitoes showed that AAPP-MetLuc was injected into the skin as a component of saliva via blood feeding. AAPP-MetLuc remained at the mosquito bite site in host skin with luciferase activity for at least 4 h after blood feeding. AAPP was also suspected of remaining at the site of injury caused by the mosquito bite and blocking platelet aggregation by binding to collagen. These results demonstrated the establishment of visualization and time-lapse analysis of mosquito saliva in living vertebrate host skin. This technique may facilitate the analysis of mosquito saliva after its injection into host skin, and the development of new drugs and disease control strategies.
Subject(s)
Anopheles/genetics , Luciferases , Skin/chemistry , Animals , Animals, Genetically Modified , Anopheles/physiology , Luminescent Proteins , Mice , Optical Imaging/methods , Saliva/chemistry , Salivary Glands/chemistry , Time-Lapse ImagingABSTRACT
Midkine (MK) is a heparin-binding growth factor and a product of a retinoic acid-responsive gene. Midkine is overexpressed in many carcinomas and thought to play an important role in carcinogenesis. However, no studies have been focussed on the role of MK in pancreatic carcinoma. This study sought to evaluate the clinical significance of MK expression in pancreatic head carcinoma, including the relationship between immunohistochemical expression and clinicopathologic factors such as prognosis. Immunohistochemical expression of MK and CD34 was evaluated in pancreatic head carcinoma specimens from 75 patients who underwent surgical resection. Midkine was expressed in 53.3% of patients. Midkine expression was significantly correlated with venous invasion, microvessel density, and liver metastasis (P=0.0063, 0.0025, and 0.0153, respectively). The 5-year survival rate was significantly lower for patients positive for MK vs patients negative for MK (P=0.0073). Multivariate analysis revealed that MK expression was an independent prognostic factor (P=0.0033). This is the first report of an association between MK expression and pancreatic head carcinoma. Midkine may play an important role in the progression of pancreatic head carcinoma, and evaluation of MK expression is useful for predicting malignant properties of pancreatic head carcinoma.
Subject(s)
Cytokines/metabolism , Pancreatic Neoplasms/metabolism , Adult , Aged , Aged, 80 and over , Blotting, Western , Female , Humans , Immunohistochemistry , Liver Neoplasms/secondary , Male , Middle Aged , Midkine , Pancreatic Neoplasms/pathology , Prognosis , Survival Analysis , Survival RateABSTRACT
DNA methylation plays an essential role in genomic imprinting observed in eutherian mammals and marsupials. In mouse, one of the two de novo DNA methyltransferases, Dnmt3a, and a related protein, Dnmt3L have been shown to be essential for imprint establishment in the parental germline. To gain insights into the evolution of imprinting mechanisms, we have identified and characterized the DNMT3 family genes in other vertebrate species. We cloned cDNAs for chicken DNMT3A and DNMT3B, whose putative protein products shared 81.5% and 48.6% amino acid sequence identity with their mouse orthologues. Using computer-assisted database searches, we also identified DNMT3A and DNMT3B orthologues in fish (fugu and zebrafish) and marsupials (opossum). We found that, while opossums had an orthologue for DNMT3L, chickens and fish did not have this gene. Thus, unlike the other DNMT3 members, DNMT3L was restricted to the species in which imprinting occurs. The acquisition of DNMT3L by a common ancestor of eutherians and marsupials might have been closely related to the evolution of imprinting.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , Evolution, Molecular , Genomic Imprinting , Vertebrates/genetics , Amino Acid Sequence , Animals , Chickens , Conserved Sequence , Fishes , Humans , Molecular Sequence Data , Opossums , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
In birds, females are heterogametic (ZW), while males are homogametic (ZZ). It has been proposed that there is no dosage compensation for the expression of Z-linked genes in birds. In order to examine if the genes are inactivated on one of the two Z chromosomes, we analyzed the allelic expression of the B4GALT1 and CHD-Z genes on Z chromosomes in male chickens. One base substitution was detected among 15 chicken breeds and lines examined for each gene, and cross mating was made between the breeds or lines with polymorphism. cDNAs were synthesized from cultured cell colonies each derived from a single cell of an F1 male embryo. The allelic expression of the B4GALT1 gene was examined by restriction fragment length polymorphism analysis of the PCR products digested with RSAI, and that of the CHD-Z gene by the single nucleotide primer extension (SNuPE) method. Both of the genes displayed biallelic expression, suggesting that these Z-linked genes were not subject to inactivation in male chickens. Comparison between expression levels in males and females by real-time quantitative PCR suggested that expression was compensated for the CHD-Z gene but not for the B4GALT1 gene.
Subject(s)
Avian Proteins , Chickens/genetics , Sex Chromosomes/genetics , Alleles , Animals , Base Sequence , Cells, Cultured , Chick Embryo , DNA/chemistry , DNA/genetics , DNA Mutational Analysis , DNA-Binding Proteins/genetics , Female , Gene Expression Regulation, Developmental , Genetic Linkage , Male , N-Acetyllactosamine Synthase/genetics , Polymorphism, Single Nucleotide , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
By screening 26 chicken breeds and lines, DNA polymorphisms were identified in the IGF2 and MPR1 genes, of which mammalian homologues are parentally imprinted, and the GAPD gene, a housekeeping control. Using the polymorphisms as genetic markers, we found that all three genes are expressed biallelically in embryonic tissues. IGF2 and MPR1 were mapped on chicken chromosomes 5 and 3, respectively, by fluorescence in situ hybridization, demonstrating conserved linkage homology between mammals and birds.
Subject(s)
Alleles , Cell Cycle Proteins/genetics , Chickens/genetics , Endopeptidases , Gene Expression Profiling , Insulin-Like Growth Factor II/genetics , Physical Chromosome Mapping , Polymorphism, Genetic/genetics , Saccharomyces cerevisiae Proteins/genetics , Animals , Base Sequence , Breeding , Chick Embryo , Conserved Sequence/genetics , Female , Gene Expression Regulation, Developmental , Genomic Imprinting/genetics , In Situ Hybridization, Fluorescence , Male , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
We have constructed approximately 1-Mb contigs of yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones covering the imprinted region in mouse chromosome band 7F4/F5. This region is syntenic to human chromosome 11p15.5, which is associated with Beckwith-Wiedemann syndrome (BWS) and certain childhood and adult tumors. These contigs provide the basis for genomic sequencing, identification of genes and their regulatory elements, and functional studies in transgenic and knockout mice, which should be of help to understand not only the mechanisms of imprinting but also the molecular events involved in the genesis of BWS and tumors.
