bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of epididymal inflammation in the induction of dominant lethal mutations in Fischer 344 rat sperm by methyl chloride.
Authors: Chellman G J , Bus J S , Working P K
Published: May 2006

This study assessed the possible relationship between methyl chloride (MeCl)-induced epididymal inflammation and the formation of dominant lethal mutations in sperm of Fischer 344 rats. Groups of 40 males were exposed to MeCl (3000 ppm 6 hr/day for 5 days), with or without concurrent treatment with the anti-inflammatory agent 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW 755C; 10 mg/kg, i.p. 1 hr pre- and postexposure); BW 755C was shown previously to inhibit MeCl-induced epididymal inflammation. Control groups (n = 20) were either untreated, injected as described above with BW 755C, or injected on the afternoon of day 5 with triethylenemelamine (0.2 mg/kg), a known dominant lethal mutagen. Each male was caged with one female weekly for 3 weeks; 12-18 days after mating, females were killed to assess dominant lethal parameters. In females bred to MeCl-exposed males, significant increases were observed in postimplantation loss at postexposure week 1 (0.84 dead implants per female vs. 0.29 in untreated controls) and in dead implants/total implants at both week 1 (0.10 vs. 0.04 control) and week 2 (0.24 vs. 0.06 control). These increases were not observed in females bred to males treated with BW 755C during MeCl exposure. Coadministration of BW 755C to males along with MeCl also reduced the percentage of mated females with two or more postimplantation losses from 31% to 8% (week 1) and 30% to 12% (week 2). Therefore, the dominant lethal mutations induced by MeCl appear to be a consequence of its induction of inflammation in the epididymis. These data demonstrate the potential genotoxicity of inflammatory processes in vivo.
Transcription attenuation in Salmonella typhimurium: the significance of rare leucine codons in the leu leader.
Authors: Carter P W , Bartkus J M , Calvo J M
Published: May 2006

The leucine operon of Salmonella typhimurium is controlled by a transcription attenuation mechanism. Four adjacent leucine codons within a 160-nucleotide leu leader RNA are thought to play a central role in this mechanism. Three of the four codons are CUA, a rarely used leucine codon within enteric bacteria. To determine whether the nature of the leucine codon affects the regulation of the leucine operon, we used oligonucleotide-directed mutagenesis to first convert one CUA of the leader to CUG and then convert all three CUA codons to CUG. CUG is the most frequently used leucine codon in enteric bacteria. A mutant having (CUA)2CUGCUC in place of (CUA)3CUC has an altered response to leucine limitation, requiring a slightly higher degree of limitation to effect derepression. Changing (CUA)3CUC to (CUG)3CUC has more dramatic effects upon operon expression. First, the basal level of expression is lowered to the point that the mutant grows more slowly than the parent in a minimal medium lacking leucine. Second, the response of the mutant to a leucine limitation is dramatically altered such that even a strong limitation elicits only a modest degree of derepression. If the mutant is grown under conditions of leucyl-tRNA limitation rather than leucine limitation, complete derepression can be achieved, but only at a much higher degree of limitation than for the wild-type operon. These results provide a clear-cut example of codon usage having a dramatic effect upon gene expression.
Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence.
Authors: Law S W , Grant S M , Higuchi K , Hospattankar A , Lackner K , Lee N , Brewer H B
Published: May 2006

Human apolipoprotein B-100 (apoB-100), the ligand on low density lipoproteins that interacts with the low density lipoprotein receptor and initiates receptor-mediated endocytosis and low density lipoprotein catabolism, has been cloned, and the complete nucleic acid and derived amino acid sequences have been determined. ApoB-100 cDNAs were isolated from normal human liver cDNA libraries utilizing immunoscreening as well as filter hybridization with radiolabeled apoB-100 oligodeoxynucleotides. The apoB-100 mRNA is 14.1 kilobases long encoding a mature apoB-100 protein of 4536 amino acids with a calculated amino acid molecular weight of 512,723. ApoB-100 contains 20 potential glycosylation sites, and 12 of a total of 25 cysteine residues are located in the amino-terminal region of the apolipoprotein providing a potential globular structure of the amino terminus of the protein. ApoB-100 contains relatively few regions of amphipathic helices, but compared to other human apolipoproteins it is enriched in beta-structure. The delineation of the entire human apoB-100 sequence will now permit a detailed analysis of the conformation of the protein, the low density lipoprotein receptor binding domain(s), and the structural relationship between apoB-100 and apoB-48 and will provide the basis for the study of genetic defects in apoB-100 in patients with dyslipoproteinemias.
Contrasting levels of transferrin gene activity in cultured rat Sertoli cells and intact seminiferous tubules.
Authors: Lee N T , Chae C B , Kierszenbaum A L
Published: May 2006

