The estimation of absorbed dose rates for non-human biota: an extended intercomparison.

An exercise to compare 10 approaches for the calculation of unweighted whole-body absorbed dose rates was conducted for 74 radionuclides and five of the ICRP's Reference Animals and Plants, or RAPs (duck, frog, flatfish egg, rat and elongated earthworm), selected for this exercise to cover a range of body sizes, dimensions and exposure scenarios. Results were analysed using a non-parametric method requiring no specific hypotheses about the statistical distribution of data. The obtained unweighted absorbed dose rates for internal exposure compare well between the different approaches, with 70% of the results falling within a range of variation of ±20%. The variation is greater for external exposure, although 90% of the estimates are within an order of magnitude of one another. There are some discernible patterns where specific models over- or under-predicted. These are explained based on the methodological differences including number of daughter products included in the calculation of dose rate for a parent nuclide; source-target geometry; databases for discrete energy and yield of radionuclides; rounding errors in integration algorithms; and intrinsic differences in calculation methods. For certain radionuclides, these factors combine to generate systematic variations between approaches. Overall, the technique chosen to interpret the data enabled methodological differences in dosimetry calculations to be quantified and compared, allowing the identification of common issues between different approaches and providing greater assurance on the fundamental dose conversion coefficient approaches used in available models for assessing radiological effects to biota.

Progesterone stimulates the expression of aminopeptidase A/angiotensinase in human choriocarcinoma cells.

In human placenta aminopeptidase A (APA), a principal enzyme that converts angiotensin II to angiotensin III, seems to be involved in angiotensin II metabolism during pregnancy. In this study, we investigated the possible effects of progesterone and estrogen on APA mRNA and protein levels in choriocarcinoma cells as a model for placenta. By RNase protection assay, progesterone induced higher APA mRNA levels than estrogen at the same concentration. Progesterone exhibited dose-dependent stimulation of APA mRNA, 1.8-fold increase at 10(-6) m for 24 h treatment. Progesterone at 10(-6) m increased APA mRNA levels within 12 h and in time-dependent fashion up to 24 h. Fluorescence-activated cell sorting analysis and measurements of APA activities revealed the induction of APA protein by progesterone. Expression of progesterone receptors (PR) and glucocorticoid receptors (GR) were determined in these cells by RT-PCR, which suggested that the progesterone's actions might be displayed through PR and/or GR. These findings may serve as a useful model to study the effects of progesterone on angiotensin II metabolism in placenta, although the physiological validity of these studies remains to be clarified.

Functional analysis of promoter activity of murine beta-1,6-N-acetylglucosaminyltransferase.

In the mouse, beta-1,6-N-acetylglucosaminyltransferase (IGnT) forms branches in poly-N-acetyllactosamines, which are good scaffolds for oncodevelopmental cell-surface antigens and recognition markers. There are two isoforms of IGnT, IGnT A and B, which are produced by alternative splicing of the IGnT gene; the unique portion is encoded by exon 1 and common portion is encoded by exons 2 and 3. Thus, the expression of each isoform is controlled by a different promoter. Here, we identified the regulatory regions of the mouse IGnT A and B genes. The promoter regions for IGnT A and B did not contain putative TATA or CAAT boxes, but each contained GT boxes. The upstream regulatory region of each gene was examined by transient luciferase reporter gene transfection experiments and gel mobility shift assay. Promoter activity for each gene was detected in F9 embryonal carcinoma cells, which express IGnT A and B, but not in N2a cells, which do not express the gene. Deletion analysis demonstrated that the regions 308 bp upstream from the transcriptional initiation site of IGnT A and 430 bp upstream from the transcriptional initiation site of IGnT B showed minimal promoter activity. Mutation of the single GT box in IGnT A and two GT boxes in IGnT B caused marked reduction of the promoter activity. These findings provided strong evidence that the GT boxes play crucial roles in transcriptional regulation of the genes.

