bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneous expression of glucokinase among pancreatic beta cells.
Authors: Jetton T L , Magnuson M A
Published: May 2006

The cellular location of glucokinase (GK), a key component of the glucose-sensing mechanism of the pancreatic islet, was determined using immunocytochemical techniques. In rat islets, GK immunoreactivity was detected only in beta cells with no immunoreactivity detected in alpha, delta, or pancreatic polypeptide-containing (PP) cells. However, within various beta cells, GK immunoreactivity varied considerably. Most beta cells displayed relatively low levels of cytoplasmic immunoreactivity whereas other beta cells stained intensely for this enzyme. Colocalization studies of GK and GLUT2, the high Km glucose transporter of beta cells, confirmed that these proteins are located in different subcellular domains of beta cells. The lack of GK immunoreactivity in glucagon- and somatostatin-secreting cells in islets suggests that these cells are not directly responsive to glucose or utilize a fundamentally different mechanism for sensing glucose fluctuations. Moreover, the differential expression of GK among pancreatic beta cells suggests that glucose phosphorylation is the probable enzymatic control point for the functional diversity of these cells.
RAR-beta 4, a retinoic acid receptor isoform is generated from RAR-beta 2 by alternative splicing and usage of a CUG initiator codon.
Authors: Nagpal S , Zelent A , Chambon P
Published: May 2006

An isoform of retinoic acid receptor beta, RAR-beta 4, has been identified. RAR-beta 4 is expressed under the control of the same retinoic acid-responsive promoter as RAR-beta 2. RAR-beta 4, which is generated by alternative splicing from the same primary transcripts as RAR-beta 2, is initiated by a non-AUG codon, CUG. The amino acid sequence of RAR-beta 4 in regions B-F is identical to that of the other RAR-beta isoforms beta 1, beta 2, and beta 3. However, the RAR-beta 4 A region is much shorter (4 amino acids long) than those of these isoforms. RAR-beta 4 exhibits a tissue-specific pattern of expression and distinct transcriptional activation properties when compared with the other RAR-beta isoforms.
Isolation of a candidate human hematopoietic stem-cell population.
Authors: Baum C M , Weissman I L , Tsukamoto A S , Buckle A M , Peault B
Published: May 2006

We have identified a rare (0.05-0.1%) subset of human fetal bone marrow cells that contains multipotent hematopoietic precursors. The population of human precursor cells that express Thy-1 and CD34 but no known lineage markers is enriched for clonogenic activity that establishes long-term, multilineage (myelomonocytic and B lymphoid) cultures on mouse marrow stromal lines. Further, the Thy-1+CD34+ subset that takes up little of the fluorescent mitochondrial dye rhodamine 123 contains virtually all the cells that establish long-term cultures. In human fetal thymus transplanted into SCID (severe combined immunodeficiency) mice, Thy-1+CD34+ fetal bone marrow cells differentiate into T lymphocytes. In two of nine cases, allogeneic Thy-1+CD34+ cells could engraft intact human fetal bone marrow grown in SCID mice, resulting in donor-derived myeloid and B cells. By extrapolation, the rare human Thy-1+Lin-CD34+ cell population contains pluripotent hematopoietic progenitors; we propose that it is highly enriched for candidate hematopoietic stem cells.
Increased apolipoprotein E and c-fms gene expression without elevated interleukin 1 or 6 mRNA levels indicates selective activation of macrophage functions in advanced human atheroma.
Authors: Salomon R N , Underwood R , Doyle M V , Wang A , Libby P
Published: May 2006

Cells found within atherosclerotic lesions can produce in culture protein mediators that may participate in atherogenesis. To test whether human atheromata actually contain transcripts for certain of these genes, we compared levels of mRNAs in carotid or coronary atheromata and in nonatherosclerotic human vessels by polymerase chain reaction (PCR) amplification of cDNAs reverse-transcribed from RNA. We measured PCR products (generated during exponential amplification) by incorporation of 32P-labeled primers. Levels of interleukin 1 alpha, 1 beta, or 6 mRNAs in plaques and controls did not differ. Compared to uninvolved vessels, plaques did contain higher levels of mRNA encoding platelet-derived growth factor A chain (42 +/- 24 vs. 12 +/- 10 fmol of product; mean +/- SD; n = 8 and 8, respectively; P = 0.007) and B chain (41 +/- 36 vs. 4 +/- 3 fmol of product, n = 14 and 6, respectively; P = 0.024). Atherosclerotic lesions consistently had much higher levels of apolipoprotein E (apoE) mRNA than did control vessels (131 +/- 71 vs. 5 +/- 3 fmol of product; n = 12 and 10, respectively; P less than 0.001). Direct RNA blot analyses confirmed elevated levels of apoE mRNA in plaque extracts. To test whether mononuclear phagocytes might be a source of the apoE mRNA, we studied a selective marker for cells of the monocytic lineage, the c-fms protooncogene, which encodes the receptor for macrophage colony-stimulating factor. Plaques also contained elevated levels of c-fms mRNA (30 +/- 17 vs. 5 +/- 3 fmol of product; n = 10 and 7, respectively; P = 0.002). Immunohistochemical colocalization demonstrated apoE protein in association with macrophages in plaques, whereas nonatherosclerotic vessels showed no immunoreactive apoE. ApoE produced locally in atheroma might modulate the functions of lesional T cells or promote "reverse cholesterol transport" by associating with high density lipoprotein particles, thus targeting them for peripheral uptake. Macrophages within the advanced human atheroma appear to exhibit a selective program of activation as they express high levels of apoE, whereas overall levels of interleukin 1 or 6 mRNAs in plaques are not elevated.
Structure-function relationship among T-cell receptors specific for lysozyme peptides bound to Ab or Abm-12 molecules.
Authors: Kobori J A , Hood L , Shastri N
Published: May 2006