Two-dimensional polyacrylamide gel electrophoresis of proteins and transfer blot hybridization of RNA have been used to study the activity and expression of the rat transferrin gene in cultured Sertoli cells and in whole testis and isolated seminiferous tubules of sexually immature and mature rats. Although the transferrin gene in cultured Sertoli cells is actively engaged in the transcription of mRNA and the mRNA is translated into a secretory product, little transferrin mRNA and transferrin protein are present in whole testes and isolated seminiferous tubules. Sertoli cells upon culturing show a time-dependent transferrin gene activation, and abundant transferrin mRNA can be detected 6 hr after plating. A similar study using intact seminiferous tubular segments from the same rats failed to show a comparable temporal activation of the transferrin gene. Results of this study, together with previous experimental data, suggest that Sertoli cells in vivo are most likely not actively engaged in the synthesis of a testicular transferrin but, instead, rely mainly on plasma transferrin contributed by the liver. In vitro, Sertoli cells, released from the physiological constraints that operate in vivo, rapidly activate the transferrin gene, resulting in abundant newly synthesized Sertoli cell transferrin product.
Interferons and tumor necrosis factors have similar catabolic effects on 3T3 L1 cells.
Authors: Patton J S , Shepard H M , Wilking H , Lewis G , Aggarwal B B , Eessalu T E , Gavin L A , Grunfeld C
Published: May 2006

The effect of a variety of cytokines on lipid metabolism in 3T3 L1 mouse fibroblasts and adipocytes was studied. Uptake of [3H]acetate by adipocytes and heparin-releasable lipoprotein lipase activity was inhibited after treatments of the cells with picomolar concentrations of recombinant human tumor necrosis factor alpha (rHuTNF-alpha), human tumor necrosis factor beta (rHuTNF-beta, also called lymphotoxin), murine interferon-gamma (rMuIFN-gamma), and a human hybrid interferon-alpha [rHuIFN-alpha 2/alpha 1 (Bgl II)]. Recombinant human interferon-gamma (rHuIFN-gamma), natural human colony-stimulating factor (HuCSF), and human interleukin 2 (HuIL-2) had no effect. Similar though less-marked suppression of [3H]acetate uptake by cytokines was seen in 3T3 L1 fibroblasts. Cytokines inhibited the incorporation of [3H]acetate into both membrane and storage lipids in the adipocytes. In addition to blocking lipid uptake and synthesis, rHuTNF-alpha and -beta, and rMuIFN-gamma stimulated the release of free fatty acid into the medium from adipocytes. Binding studies suggest that rHuTNF-alpha and rHuTNF-beta compete for the same cell-surface receptor on 3T3 L1 adipocytes, while rMuIFN-gamma binds to a separate receptor. The binding of rTNF-alpha to both adipocytes and fibroblasts can be significantly enhanced by preexposure of the cells to rMuIFN-gamma. There appear to be both high- and low-affinity receptors for rHuTNF-alpha on adipocytes, whereas fibroblasts exhibit a single class of high-affinity receptors. These results suggest that a variety of structurally distinct cytokines possess lipid mobilization activity, which may be of critical importance to the host in defense against infection or malignancy.
Genetic basis for the cross-reactive idiotypes on the light chains of human IgM anti-IgG autoantibodies.
Authors: Chen P P , Albrandt K , Orida N K , Radoux V , Chen E Y , Schrantz R , Liu F T , Carson D A
Published: May 2006

The role of immunoglobulin structural genes in the generation of autoantibodies in humans has not been elucidated. Human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors, RFs) from unrelated people often share idiotypic antigens. Antibodies against synthetic peptides have localized two of the shared idiotypic determinants to the second and third complementarity-determining regions of the kappa light chain. The reported sequences of several human RF light chains are remarkably homologous in these regions. Animal studies have shown that some shared idiotypic antigens represent serological markers for immunoglobulin variable (V)-region genes. Therefore, we hypothesized that human RF light chains derived from a single germ-line gene, designated V kappa-(RF), or from a small family of very closely related genes. In the present experiments, we have isolated and sequenced two human V kappa germ-line genes that encode kappa light chains, which are identical or closely related to the light chains of human RF. The data indicate that the shared idiotypic antigens on RF are phenotypic markers for a kappa V-region gene that is highly conserved in the human population. The results also imply that the light chains of IgM anti-IgG autoantibodies can be encoded by germ-line genes without any somatic mutation.
Platelet glycoproteins IIb and IIIa: evidence for a family of immunologically and structurally related glycoproteins in mammalian cells.
Authors: Charo I F , Fitzgerald L A , Steiner B , Rall S C , Bekeart L S , Phillips D R
Published: May 2006