Metallosphaera sedula TA-2, a calditoglycerocaldarchaeol deletion strain of a thermoacidophilic archaeon.

A spherical thermoacidophilic archaeon, strain TA-2, was obtained from acidic hot springs located in Ohwaku Valley, Hakone, Japan. This isolate is an obligate aerobic chemoorganoheterotroph that grows optimally at about 75 degrees C, pH 2.8. The G + C content of DNA from TA-2 is 47 mol%. The 16S rRNA gene from TA-2 showed more than 99% similarity with those of Metallosphaera sedula and Metallosphaera prunae and less than 92% similarity with other members of the order Sulfolobales. DNA-DNA hybridization experiments showed more than 93% genomic DNA homology among TA-2, M. sedula DSM5348T, and M. prunae DSM10039T. However, TA-2 lacks calditoglycerocaldarchaeol derivatives, which are usually found in the membrane lipids of members of the order Sulfolobales. Therefore, calditoglycerocaldarchaeol may not be essential for survival in thermophilic and acidophilic environments. The isolate was deposited as Metallosphaera sedula TA-2 (JCM 9064, IFO 15160).

Colocalization of neuropilin-1 and Flk-1 in retinal neovascularization in a mouse model of retinopathy.

PURPOSE: To investigate the mechanisms of the development of retinal neovascularization, the localizations of vascular endothelial (VEGF) receptors Flk-1 and neuropilin (NP)-1 mRNAs were examined. METHODS: The model of retinopathy of prematurity (ROP) was produced by ischemia-induced ocular neovascularization, by exposing postnatal day-7 mice to 75% oxygen for 5 days and then returning them to room air for 5 days. Retinal neovascularization was visualized by injection of fluorescein-dextran. Expression of Flk-1 and NP-1 mRNAs were examined by in situ hybridization with flatmount and serial sections of the retina. The localization of NP-1 was also confirmed by immunohistochemistry. Blood vessel patterns were characterized by immunohistochemical localization of von Willebrand factor (vWF). RESULTS: Flatmount in situ hybridization showed intense expression of NP-1 and Flk-1 mRNAs colocalized in the area of neovascularization. In situ hybridization of serial sections of the retina revealed that expression of Flk-1 and NP-1 was restricted to neovascularized vessels of the retina from ROP mice. CONCLUSIONS: The restricted expression of Flk-1 and NP-1 on neovascularized vessels suggests that these molecules may play important roles in retinal neovascularization. This is the first report of the colocalization of NP-1 and Flk-1 on neovascularized vessels of the retina from ROP mice.

Glypican-2 binds to midkine: the role of glypican-2 in neuronal cell adhesion and neurite outgrowth.

Cell-surface heparan sulfate proteoglycans participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study was performed to elucidate whether glypican-2 plays a role in interactions of neurons with midkine (MK), a heparin-binding neuroregulatory factor. MK bound to heparan sulfate chains of glypican-2 in a manner similar to syndecan-3. Microbeads coated with MK or poly-L-lysine induced clustering of glypican-2 as well as syndecan-3. Substratum-bound MK or poly-L-lysine induced cell adhesion of N2a neuroblastoma cells, while only MK promoted neurite outgrowth of these cells. Ligation of cell-surface glypican-2 with MK or an antibody against epitope-tagged glypican-2 induced cell adhesion and promoted neurite outgrowth. These results verified that cell-surface glypican-2 bound to MK and suggested that MK-glypican-2 interactions participate in neuronal cell migration and neurite outgrowth. In addition, we observed different localization of epitope-tagged glypican-2 and syndecan-3 on the surface of N2a cells; the result suggested that they may play different roles in MK-mediated neural function.

Feline embryo transfer during the non-breeding season.