The alpha beta T-cell receptor (TCR) recognizes antigenic peptides bound to major histocompatibility complex (MHC) molecules. In contrast to the antibody combining site, for which the antigen contact or complementarity-determining residues (CDRs) have been precisely defined, the location and function of the corresponding CDR regions of the alpha and beta TCR chains are not known. To develop a model system for systematic analysis of the CDRs of the alpha beta TCR, we isolated a panel of murine T-cell clones that recognize a lysozyme peptide containing residues 74-88 bound to either Ab or Abm-12 MHC class II molecules. Although these two MHC molecules differ by only three amino acid residues within the A beta chain, each of the T-cell clones was specific for peptide bound to the self-MHC molecule and did not recognize the same peptide bound to the other MHC molecule. The structural basis for this exquisite ligand specificity of the TCRs was analyzed by isolation and characterization of alpha and beta chain genes from five closely related T-cell clones. Comparison of predicted amino acid sequences mapped the ligand specificity differences to residues present within the alpha chain variable region segment and the alpha and beta chain variable-joining region junction regions. Thus with current models of TCR-ligand interactions, the results suggest that residues 26-30 of the alpha chain variable region may constitute one of the CDR regions of the TCR.
Molecular associations between the T-lymphocyte antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5.
Authors: Beyers A D , Spruyt L L , Williams A F
Published: May 2006

The T-cell antigen receptor (TCR) complex is the key structure involved in signal transduction in T cells. To analyze associations between the TCR complex and other molecules, immunoprecipitations were carried out, followed by phosphorylation of molecules in vitro by tyrosine kinases associated with the precipitated molecules. This provided a sensitive assay for molecular complexes, and associations were demonstrated between the TCR complex and the surface antigens CD2, CD4, or CD8 and CD5 in normal rat T cells. The complexes were readily seen in immunoprecipitates from Brij 96 but not Nonidet P-40 detergent extracts. The multimolecular complexes are associated with the internal tyrosine kinases p56lck and p59fyn. The presence of p56lck associated with CD4 or CD8 was also examined in early thymocytes, natural killer cells, and macrophages. The kinase was present in all cases except that of normal macrophages.
Interaction of the pertussis toxin peptide containing residues 30-42 with DR1 and the T-cell receptors of 12 human T-cell clones.
Authors: De Magistris M T , Di Tommaso A , Domenighini M , Censini S , Tagliabue A , Oksenberg J R , Steinman L , Judd A K , O'Sullivan D , Rappuoli R
Published: May 2006

The interaction of the immunodominant pertussis toxin peptide containing residues 30-42 (p30-42) with soluble DR1 molecules and the T-cell receptor (TCR) of 12 DR1-restricted human T-cell clones has been analyzed. Peptide analogues of p30-42 containing single alanine substitutions were used in DR1-binding and T-cell proliferation assays to identify the major histocompatibility complex and TCR contact residues. Each T-cell clone was found to recognize p30-42 with a different fine specificity. However, a common core comprising amino acids 33-39 was found to be important for stimulation of all T-cell clones. Within this core two residues, Leu33 and Leu36, interact with the DR1 molecule, whereas Asp34, His35, Thr37, and Arg39 are important for TCR recognition in most of the clones. Computer modeling of the structure of p30-42 showed that an alpha-helical conformation is compatible with the experimental data. The analysis of TCR rearrangement revealed that the peptide was recognized by T-cell clones expressing different variable region alpha (V alpha) and variable region beta (V beta) chains, although a preferential use of V alpha 8-V beta 13 and V alpha 11-V beta 18 combinations was found in clones from the same donor. Understanding the details of the interaction of antigenic peptides with the major histocompatibility complex and TCR molecules should provide the theoretical basis to design T-cell epitopes and obtain more immunogenic vaccines.
DNA damage and mutation in human cells exposed to nitric oxide in vitro.
Authors: Nguyen T , Brunson D , Crespi C L , Penman B W , Wishnok J S , Tannenbaum S R
Published: May 2006