Human and bovine cultured cell lines and circulating leukocytes were examined for the presence of surface proteins similar to platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human endothelial cells, smooth muscle cells, and MG-63 fibroblast-like cells were found to have surface proteins that cross-reacted with platelet GPIIb and GPIIIa antibodies, existed as complexes, and had molecular weights similar to those of the corresponding platelet glycoproteins. Bovine endothelial cells and smooth muscle cells also expressed GPIIb- and GPIIIa-like surface proteins. Metabolic labeling studies with [35S]methionine demonstrated that the cultured cells synthesized these glycoproteins. The GPIIIa-like protein in human endothelial and smooth muscle cells had the same isoelectric point as platelet GPIIIa, whereas their GPIIb alpha-like protein was slightly more acidic than platelet GPIIb alpha (pI = 5.2-5.3 versus 5.5). Platelet and endothelial cell GPIIb alpha (but not GPIIIa) showed an increased electrophoretic mobility in Ca2+ -containing versus EDTA-containing gels, implying a Ca2+ -GPIIb alpha interaction. The amino acid sequence of the amino termini of platelet GPIIb alpha and GPIIb beta and of the alpha chains of the leukocyte LFA-1 and Mac-1 glycoprotein complexes had significant sequence homology. These data indicate that glycoproteins that have either immunological cross-reactivity or amino-terminal sequence homology with the platelet GPIIb-IIIa complex are widely distributed in human and non-human adherent cells and circulating leukocytes and suggest that these proteins may be the products of a large gene family whose expression is cell specific.
Localization of sodium channels in axon hillocks and initial segments of retinal ganglion cells.
Authors: Wollner D A , Catterall W A
Published: May 2006

Affinity-purified antibodies against the sodium channel from rat brain were employed to localize sodium channels in the retina by immunocytochemical procedures. In rat retina, intense staining was observed in the ganglion cell axon layer and light staining was detected in fibers of the inner plexiform layer. In frog retina, only the ganglion cell axon layer was stained. Examination at higher magnification revealed that axon hillocks and initial segments of ganglion cells had a high density of immunoreactive sodium channels, whereas the cell bodies were devoid of stain. The sharply defined region of high sodium channel density at the axon hillock is likely to be responsible for the low threshold for action potential initiation in this region of vertebrate central neurons.
Structure, evolution, and polymorphisms of the human apolipoprotein A4 gene (APOA4).
Authors: Karathanasis S K , Oettgen P , Haddad I A , Antonarakis S E
Published: May 2006

The genes coding for three proteins of the plasma lipid transport system--apolipoproteins A1 (APOA1), C3 (APOC3), and A4 (APOA4)--are closely linked and tandemly organized on the long arm of human chromosome 11. In this study the human APOA4 gene has been isolated and characterized. In contrast to APOA1 and APOC3 genes, which contain three introns, the APOA4 gene contains only two. An intron interrupting the 5' noncoding region of the APOA1 and APOC3 mRNAs is absent from the corresponding position of the APOA4 mRNA. However, similar to APOA1 and APOC3 genes, the introns of the APOA4 gene separate nucleotide sequences coding for the signal peptide and the amphipathic domains in APOA4. These results suggest that the APOA1, APOC3, and APOA4 genes were derived from a common evolutionary ancestor and indicate that during evolution the APOA4 gene lost one of its ancestral introns. Two restriction endonuclease sites, an Xba I located in the second intron of the APOA4 gene and a different Xba I located 9 kilobases 3' to the APOA4 gene, are polymorphic in Mediterranean and Northern European populations. Haplotype analysis indicated that even though these polymorphic sites are located within 9 kilobases they do not display significant nonrandom association. Finally, restriction mapping analysis of DNA from a patient with combined APOA1-APOC3 deficiency and premature coronary artery disease indicated that this patient has a structurally normal APOA4 gene.
Cell-type-specific transcription of an immunoglobulin kappa light chain gene in vitro.
Authors: Mizushima-Sugano J , Roeder R G
Published: May 2006