To obtain normal kits by embryo treansfer (ET) during the non-breeding season, maintenance of pregnancy was carried out by administration of sustained action progesterone (P4) in queens. Embryos were recovered six days after mating from five donor queens in which ovulation was induced by administration of eCG and hCG. The number of embryos recovered ranged from 24 to 53 (mean: 37.2 +/- 6.4) per animal and most embryos were compacted morulae. The yield of embryos was 49.0-93.3% (mean: 73.8 +/- 9.6%). As for recipients, porcine pituitary gland preparation and hCG were administered to 19 queens and estrus and ovulation were induced in 18 queens (94.7%). These queens underwent intrauterine ET of five compacted morulae and 17 cats (94.4%) were impregnated. The number of implantations was 2-5 (mean: 3.7 +/- 0.3). Among these impregnated queens, 15 cats received P4 adminstration starting on day 24 of gestation and 1-5 newborns (mean: 3.4 +/- 0.3) were obtained by normal delivery or caesarean section on day 64-69 of gestation. However, two animals that were not treated with P4 underwent spontaneous abortion about the mid gestational period. Therefore, it is possible to obtain normal kits from queens in the non-breeding season by ET with maintenance of pregnancy by P4 administration.

Genomic organization and transcriptional regulation of the mouse GD3 synthase gene (ST8Sia I): comparison of genomic organization of the mouse sialyltransferase genes.

The genomic organization of the gene encoding the mouse GD3 synthase (ST8Sia I) has been determined. The mouse ST8Sia I gene spans over 100 kilobases of genomic DNA with a unique genomic structure of 5 exons. Analysis of the sequence immediately upstream of the transcription initiation site revealed that the ST8Sia I promoter contained no canonical TATA- or CCAAT-box, but contained a putative Sp1 binding site. Transient transfection experiments demonstrated functional promoter activity of the ST8Sia I promoter in an ST8Sia I-expressing cell line, P19, but not in an ST8Sia I-nonexpressing cell line, NIH3T3. Mobility shift assay and mutation analysis of the promoter region indicated that the Sp1 binding site is involved in the transcriptional regulation of the ST8Sia I gene in P19 cells. Here, the genomic structural analyses of mouse sialyltransferase genes are summarized and the genomic structures of these genes are compared.

A novel variant form of murine beta-1, 6-N-acetylglucosaminyltransferase forming branches in poly-N-acetyllactosamines.

A novel form of murine beta-1,6-N:-acetylglucosaminyltransferase that forms branches in poly-N:-acetyllactosamines (designated as IGnT B) was cloned based on sequence homology to the known IGnT (designated as IGnT A). When expressed as proteins, IGnT B showed higher specific activity than IGnT A. The C-terminal 1/4 of IGnT B was identical to that of IGnT A, while the rest of the predicted sequences showed 63% identity. Genomic analysis indicated that IGnT A and IGnT B were derived by alternative splicing; the unique portion was encoded by exon 1, and the common portion was encoded by exons 2 and 3. IGnT B showed an expression profile closely related to that of IGnT A and was strongly expressed in the liver, kidney and intestine, and moderately in the mammary gland, submaxially gland, embryonic stem cells, and embryonal carcinoma cells. The specificity of IGnT B examined using various substrates was indistinguishable from that of IGnT A, which is classified as the central acting IGnT (cIGnT). Thus, IGnT B acted on Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, but not on GlcNAcbeta1-3Galbeta1-4Glc. It formed branches in both of the internal galactosyl residues of Galbeta1-4Glc-NAcbeta1-3Galbeta1-4GlcNAcbeta1-++ +3Galbeta1-4Glc, and prolonged incubation resulted in production of the di-branched oligosaccharide. Although addition of sialic acid to the terminal galactosyl residue did not abolish the acceptor activity, alpha2-6 sialylation was a preferred one as compared to alpha2-3 sialylation.

Sequence analysis of three family B DNA polymerases from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis.