Nitric oxide (NO.) is a physiological messenger formed by several cell types. Reaction with O2 forms oxides that nitrosate amines at pH values near 7. We now report experiments in which NO. was added to intact human cells and to aerobic solutions of DNA, RNA, guanine, or adenine. TK6 human lymphoblastoid cells were mutated 15- to 18-fold above background levels at both the HPRT and TK gene loci. Xanthine and hypoxanthine, from deamination of guanine and adenine, respectively, were formed in all cases. NO. induced dose-responsive DNA strand breakage. Yields of xanthine ranged from nearly equal to about 80-fold higher than those of hypoxanthine. Yields of xanthine and hypoxanthine from nucleic acids were higher than those from free guanine and adenine. This was most pronounced for xanthine; 0.3 nmol/mg was formed from free guanine vs. 550 nmol/mg from calf thymus RNA. Nitric oxide added to TK6 cells produced a 40- to 50-fold increase in hypoxanthine and xanthine in cellular DNA. We believe that these results, plus the expected deaminations of cytosine to uracil and 5-methylcytosine to thymine, account for the mutagenicity of nitric oxide toward bacteria and mammalian cells.
Isolation and sequence analysis of the cDNA for pig kidney fructose 1,6-bisphosphatase.
Authors: Williams M K , Kantrowitz E R
Published: May 2006

A full-length clone of pig kidney fructose 1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) was isolated by screening a cDNA library for complementation of an Escherichia coli fbp deletion mutation. The open reading frame of 1011 bases corresponds to 337 amino acids, two more than have been previously reported [Marcus, F., Edelstein, I., Reardon, I. & Heinrikson, R. L. (1982) Proc. Natl. Acad. Sci. USA 79, 7161-7165]. The extra two amino acids (Ala-Lys) are located at the C-terminal end of the protein as an extension. Comparison of the deduced amino acid sequence with the reported (see above) and revised amino acid sequence [Harrsch, P. B., Kim, Y., Fox, J. L. & Marcus, F. (1985) Biochem. Biophys. Res. Commun. 133, 520-526] indicates three differences in addition to the C-terminal extension. Gln-20, Thr-96, and Asn-199 in the amino acid sequence are found to be Glu, Ser, and Asp, respectively. Since the x-ray structure of the pig kidney enzyme has been reported, the cDNA clone will allow the construction of site-specific mutants to help test possible structure-function relationships in this important metabolic enzyme.
A growth-regulated protease activity that is inhibited by the anticarcinogenic Bowman-Birk protease inhibitor.
Authors: Billings P C , Habres J M
Published: May 2006

The Bowman-Birk protease inhibitor (BBI) has been shown to be an effective suppressor of carcinogenesis in vivo and in vitro. To elucidate the mechanism(s) by which BBI suppresses carcinogenesis, we believe it will be necessary to identify and characterize the target enzymes that specifically interact with the BBI. We have shown previously that several cellular proteins in C3H/10T1/2 mouse embryo fibroblast cells specifically bind to a BBI affinity resin. In the current report, we demonstrate that one of these proteins has proteolytic activity as judged by its ability to degrade gelatin. The enzyme has a mass of 45 kDa and subcellular fractionation experiments demonstrate that this enzyme is located in the cytosol. Furthermore, the proteolytic activity was inhibited by diisopropylfluorophosphate but was not affected by EDTA, indicating that this enzyme is a serine protease. Higher levels of protease activity were found in logarithmic-phase C3H/10T1/2 cells compared with nondividing (confluent) cells, suggesting that this protease activity is growth regulated. Similar levels of this activity were present in nontransformed and in radiation-transformed C3H/10T1/2 cells. Treatment of nontransformed C3H/10T1/2 cells with phorbol 12-myristate 13-acetate increased the specific activity of this protease 5- to 10-fold. Our results suggest that this protease is a target enzyme of the BBI in these cells.
Developmental and environmental regulation of a mammary gland-specific nuclear factor essential for transcription of the gene encoding beta-casein.
Authors: Schmitt-Ney M , Happ B , Ball R K , Groner B
Published: May 2006

During the lactation period, mammary epithelial cells secrete large amounts of milk proteins. The coordinate regulation of milk protein expression is effected by the lactogenic hormones. We have investigated the activity of a mammary gland-specific transcription factor (MGF), which mediates hormonal influences at the level of a milk protein gene promoter. MGF-binding sites are present in the promoters of the most abundantly expressed milk protein genes. Mutation of the MGF-binding site in the beta-casein gene promoter completely abolishes responsiveness of the promoter to lactogenic hormones in cultured mammary epithelial cells. MGF activity is closely controlled in vivo. High MGF levels were found in mouse mammary gland nuclear extracts toward the end of pregnancy and during lactation. Withdrawal of suckling pups from their mothers during the lactation period caused a strong and rapid decrease of MGF activity. Readdition of pups to their mothers restored maximal MGF levels within 4 hr. We investigated MGF phosphorylation as a possible posttranslational modification responsible for regulation of the DNA-binding activity of MGF. Treatment of nuclear extracts from lactating mammary glands with potato acid phosphatase abolished MGF-binding activity. Casein kinase II phosphorylation of nuclear extracts from animals withdrawn from their pups for 24 hr enhanced MGF-binding activity. These results suggest that the reversible activation of MGF by suckling and withdrawal might be mediated by the action of kinases and phosphatases.
Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.
Authors: Suchi M , Dinur T , Desnick R J , Gatt S , Pereira L , Gilboa E , Schuchman E H
Published: May 2006