We have established a cell-free system, derived from a human B-cell lymphoma, in which immunoglobulin kappa light chain gene promoters are both accurately transcribed and regulated in a cell-type-specific manner. Thus, accurate transcription from the T1 kappa light chain gene promoter was much more efficient in B-cell extracts than in HeLa cell extracts, whereas control promoters (adenovirus major late and histone H2B) were transcribed equally well in either extract. More important, the increased kappa light chain gene transcription in B-cell extracts was dependent upon upstream sequences (containing the conserved decanucleotide element) previously shown to be necessary for B-cell-specific transcription in vivo; in contrast, removal of these sequences had no effect on the low level of kappa transcription in HeLa extracts. The maximal level of upstream sequence-mediated transcription was dependent upon template topology. These studies show that there is at least one B-cell-specific factor that stimulates transcription from purified DNA templates, and they further suggest that the in vivo action of the factor(s) on other components of the transcription machinery is direct rather than indirect (e.g., via the maintenance of an open chromatin structure). The cell-free system described here should facilitate both purification and functional studies of the B-cell-specific factor(s).
GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5' TGACTC 3' sequences.
Authors: Arndt K , Fink G R
Published: May 2006

The GCN4 gene is required for the general amino acid control derepression response in yeast. GCN4 protein protects a repeated sequence motif in the 5'-untranslated region of HIS4, HIS3, ILV1, and ILV2 genes subject to general control. At low concentrations of GCN4, only certain repeats in these genes are bound. The repeats differ slightly from the 5' TGACTC 3' consensus core sequence, and the selective binding of some sites at low GCN4 concentrations is related to the relative affinity of these sites to GCN4. Using purified GCN4 protein obtained from an overproducing strain of Escherichia coli, we were able to obtain complete protection of all of the repeat elements in these four genes at high GCN4 concentrations. Analysis of the relative binding constant to the 15 repeated sequences protected by GCN4 shows that the optimal binding site for GCN4 is 5' RRTGACTC 3' followed by a short stretch of thymidines. Another protein, present mostly in yeast nuclear extracts, binds to the HIS4 promoter at a site overlapping one of the GCN4 binding sites. This protein is displaced from its binding site at high GCN4 concentrations.
Phosphatidylinositol 4,5-bisphosphate turnover is transient while phosphatidylinositol turnover is persistent in thyrotropin-releasing hormone-stimulated rat pituitary cells.
Authors: Imai A , Gershengorn M C
Published: May 2006

Stimulated inositolphospholipid turnover has been proposed to be initiated and sustained by hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which may be replenished by an enhanced flux of phosphatidylinositol (PtdIns) to PtdIns 4-phosphate (PtdIns4P) to PtdIns(4,5)P2. To determine whether there is continued hydrolysis and resynthesis of PtdIns(4,5)P2 in rat pituitary cells (GH3 cells) during stimulation by thyrotropin-releasing hormone (TRH), we investigated the turnover kinetics of the inositolphospholipids and of phosphatidic acid (PtdOH). In cells incubated with 32Pi for 1 min, TRH rapidly and persistently (for at least 30 min) enhanced the rate of 32P-labeling of PtdOH. After a lag time of 1 min, TRH markedly and persistently increased 32P-labeling of PtdIns also. In contrast, TRH caused only a transient increase in 32P-labeling of PtdIns(4,5)P2 that lasted less than 2 min. There was no rapid (before 10 min) effect of TRH on 32P-labeling of PtdIns4P. By 2 min of TRH stimulation, specific 32P radioactivity in PtdOH increased from 3.6% (control) of that in the gamma-phosphate of ATP to 15%; in PtdIns, from 0.07% to 1.3%; and in PtdIns(4,5)P2, from 3.8% to 5.4% (specific 32P radioactivity in PtdIns4P was 1.7% of that in ATP in control and TRH-stimulated cells). In cells exposed to TRH for 4 min and then to 32Pi, 32P-labeling of PtdOH and PtdIns increased, but that of PtdIns(4,5)P2 was not affected. Last, persistent turnover of PtdOH and PtdIns was not caused by initial hydrolysis of PtdIns(4,5)P2 because the turnover of PtdOH and PtdIns could be terminated by displacement of TRH from its receptor by chlordiazepoxide and restarted by reoccupying the receptors with TRH. These data demonstrate that turnover of PtdIns(4,5)P2 is stimulated only transiently, whereas turnover of PtdIns and PtdOH is stimulated persistently by TRH in GH3 cells. Hence, inositolphospholipid turnover in GH3 cells does not occur via continued hydrolysis of PtdIns(4,5)P2 accompanied by enhanced flux of PtdIns to PtdIns4P to PtdIns(4,5)P2, but there is direct and persistent hydrolysis of PtdIns. The dissociation of these actions suggests that there are separate mechanisms involved in coupling TRH-receptor complexes to stimulation of PtdIns(4,5)P2 and PtdIns hydrolysis in GH3 cells.
Sequence of mRNA coding for bindin, a species-specific sea urchin sperm protein required for fertilization.
Authors: Gao B , Klein L E , Britten R J , Davidson E H
Published: May 2006