Three family B DNA polymerase genes, designated B1, B2, and B3, were cloned from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis, and sequenced. Deduced amino acid sequences of B1 and B3 DNA polymerases have all exonuclease and polymerase motifs which include critical residues for catalytic activities. Furthermore, a YxGG/A motif, which is located between 3'-5' exonuclease and polymerization domains of family B DNA polymerases, was also found in each of the B1 and B3 sequences. These findings suggested that S. ohwakuensis B1 and B3 DNA polymerases have both exonuclease and polymerase activities. However, amino acid sequence of the B2 DNA polymerase of this organism contains several amino acid substitutions in Pol-motifs, and also lacks Exo-motif I and Exo-motif II. These substitutions and lack of certain motifs raise questions about polymerase and exonuclease activities of the corresponding gene product. The B3 sequence of S. ohwakuensis is more closely related to Pyrodictium, Aeropyrum, and Archaeoglobus DNA polymerase B3 sequences than to the Sulfolobus B3 sequences. Phylogenetic analysis showed that crenarchaeal B1 DNA polymerases are closely related to each other, and suggested that crenarchaeal B3, euryarchaeal family B, and eukaryal epsilon DNA polymerases may be orthologs.

Diversity of N-acetylglucosamine-6-O-sulfotransferases: molecular cloning of a novel enzyme with different distribution and specificities.

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) transfers sulfate to the C-6 position of non-reducing N-acetylglucosamine (GlcNAc) residues. We cloned human and mouse cDNAs encoding a novel GlcNAc6ST, designated as GlcNAc6ST-4, which showed sequence identities of 26 to 41% to other GlcNAc6STs. Human organs with strong expression of the enzyme mRNA were the heart, spleen, and ovary, while in the mouse strong expression was detected in the kidney. The enzyme expressed in CHO cells preferentially acted on mannose-linked GlcNAc, while a core 2 mucin-type oligosaccharide and an N-acetyllactosamine oligomer also served as acceptors. The distribution and the specificity of GlcNAc6ST are different from those of GlcNAc6STs identified previously.

The ratio of splicing variants of MGC-24/CD164, a sialomucin, correlates with the metastatic potential of colorectal carcinomas.

MGC-24/CD164 is a sialomucin expressed in many normal and cancerous tissues. In humans, soluble and transmembrane forms of MGC-24 are produced by alternative splicing. The total MGC-24 RNA level was found to be lower in human colorectal carcinomas as compared with the adjacent normal mucosal tissues. Lower MGC-24 mRNA levels in colon carcinomas and in the adjacent normal mucosa epithelium correlate with lymphatic vessel invasion by the carcinoma. The ratio of the soluble form to the transmembrane form of the mRNA in colorectal carcinomas was determined by ribonuclease protection assay. Higher ratios were correlated with less venous invasion and less remote metastasis, which became evident during postoperative observation.

Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney.

Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases. The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.

Molecular cloning and genomic analysis of mouse GalNAc alpha2, 6-sialyltransferase (ST6GalNAc I).

cDNA clones encoding mouse GalNAc alpha2,6-sialyltransferase (ST6GalNAc I) were isolated from a mouse submaxillary gland cDNA library. The deduced amino acid sequence of cDNA clones is 526 amino acids in length and has highly conserved motifs among sialyl transferases, sialyl motifs L, S, and VS. The expressed recombinant enzyme exhibited similar substrate specificity to chicken ST6GalNAc I. The mouse ST6GalNAc I gene was expressed in submaxillary gland, mammary gland, colon, and spleen. The mouse ST6GalNAc I gene was also cloned from a mouse genomic library, which was divided into 9 exons spanning over 8 kilobases of genomic DNA. The genomic structure of the mouse ST6GalNAc I gene was similar to that of the mouse ST6GalNAc II gene. Unlike the ST6GalNAc II gene, however, which has a housekeeping gene-like promoter with GC-rich sequences, the ST6GalNAc I gene has two promoters and they do not contain GC-rich sequences but contain putative binding sites for tumor-associated transcription factors such as c-Myb, c-Myc/Max, and c-Ets. Analysis of the 5'-RACE PCR products suggested that the mouse ST6GalNAc I gene expression is regulated by these two promoters in tissue-specific manners.