Types A and B Niemann-Pick disease (NPD) result from inherited deficiencies of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12). To evaluate the feasibility of somatic gene therapy for the treatment of these disorders, retroviral-mediated gene transfer was used to introduce the full-length ASM cDNA into cultured fibroblasts from two unrelated type A NPD patients. The ASM activities in these cells were less than 4% of mean normal levels, and, consequently, they accumulated approximately 3-fold elevated levels of sphingomyelin. After retroviral-mediated transfer of the ASM cDNA, ASM activities in the NPD cells increased to levels up to 16-fold those found in normal fibroblasts. In addition, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies also were undertaken to evaluate the effects of retroviral-mediated gene transfer on the pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a type A NPD patient by using apolipoprotein E-mediated endocytosis, only approximately 6% of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced (i.e., "corrected") by retroviral-mediated gene transfer could degrade approximately 80% of the delivered sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the pathology of NPD cells. Cell-loading studies were also used to develop a selection system for discriminating between NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene-labeled sphingomyelin, was 3- to 5-fold that of normal or transduced cells. As a consequence, the NPD and transduced cells could be efficiently sorted by flow cytometry with a fluorescence-activated cell sorter. In addition, the NPD cells could be selectively killed by photosensitization after irradiation with a long-wavelength UV light. These results should permit direct selection of ASM-expressing cells after retroviral-mediated gene transfer without the need to preselect for a cotransferred marker gene.
Characterization of the murine BEK fibroblast growth factor (FGF) receptor: activation by three members of the FGF family and requirement for heparin.
Authors: Mansukhani A , Dell'Era P , Moscatelli D , Kornbluth S , Hanafusa H , Basilico C
Published: May 2006

The bek gene encodes a member of the high-affinity fibroblast growth factor receptor family. The BEK/FGFR-2 receptor is a membrane-spanning tyrosine kinase with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 32D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not bind keratinocyte growth factor or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike the BEK-expressing Chinese hamster ovary cells, 32D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands.
The gene for a tRNA modifying enzyme, m5U54-methyltransferase, is essential for viability in Escherichia coli.
Authors: Persson B C , Gustafsson C , Berg D E , Björk G R
Published: May 2006

One of the most abundant modified nucleosides in tRNA is 5-methyluridine (m5U or rT, ribothymidine). The enzyme tRNA(m5U54)methyltransferase [S-adenosyl-L-methionine:tRNA (uracil-5-)-methyltransferase, EC 2.1.1.35] (the trmA gene product) catalyzes S-adenosylmethionine-dependent methylation of the uracil in position 54 (T psi C loop) in all Escherichia coli tRNAs to form m5U. Hitherto no modified nucleoside in tRNA has been shown to be essential for growth, although their importance in fine tuning the function of tRNA is well established. In this paper, we show that the structural gene trmA is essential for viability, although the known catalytic activity of the tRNA(m5U54)methyltransferase is not.
Treatment with 2,4-dinitrophenol mimics ontogenetic and phylogenetic changes in a hydractiniid hydroid.
Authors: Blackstone N W , Buss L W
Published: May 2006

Clonal plants, fungi, and invertebrate animals often display both intra- and interspecific heterochronic variation in the relative proportions of ramets (e.g., polyps, plantlets) and stolons (or rhizomes, hyphae). Treatment of developing colonies of Podocoryne carnea, a hydractiniid hydroid, with dilute solutions of 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, accelerates the usual ontogenetic trajectory of polyp and stolon production. This morphological heterochrony can be related to the hydrodynamic behavior of the gastrovascular system under the conditions of "loose-coupling" of oxidative phosphorylation produced by DNP. In its normal ontogeny, Hydractinia symbiolongicarpus, a closely related hydractiniid hydroid, not only shows morphological heterochrony similar to that induced in P. carnea by DNP, but also shows a pattern of gastrovascular flow similar to that observed in P. carnea under treatment with DNP. These results support hypotheses linking metabolic activity to heterochronic variation and provide a potentially widely applicable experimental basis for the study of such variation.
Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults.
Authors: Broxmeyer H E , Hangoc G , Cooper S , Ribeiro R C , Graves V , Yoder M , Wagner J , Vadhan-Raj S , Benninger L , Rubinstein P
Published: May 2006