Bindin, a major protein of the sea urchin acrosome granule, mediates the species-specific adhesion and binding of sperm to egg required to effect fertilization. We report the isolation and sequence of bindin cDNA clones prepared from Strongylocentrotus purpuratus testis RNA. The bindin gene appears to be productively expressed only in males and only in testes. The protein is produced from a 51-kDa precursor, which is subsequently processed to yield the mature 24-kDa bindin protein.
Regulation of expression of human granulocyte/macrophage colony-stimulating factor.
Authors: Chan J Y , Slamon D J , Nimer S D , Golde D W , Gasson J C
Published: May 2006

Colony-stimulating factors (CSFs) are glycoproteins that stimulate the growth of hematopoietic progenitors and enhance the functional activity of mature effector cells. Human granulocyte/macrophage colony-stimulating factor (GM-CSF) is a 22-kDa glycoprotein that stimulates the growth of myeloid and erythroid progenitors in vitro and increases the responsiveness of neutrophils, monocytes, and eosinophils to physiologic stimuli. Elucidation of the cell and tissue sources of CSFs, as well as study of their regulation of expression, is required to understand their role in physiologic and pathophysiologic states. An extensive survey of normal and neoplastic human tissues did not reveal constitutive production of detectable levels of GM-CSF mRNA in any of the 64 samples studied. Antigen- or lectin-activated T lymphocytes have been shown to produce GM-CSF; therefore, to elucidate the genetic sequences required, we constructed recombinant plasmids containing 5' flanking DNA of the GM-CSF gene linked to the marker chloramphenicol acetyltransferase gene. The recombinant constructs were transfected into a human T-cell leukemia virus type I (HTLV)-infected T-lymphoblast cell line that can be stimulated to produce high levels of GM-CSF. We show here that the 5' flanking sequences of the GM-CSF gene can direct increased expression of the chloramphenicol acetyltransferase gene in activated T-lymphoblast cells.
Interleukin 1 induces leukocyte infiltration and cartilage proteoglycan degradation in the synovial joint.
Authors: Pettipher E R , Higgs G A , Henderson B
Published: May 2006

Interleukin 1 (IL-1) is a polypeptide released by activated macrophages and is thought to be a key mediator of host responses to infection and inflammation. The availability of highly purified and recombinant material has now permitted the evaluation of IL-1 as a mediator of chronic inflammatory processes in vivo. We have demonstrated that intraarticular injection of IL-1 into rabbit knee joints induces the accumulation of polymorphonuclear and mononuclear leukocytes in the joint space and the loss of proteoglycan from the articular cartilage. The effects on cartilage could not be explained solely by the presence of leukocytes, since injections of endotoxin also stimulated leukocyte accumulation in the joint but had no effect on proteoglycan loss. Responses to IL-1 were not associated with increased production of the icosanoids prostaglandin E2 or leukotriene B4 and were not reduced by an inhibitor of their synthesis. The pattern of leukocyte infiltration and cartilage breakdown 24 hr after IL-1 injection was similar to that seen in animals with antigen-induced arthritis of 1 week's duration. These observations support the hypothesis that IL-1 acts directly to mediate the erosive processes of chronic arthritis.
Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors.
Authors: Coca-Prados M , Wax M B
Published: May 2006