Comparative analysis of the genomic structures and promoter activities of mouse Siaalpha2,3Galbeta1,3GalNAc GalNAcalpha2, 6-sialyltransferase genes (ST6GalNAc III and IV): characterization of their Sp1 binding sites.

The genomic organization of the genes encoding the mouse N-acetylgalactosamine alpha2,6-sialyltransferase specific for Siaalpha2,3Galbeta1,3GalNAc (ST6GalNAc III and IV) has been determined. The ST6GalNAc III gene spans over 120 kilobases of genomic DNA with 5 exons; on the other hand, the ST6GalNAc IV gene spans over 12 kilobases of genomic DNA with 6 exons. But the exon-intron boundaries of these genes are very similar. The 5'-flanking regions of these genes do not contain a TATA- or CAAT-box but have three putative Sp1 binding sites for each promoter. Transient transfection experiments demonstrated functional promoter activity in an ST6GalNAc III-expressing cell line, P19, for the ST6GalNAc III promoter, and in an ST6GalNAc IV-expressing cell line, NIH3T3, for the ST6GalNAc IV promoter. Mobility shift assaying and mutational analysis of the promoter region indicated that two of the three Sp1 binding sites are involved in the transcriptional regulation of the ST6GalNAc III gene in P19 cells, while all three Sp1 binding sites are involved in the transcriptional regulation of the ST6GalNAc IV gene in NIH3T3 cells.

Midkine binds specifically to sulfatide the role of sulfatide in cell attachment to midkine-coated surfaces.

Midkine is a heparin-binding polypeptide which is implicated in the control of development and repair of various tissues. Recognition of sulfate groups in glycosaminoglycans is important for its function. To elucidate further its mechanism of action, the interactions of midkine with sulfated glycolipids were studied. Of various glycolipids and lipids examined, midkine bound strongly to sulfatide and cholesterol-3-sulfate (CHO-3-SO4) in a dose-dependent manner but failed to bind to other standard glycolipids and lipids. The properties of midkine binding to sulfatide and to CHO-3-SO4 differed in their sensitivity to inhibition by anionic polysaccharides, salt concentration and unlabeled midkine. Heparin inhibited midkine binding to sulfatide but weakly inhibited its binding to CHO-3-SO4. Liposomes bearing sulfatide carried out significant interactions with immobilized midkine, whereas those bearing CHO-3-SO4 did not. Incorporation of sulfatide into 32D cells and trypsinized COS cells enhanced 125I-labelled midkine binding, whereas incorporation of ganglioside or galactosylceramide had no effect. Furthermore, sulfatide-incorporated cells enhanced cell attachment to midkine-coated coverslips. These results indicate that midkine binds to sulfatide under physiological conditions and the midkine-sulfatide interaction may be important in controlling cell attachment.

Phylogenetic analysis of archaeal PCNA homologues.

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27kDa) and 248 aa (27kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages.

Partial purification and characterization of thermostable acid phosphatase from thermoacidophilic archaeon Sulfolobus acidocaldarius.

Thermostable acid phosphatase (APase) from thermoacidophilic archaeon Sulfolobus acidocaldarius was isolated, partially purified, and characterized. The optimum pH and temperature of the enzyme for p-nitrophenylphosphate (pNPP) as a substrate were 5.0 and 70 degrees C, respectively. The apparent K(m) value was 1.9 mM. This APase showed a native molecular mass of 20 kDa on a gel filtration chromatography. Of the APase activity, 60% remained after 60 min of heat treatment at 75 degrees C. To confirm whether the APase is active in the monomeric form, we attempted to elute the enzyme from SDS-polyacrylamide gels with Disk electrophoresis apparatus and renature the enzyme. The APase activity was recovered up to 50% in the 14- to 35-kDa range, and maximum around 25 kDa. These results suggest that this APase is monomeric protein.