We estimated whether single collections of cord blood contained sufficient cells for hematopoietic engraftment of adults by evaluating numbers of cord blood and adult bone marrow myeloid progenitor cells (MPCs) as detected in vitro with steel factor (SLF) and hematopoietic colony-stimulating factors (CSFs). SLF plus granulocyte-macrophage (GM)-CSF detected 8- to 11-fold more cord blood GM progenitors [colony-forming units (CFU)-GM] than cells stimulated with GM-CSF or 5637 conditioned medium (CM), growth factors previously used to estimate cord blood CFU-GM numbers. SLF plus erythropoietin (Epo) plus interleukin 3 (IL-3) enhanced detection of cord blood multipotential (CFU-GEMM) progenitors 15-fold compared to stimulation with Epo plus IL-3. Under the same conditions, bone marrow CFU-GM and CFU-GEMM were only enhanced in detection 2- to 4- and 6- to 8-fold. Increased detection of cord blood CFU-GEMM correlated directly with decreased detection of cord blood erythroid burst-forming units (BFU-E). In contrast, adult bone marrow CFU-GEMM and BFU-E numbers were both enhanced by SLF plus Epo plus IL-3. This suggests that most cord blood BFU-E may actually be CFU-GEMM. Cord blood collections (n = 17) contained numbers of MPCs (especially CFU-GM) similar to the number found in nine autologous bone marrow collections. To assess additional sources of MPCs, the peripheral blood of 1-day-old infants was assessed. However, average concentrations of MPCs circulating in these infants were only 30-46% that in their cord blood. Expansion of cord blood MPCs was also evaluated. Incubation of cord blood cells for 7 days with SLF resulted in 7.9-, 2.2-, and 2.7-fold increases in numbers of CFU-GM, BFU-E, and CFU-GEMM compared to starting numbers; addition of a CSF with SLF resulted in even greater expansion of MPCs. The results suggest that cord blood contains a larger number of early profile MPCs than previously recognized and that there are probably sufficient numbers of cells in a single cord blood collection to engraft an adult. Although the expansion data must be considered with caution, as human marrow repopulating cells cannot be assessed directly, in vitro expansion of cord blood stem and progenitor cells may be feasible for clinical transplantation.
Determination of transmembrane protein structure by disulfide cross-linking: the Escherichia coli Tar receptor.
Authors: Pakula A A , Simon M I
Published: May 2006

We have devised a generally applicable strategy for analysis of protein structure and have applied it to examine the structure of the transmembrane portion of the Tar receptor of Escherichia coli. The basis of our approach is the use of disulfide cross-linking to identify residues that are within close proximity. To generate and test large numbers of cysteine pairs, we used an unusual method of mutagenesis by which cysteine substitutions can be created randomly at a number of targeted codons. Cysteine-substituted proteins encoded by mutagenized genes may be screened directly for disulfide formation within oligomers or, alternatively, different pools of genes may be randomly recombined to generate gene populations with substitutions in multiple regions. Thus, it is possible to detect a variety of disulfide cross-links between and within individual protein molecules. Interactions between the four membrane-spanning stretches of the Tar dimer were probed by measuring the tendency of 48 cysteine substitutions throughout this region to form disulfide cross-links with one another. We have interpreted these data to suggest a helical-bundle structure for the transmembrane region. The four helices of this bundle are not structurally equivalent: the two TM1 helices interact closely, whereas the TM2 helices are more peripherally located.
Splanchnic metabolism of dietary arginine in relation to nitric oxide synthesis in normal adult man.
Authors: Castillo L , deRojas T C , Chapman T E , Vogt J , Burke J F , Tannenbaum S R , Young V R
Published: May 2006

Urinary nitrate (NO3) is the stable end product of nitric oxide, which is formed, in turn, from a guanidino nitrogen of arginine. We have conducted two experiments, each in four healthy adult men receiving a low nitrate diet for 7-10 days, to investigate the in vivo conversion of arginine to nitrate. In the first study [guanidino-15N2, 5,5-2H2]arginine was given on day 7 via a primed continuous intravenous infusion for 8 h. In the second study, the labeled arginine was given for 8 h by the intragastric route on day 7 and by the intravenous route on day 10. Measurement of 15NO3 output in urine collected for 24 h beginning at the time of the arginine tracer infusion revealed a more extensive transfer of 15N when the arginine tracer was given intragastricly. From the comparative labeling of 15NO3 after administration of the tracer arginine via the intragastric and intravenous routes, we estimate that 16% +/- 2% of the daily production of nitrate arises from the metabolism of dietary arginine that is taken up during its "first pass" in the splanchnic region. Hence, nitric oxide production occurs, to a measurable extent, in this area in healthy subjects, raising the question as to how various pathophysiological states might alter the relations between exogenous and endogenous sources of arginine as precursors of NO. and the relative contributions made by various organs to whole body (NO.) NO3 formation. These results also raise important questions about the use of nitric oxide synthase inhibitors in animal and human studies.
An apolipoprotein CIII haplotype protective against hypertriglyceridemia is specified by promoter and 3' untranslated region polymorphisms.
Authors: Dammerman M , Sandkuijl L A , Halaas J L , Chung W , Breslow J L
Published: May 2006

Five DNA polymorphisms were detected in the promoter of the apolipoprotein CIII gene of a type III hyperlipidemic subject with severe hypertriglyceridemia (HTG). The polymorphic sites were C-641-->A, G-630--> A, T-625-->deletion, C-482-->T, and T-455-->C, with the previously reported base at each site designated allele 1 and the variant base designated allele 2. The sites were in strong linkage disequilibrium with each other and with a polymorphic Sst I site in the apolipoprotein CIII 3' untranslated region whose presence (S2 allele) has previously been shown to be associated with HTG. The distribution of haplotypes of the form -625 -482 Sst I among 78 normolipidemic adults and 79 adults with severe HTG was estimated by maximum likelihood analysis. The 211 haplotype was estimated to be 3.8-fold more common in normal subjects than in HTG subjects (estimated proportions, 0.186 and 0.049, respectively). This haplotype was associated with reduced HTG risk (relative risk, 0.28; P = 0.005) when compared with other haplotypes lacking the Sst I site (S1 allele). The 222 haplotype was estimated to be present on 48 of the 54 S2-containing chromosomes observed and was associated with increased risk for HTG (relative risk, 3.14; P < 0.0025). These results support the existence of apolipoprotein CIII promoter/Sst I haplotypes conferring either protection against or susceptibility to severe HTG.
Molecular cloning of olfactomedin, an extracellular matrix protein specific to olfactory neuroepithelium.
Authors: Yokoe H , Anholt R R
Published: May 2006