Ciliary epithelial cells derived from human eye were successfully propagated through many generations after transformation with simian virus 40. The cell clone 8-SVHCE was isolated and characterized by immunoprecipitation and pharmacological studies that demonstrated the presence of several functional properties observed in the parent cells of this tissue. Immunoprecipitation revealed the presence of large tumor (T) antigen, and Southern blot analysis showed the incorporation of viral DNA into high molecular weight ciliary epithelial cell DNA. The presence of beta-adrenergic receptors was demonstrated by direct binding of a radiolabeled antagonist, [125I]iodopindolol, to membrane preparations of 8-SVHCE cells (Kd = 41.8 pM and Bmax = 67.1 fmol/mg of protein). Competition experiments with [125I]iodopindolol and selective drugs suggested that the receptors are of the beta 2-adrenergic subtype. Studies of catecholamine-stimulated cellular cAMP production and of isoproterenol-dependent protein phosphorylation of vimentin in 8-SVHCE indicated the functional conservation of beta-adrenergic receptor-mediated processes that are thought to be important in the regulation of aqueous humor production by the ciliary epithelium in vivo.
Immunoglobulin and T-cell receptor gene rearrangement and expression in human lymphoid leukemia cells at different stages of maturation.
Authors: Davey M P , Bongiovanni K F , Kaulfersch W , Quertermous T , Seidman J G , Hershfield M S , Kurtzberg J , Haynes B F , Davis M M , Waldmann T A
Published: May 2006

The use of probes to genes (IG and TCRB) encoding immunoglobulins (IG) and the beta chain of the T-cell antigen receptor (TCRB), respectively, have become a sensitive means to assess clonality and lineage in lymphoid malignancies. It has become apparent that some individual cases show rearrangements of both IG and TCRB genes. In an attempt to more accurately define cell lineage we have analyzed cells from patients with B- or T-cell leukemia (n = 26) at various stages of maturation with probes to two additional TCR genes, TCRG and TCRA (encoding the TCR gamma and alpha chains, respectively), as well as the IG heavy chain joining region (IGHJ) and TCRB genes. On Southern blot analysis, the mature T-cell leukemia cells studied had rearranged TCRG and TCRB while IGHJ remained as in the germ line. The mature B-cell leukemia cells studied had rearranged IGHJ with germ-line TCRG and TCRB. These data suggest that, in the majority of more mature leukemias, cells have rearranged IG or TCR genes but not both. In contrast, cells from five of nine precursor B-cell leukemia patients and cell lines from one of four precursor T-cell leukemia patients had rearranged both IGHJ and TCR genes. TCRG and TCRB mRNAs were expressed in the cells of precursor T- but not B-cell leukemia patients studied. The spectrum of leukemia cells studied within the T-cell series permitted an assessment of the order of TCR gene rearrangements. Two of 13 patients had cells with germ-line TCRG and TCRB, 2 patients had cells with rearranged TCRG alone, and the remainder had cells with rearranged TCRG and TCRB. TCRG and TCRB mRNAs were expressed in precursor T-cell leukemia cells, whereas TCRB and TCRA were expressed in mature T-cell leukemia cells. These results parallel observations from mouse studies on gene expression and support the view of a hierarchy of TCR gene rearrangements in T-lymphocyte ontogeny. TCRG genes are rearranged first, subsequently TCRB genes are rearranged, followed by TCRA gene activation.
Combination of a long-acting delivery system for luteinizing hormone-releasing hormone agonist with Novantrone chemotherapy: increased efficacy in the rat prostate cancer model.
Authors: Schally A V , Kook A I , Monje E , Redding T W , Paz-Bouza J I
Published: May 2006

The combination of hormonal treatment based on a long-acting delivery system for the agonist [6-D-tryptophan]luteinizing hormone-releasing hormone ([D-Trp6]-LH-RH) with the chemotherapeutic agent Novantrone (mitoxantrone dihydrochloride) was studied in the Dunning R3327H rat prostate cancer model. Microcapsules of [D-Trp6]-LH-RH formulated from poly(DL-lactide-co-glycolide) and calculated to release a controlled dose of 25 micrograms/day were injected intramuscularly once a month. Novantrone (0.25 mg/kg) was injected intravenously once every 3 weeks. Three separate experiments were carried out. When the therapy was started 45 days after transplantation and continued for 70 days, tumor volume in the presence of the microcapsules (966 +/- 219 mm3) or Novantrone (3606 +/- 785 mm3) given alone was significantly decreased compared to controls (14,476 +/- 3045 mm3). However, the combination of microcapsules and Novantrone caused a greater inhibition of tumor growth (189 +/- 31 mm3) than the single agents. Similar effects were seen when the percent increase in tumor volume was examined. Tumor volume increased 10,527 +/- 1803% for the control group. The inhibition of growth caused by the [D-Trp6]LH-RH microcapsules alone (672 +/- 153% increase in volume) was again greater than that caused by Novantrone alone (2722 +/- 421% increase). The combination of the two agents was again the most effective, resulting in an increase in tumor volume of only 105 +/- 29%. Control tumors weighed 30.0 +/- 6.5 g. Tumor weights were much less in the groups treated with either microcapsules (3.28 +/- 0.69 g) or Novantrone (19.53 +/- 3.3 g) alone. The lowest tumor weights after 70 days of treatment were obtained in the group that received the combination of [D-Trp6]LH-RH microcapsules and Novantrone (1.02 +/- 0.2 g). Testes and ventral prostate weights were significantly diminished by the administration of microcapsules of [D-Trp6]LH-RH alone or in combination with Novantrone. In both of these groups, luteinizing hormone and prolactin levels were reduced and serum testosterone was suppressed to undetectable levels. Similar results were obtained in two other experiments in which the duration of treatment was 60 or 105 days. These results suggest that the overall response could reflect the inhibition of proliferation of hormone-independent cancer cells by Novantrone in addition to the suppressive effect of [D-Trp6]LH-RH on the growth of androgen-dependent tumor cells. The administration of Novantrone in combination with microcapsules of [D-Trp6]LH-RH might produce a better clinical response than LH-RH analog alone in patients with advanced prostate carcinoma.
Human class II (pi) alcohol dehydrogenase has a redox-specific function in norepinephrine metabolism.
Authors: Mårdh G , Dingley A L , Auld D S , Vallee B L
Published: May 2006