The extracellular mucous matrix of olfactory neuroepithelium is a highly organized structure in intimate contact with chemosensory cilia that house the olfactory transduction machinery. Here we describe the molecular cloning and primary structure of olfactomedin, which is the major component of this extracellular matrix. Olfactomedin is expressed exclusively in olfactory neuroepithelium and its amino acid sequence shows no homologies to any known protein. This olfactory tissue-specific glycoprotein contains cysteines which form disulfide-linked polymers that constitute the primary architecture of the olfactory extracellular matrix. By analogy to other extracellular matrix proteins of the nervous system, olfactomedin may influence the maintenance, growth, or differentiation of chemosensory cilia on the apical dendrites of olfactory neurons.
Molecular basis of a multiple lymphokine deficiency in a patient with severe combined immunodeficiency.
Authors: Castigli E , Pahwa R , Good R A , Geha R S , Chatila T A
Published: May 2006

We have previously reported that the T lymphocytes of a child with severe combined immunodeficiency are defective in the transcription of several lymphokine genes that include IL2, IL3, IL4, and IL5, which encode interleukins 2, 3, 4, and 5 (IL-2, -3, -4, and -5). To determine whether the defect in the patient's T lymphocytes involved a trans-acting factor common to the affected lymphokine genes, we examined the ability of nuclear factors from the patient's T lymphocytes to bind response elements present in the regulatory region of IL2. Nuclear factor NF-kB, activation protein 1 (AP-1), OCT-1, and NF-IL-2B binding activity were normal. In contrast, the binding of the nuclear factor of activated T cells (NF-AT) to its response element in the IL2 enhancer and to an NF-AT-like response element present in the IL4 enhancer was abnormal. To ascertain whether the abnormal NF-AT binding activity was related to an impaired function, we transfected patient and control T lymphocytes with constructs containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) under the control of the entire IL2 regulatory region or of multimers of individual enhancer sequences. CAT expression directed by the IL2 regulatory region or by a multimer of the NF-AT-binding site was markedly lower in the patient relative to controls. In contrast, CAT gene expression directed by a multimer of the OCT-1 proximal (OCT-1p)-binding site was equivalent in patient and controls. These results indicate that an abnormality of/or influencing NF-AT may underlie the multiple lymphokine deficiency in this patient.
Structure and expression of the human trithorax-like gene 1 involved in acute leukemias.
Authors: Parry P , Djabali M , Bower M , Khristich J , Waterman M , Gibbons B , Young B D , Evans G
Published: May 2006

The human trithorax-like gene 1 (Htrx1 gene) is disrupted in 11q23 translocations that are associated with acute leukemias. Sequencing of a partial human cDNA revealed an open reading frame encoding 1012 amino acids with extensive homology to the Drosophila trithorax protein, particularly in the zinc finger-like domains. Htrx1 gene appears to be unique in the human genome and has been conserved during evolution. Use of the human cDNA as a probe demonstrates that this gene is interrupted in both infant and adult acute myeloid (AML) and lymphoid (ALL) leukemia patients with 11q23 translocations. The structure of the Htrx1 gene around the breakpoints shows that this part of the human gene is interrupted by nine introns. As a result of the rearrangement, zinc finger domains are translocated in both ALL and AML patients. Expression studies reveal that the Htrx1 gene differentially expresses three transcripts within the normal lymphocyte cell lineage.
Unusual organization of the human T-cell receptor beta-chain gene complex is linked to recombination hotspots.
Authors: Seboun E , Houghton L , Hatem C J , Lincoln R , Hauser S L
Published: May 2006

Two rare Sfi I polymorphisms of 360 kb and 280 kb present within the human T-cell antigen receptor beta-chain gene complex were revealed by pulsed-field gel electrophoresis. They represent allelic variants of the polymorphic 330- and 300-kb Sfi I fragments previously described. The 360-kb polymorphism results from duplication of the 30-kb DNA fragment responsible for the 330/300-kb insertion/deletion-related polymorphism. The 280-kb polymorphism results from a 20-kb deletion from the 300-kb SfiI allele. The rare polymorphisms also map on either side of a Sal I site located near a recombination hotspot, suggesting that germline duplications and deletions arose from nonhomologous crossover events.
Molecular cloning, functional expression, and pharmacological characterization of an N-methyl-D-aspartate receptor subunit from human brain.
Authors: Planells-Cases R , Sun W , Ferrer-Montiel A V , Montal M
Published: May 2006