Studies of the function of human alcohol dehydrogenase (ADH) have revealed substrates that are virtually unique for class II ADH (pi ADH). It catalyzes the formation of the intermediary glycols of norepinephrine metabolism, 3,4-dihydroxyphenylglycol and 4-hydroxy-3-methoxyphenylglycol, from the corresponding aldehydes 3,4-dihydroxymandelaldehyde and 4-hydroxy-3-methoxymandelaldehyde with Km values of 55 and 120 microM and kcat/Km ratios of 14,000 and 17,000 mM-1 X min-1; these are from 60- to 210-fold higher than those obtained with class I ADH isozymes. The catalytic preference of class II ADH also extends to benzaldehydes. The kcat/Km values for the reduction of benzaldehyde, 3,4-dihydroxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde by pi ADH are from 9- to 29-fold higher than those for a class I isozyme, beta 1 gamma 2 ADH. Furthermore, the norepinephrine aldehydes are potent inhibitors of alcohol (ethanol) oxidation by pi ADH. The high catalytic activity of pi ADH-catalyzed reduction of the aldehydes in combination with a possible regulatory function of the aldehydes in the oxidative direction leads to essentially "unidirectional" catalysis by pi ADH. These features and the presence of pi ADH in human liver imply a physiological role for pi ADH in the degradation of circulating epinephrine and norepinephrine.
Interferon-stimulated transcription: isolation of an inducible gene and identification of its regulatory region.
Authors: Levy D , Larner A , Chaudhuri A , Babiss L E , Darnell J E
Published: May 2006

A human gene (termed ISG-54K) that is induced from near undetectable levels to high transcriptional activity by alpha- and beta-interferons has been cloned. The genomic structure and nucleotide sequence of the coding region were determined and the RNA initiation site was identified. The 5' portion of the gene was fused with a heterologous gene lacking an active promoter in recombinant plasmid and adenoviral vectors. These fusion genes were used to assess the activity of the ISG-54K promoter in response to interferon. RNA was formed in HeLa cells from recombinant plasmids only in response to interferon. Furthermore, in human diploid fibroblasts, infection with the recombinant adenovirus vector resulted in a 50-fold increase in specific RNA in response to interferon, followed by a subsequent decrease, imitating the natural regulated transcriptional cycle of the endogenous gene.
Indole-3-acetic acid and fusicoccin cause cytosolic acidification of corn coleoptile cells
Authors: Felle Hubert , Brummer Benno , Bertl Adam , Parish Roger W.
Published: May 2006