A cDNA encoding a full-length N-methyl-D-aspartate (NMDA) receptor subunit 1, hNR1, was isolated from a human brain cDNA library. The hNR1 cDNA encodes an open reading frame of approximately 2.7 kb that shares high homology with the rat brain NMDA receptor subunit 1 and the mouse zeta 1 subunit. The hNR1 sequence, however, diverges from the rodent and murine homologs near the C terminus, suggesting that they represent alternatively spliced messages of the same gene. Oocytes injected with cRNA synthesized from the hNR1 cDNA express glutamate and NMDA-activated currents in the presence of glycine. Currents are blocked by the NMDA-receptor-specific antagonists 2-amino-5-phosphovaleric acid and 7-chlorokynurenate, and the open channel blockers MK-801 and phencyclidine, by Mg2+ ions in a voltage-dependent manner, and by Zn2+. Expressed hNR1 homomeric receptor channels exhibit the high Ca2+ permeability characteristic of neuronal NMDA receptors. Therefore, the cDNA clone hNR1 codes for a human brain NMDA receptor subunit cognate to the rodent and murine brain NR1 subunits.
RNS2: a senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation.
Authors: Taylor C B , Bariola P A , delCardayré S B , Raines R T , Green P J
Published: May 2006

Several self-compatible species of higher plants, such as Arabidopsis thaliana, have recently been found to contain S-like RNases. These S-like RNases are homologous to the S-RNases that have been hypothesized to control self-incompatibility in Solanaceous species. However, the relationship of the S-like RNases to the S-RNases is unknown, and their roles in self-compatible plants are not understood. To address these questions, we have investigated the RNS2 gene, which encodes an S-like RNase (RNS2) of Arabidopsis. Amino acid sequence comparisons indicate that RNS2 and other S-like RNases make up a subclass within an RNase superfamily, which is distinct from the subclass formed by the S-RNases. RNS2 is most similar to RNase LE [Jost, W., Bak, H., Glund, K., Terpstra, P., Beintema, J. J. (1991) Eur. J. Biochem. 198, 1-6.], an S-like RNase from Lycopersicon esculentum, a Solanaceous species. The fact that RNase LE is more similar to RNS2 than to the S-RNases from other Solanaceous plants indicates that the S-like RNases diverged from the S-RNases prior to speciation. Like the S-RNase genes, RNS2 is most highly expressed in flowers, but unlike the S-RNase genes, RNS2 is also expressed in roots, stems, and leaves of Arabidopsis. Moreover, the expression of RNS2 is increased in both leaves and petals of Arabidopsis during senescence. Phosphate starvation can also induce the expression of RNS2. On the basis of these observations, we suggest that one role of RNS2 in Arabidopsis may be to remobilize phosphate, particularly when cells senesce or when phosphate becomes limiting.
Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.
Authors: Pfeifer A M , Cole K E , Smoot D T , Weston A , Groopman J D , Shields P G , Vignaud J M , Juillerat M , Lipsky M M , Trump B F
Published: May 2006

Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma.
A gamma-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvesicles of pancreatic beta cells.
Authors: Thomas-Reetz A , Hell J W , During M J , Walch-Solimena C , Jahn R , De Camilli P
Published: May 2006

A variety of peptide-secreting endocrine cells contain a population of recycling microvesicles that share several major membrane polypeptides with neuronal synaptic vesicles (SVs). The function of these synaptic-like microvesicles (SLMVs) remains to be elucidated. It was previously suggested that SLMVs of pancreatic beta cells may store and secrete gamma-aminobutyric acid (GABA). GABA, the major nonpeptide inhibitory neurotransmitter of the central nervous system, is stored in and secreted from SVs. GABA uptake into SVs is mediated by a transporter that is driven by a vacuolar proton ATPase. GABA is also present at high concentration in the endocrine pancreas where it is selectively localized in insulin-secreting beta cells, the core cells of pancreatic islets. GABA is not present in peripheral islet cells (mantle cells), represented primarily by glucagon-secreting alpha cells. In this study, an immunoisolation procedure was used to purify SLMVs from cell lines derived from mouse beta cells and alpha cells. SLMVs obtained from the beta-cell line, but not those obtained from the alpha-cell line, displayed a GABA-transport activity dependent upon a proton electrochemical gradient generated by a vacuolar proton ATPase. These data support the hypotheses that (i) SLMVs have a secretory function similar to that of SVs and (ii) beta-cell SLMVs are involved in the secretion of GABA, which in turn may have a paracrine function on mantle cells of the islet.
Growth inhibition by transforming growth factor beta (TGF-beta) type I is restored in TGF-beta-resistant hepatoma cells after expression of TGF-beta receptor type II cDNA.
Authors: Inagaki M , Moustakas A , Lin H Y , Lodish H F , Carr B I
Published: May 2006

The growth of human hepatoma Hep 3B cells is potently inhibited by TGF-beta 1 (ID50 = 0.2 ng/ml, 8 pM). A mutant cell line was derived that was not inhibited in growth by TGF-beta 1 at 5 ng/ml (200 pM) and that lacked TGF-beta receptor type II (TGF-beta RII) gene. Transfection of the cloned cDNA for human TGF-beta RII to this mutant cell line restored receptor expression as well as the inhibition in growth by TGF-beta 1. In both wild-type and mutant cells stably transfected with TGF-beta RII cDNA, TGF-beta RII coimmunoprecipitated with TGF-beta receptor type I in the presence of ligand. These experiments provide direct evidence for the role of TGF-beta RII in the inhibitory effect of TGF-beta on growth and suggest that TGF-beta RII acts by means of a heteromeric surface complex with TGF-beta receptor type I.
Functional interchangeability of the structurally similar tetranucleotide loops GAAA and UUCG in fission yeast signal recognition particle RNA.
Authors: Selinger D , Liao X , Wise J A
Published: May 2006

Signal recognition particle (SRP) RNA exhibits significant primary sequence conservation only in domain IV, a bulged hairpin capped by a GNRA (N, any nucleotide; R, purine) tetranucleotide loop except in plant homologs. Tetraloops conforming to this sequence or to the consensus UNCG enhance the stability of synthetic RNA hairpins and have strikingly similar three-dimensional structures. To determine the biological relevance of this similarity, as well as to assess the relative contributions of sequence and structure to the function of the domain IV tetraloop, we replaced the GAAA sequence in fission yeast SRP RNA with UUCG. Haploid strains harboring this substitution are viable, providing experimental evidence for the functional equivalence of the two tetraloops. We next tested the two sequences found in plant SRP RNAs at this location for function in the context of the Schizosaccharomyces pombe RNA. While substitution of CUUC does not allow growth, a viable strain results from replacing GAAA with UUUC. Although the viable tetraloop substitution mutants exhibit wild-type growth under normal conditions, all three express conditional defects. To determine whether this might be a consequence of structural perturbations, we performed enzymatic probing. The results indicate that RNAs containing tetraloop substitutions exhibit subtle differences from the wild type not only in the tetraloop itself, but also in the 3-base pair adjoining stem. To directly assess the importance of the latter structure, we disrupted it partially or completely and made the compensatory mutations to restore the helix. Surprisingly, mutant RNAs with as little as one Watson-Crick base pair can support growth.
Lithium phthalocyanine: a probe for electron paramagnetic resonance oximetry in viable biological systems.
Authors: Liu K J , Gast P , Moussavi M , Norby S W , Vahidi N , Walczak T , Wu M , Swartz H M
Published: May 2006

Lithium phthalocyanine (LiPc) is a prototype of another generation of synthetic, metallic-organic, paramagnetic crystallites that appear very useful for in vitro and in vivo electron paramagnetic resonance oximetry. The peak-to-peak line width of the electron paramagnetic resonance spectrum of LiPc is a linear function of the partial pressure of oxygen (pO2); this linear relation is independent of the medium surrounding the LiPc. It has an extremely exchange-narrowed spectrum (peak-to-peak line width = 14 mG in the absence of O2). Physicochemically LiPc is very stable; its response to pO2 does not change with conditions and environments (e.g., pH, temperature, redox conditions) likely to occur in viable biological systems. These characteristics provide the sensitivity, accuracy, and range to measure physiologically and pathologically pertinent O2 tensions (0.1-50 mmHg; 1 mmHg = 133 Pa). The application of LiPc in biological systems is demonstrated in measurements of pO2 in vivo in the heart, brain, and kidney of rats.
Interleukin 2 regulates Raf-1 kinase activity through a tyrosine phosphorylation-dependent mechanism in a T-cell line.
Authors: Turner B C , Tonks N K , Rapp U R , Reed J C
Published: May 2006

Previously we found that interleukin 2 (IL-2) induces tyrosine phosphorylation and activation of the serine/threonine-specific kinase encoded by the raf-1 protooncogene in a T-cell line, CTLL-2. Here we extended these findings by exploring the effects of selective removal of phosphate from tyrosines in p72-74-Raf-1 kinase that had been immunoprecipitated from IL-2-stimulated CTLL-2 cells. Treatment in vitro of IL-2-activated Raf-1 with the tyrosine-specific phosphatases CD45 and TCPTP (formerly called T-cell protein tyrosine phosphatase) reduced Raf kinase activity to nearly baseline levels. This effect was completely inhibited by the phosphatase inhibitor sodium orthovanadate. In contrast, treatment of Raf-1 with a serine/threonine-specific phosphatase, protein phosphatase 1 (PP-1), resulted in a more modest decrease in Raf in vitro kinase activity, and this effect was prevented by okadaic acid. Two-dimensional phosphoamino acid analysis confirmed the selective removal of phosphate from tyrosine by CD45 and from serine and threonine by PP-1. The immunoreactivity of p72-74-Raf-1 with anti-phosphotyrosine antibodies was also completely abolished by treatment with CD45 in the absence but not in the presence of sodium orthovanadate. These findings provide evidence that the IL-2-stimulated phosphorylation of Raf-1 on tyrosines plays an important role in upregulating the activity of this serine/threonine-specific kinase in CTLL-2 cells and, as such, provides a model system for studying the transfer of growth factor-initiated signals from protein tyrosine kinases to serine/threonine-specific kinases.