Microelectrodes were used to measure simultaneously the effects of indole-3-acetic acid (IAA) on membrane potential and cytosolic pH of corn coleoptile cells. IAA caused an initial depolarization followed by hyperpolarization, the latter displaying rhythmic oscillations. The extent of the changes in membrane potential was dependent on IAA concentration, and hyperpolarization, but not depolarization, could be detected with concentrations of IAA as low as 10 nM. Membrane hyperpolarization was preceded by a decrease in cytosolic pH. The decrease commenced ≈5 min after adding IAA and continued for 15-20 min before reaching a new steady state ≈0.1 pH unit lower than the original pH. The decrease in pH was readily detectable after treatment with 0.1 μM IAA. Fusicoccin and acetate, which, like IAA, induce elongation growth, caused a similar drop in cytosolic pH and subsequent membrane hyperpolarization, the decrease in pH commencing within seconds. The addition of fusicoccin to IAA-treated cells resulted in a further cytosolic acidification and membrane hyperpolarization. The two substances probably change cytosolic pH via different mechanisms. The results imply that one of the primary effects of auxins in coleoptiles is to lower cytosolic pH.
MB1, a quail leukocyte-endothelium antigen: partial characterization of the cell surface and secreted forms in cultured endothelial cells.
Authors: Labastie M C , Poole T J , Péault B M , Le Douarin N M
Published: May 2006

We describe here conditions allowing the selective growth in culture of embryonic capillary endothelial cells from quail yolk sac. Such cultures were set up to characterize an antigen present on the endothelial cell surface and to study whether it was secreted in the culture medium. This antigen, MB1, was previously evidenced by a monoclonal antibody raised to quail IgM heavy chain. It is present at the surface of all endothelial and hemopoietic cells (except mature erythrocytes) starting from the hemangioblast, the early mesodermal precursor of blood and vascular endothelial cells. The MB1 epitope is also found on quail plasma molecules of 80 and 125-200 kDa. By immunoprecipitation of either surface or metabolically labeled endothelial cellular material, we have chemically characterized MB1-bearing components as glycoproteins of apparent molecular mass ranging from 80 to 200 kDa and provided evidence for their release into the culture medium. This is consistent with the hypothesis that, in the quail, vascular endothelium participates in the secretion of the alpha-MB1-positive plasmatic components.
A guanine nucleotide-binding protein mediates the inhibition of voltage-dependent calcium current by somatostatin in a pituitary cell line.
Authors: Lewis D L , Weight F F , Luini A
Published: May 2006

Somatostatin reduces voltage-dependent Ca2+ current (ICa) and intracellular free Ca2+ concentration in the AtT-20/D16-16 pituitary cell line. We tested whether guanine nucleotide-binding proteins (G or N proteins) are involved in the signal transduction mechanism between the somatostatin receptor and voltage-dependent Ca2+ channels. Treatment of the cells with pertussis toxin, which selectively ADP ribosylates the GTP binding proteins Gi and Go and suppresses the ability of Gi to couple inhibitory receptors to adenylate cyclase, abolished the action of somatostatin on both ICa and intracellular free Ca2+. Intracellular application of the nonhydrolyzable guanine nucleotide analog guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), which irreversibly activates G proteins, changed the somatostatin effect on ICa from a reversible to an irreversible inhibition. Intracellular GTP[gamma S] alone caused a very slowly developing inhibition of ICa. When ICa was inhibited by GTP[gamma S] (alone or with somatostatin), it failed to respond to subsequent applications of somatostatin. The effect of GTP[gamma S] on the inhibition of ICa by somatostatin was not altered by the intracellular application of cAMP and 3-isobutyl-1-methylxanthine. The results suggest that a GTP-binding protein is directly involved in the cAMP-independent receptor-mediated inhibition of voltage-dependent Ca2+ channels.
Interferon-gamma regulates an antigen specific for endothelial cells involved in lymphocyte traffic.
Authors: Duijvestijn A M , Schreiber A B , Butcher E C
Published: May 2006

One of the most striking examples of localized vascular differentiation is exhibited by specialized lymphoid organ venules that mediate the extravasation of circulating lymphocytes from the blood. These vessels are characterized by cuboidal or "high" endothelial cell morphology and are unique in their functional capacity to interact with migrating lymphocytes, regulating both the rate and specificity of lymphocyte traffic through particular regions of the body. We describe here a monoclonal antibody, MECA-325, that defines an endothelial cell differentiation antigen selectively expressed on high endothelium in the mouse. Thus an antigen defining a specific functional subset of endothelial cells has been found. Furthermore, we demonstrate that the MECA-325 antigen can be induced in mouse lung or bone marrow-derived endothelial cell lines in vitro by interferon-gamma but not by interferon-beta, interleukin-1, or endothelial cell mitogens. The results define a unique marker associated with differentiated endothelial cells mediating lymphocyte traffic from the blood, and they provide evidence that the specialized phenotype of these high endothelial cells may be induced and controlled by local factors associated with immune activity.
Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.
Authors: Sangameswaran L , Fales H M , Friedrich P , De Blas A L
Published: May 2006

An endogenous brain substance that binds to the central-type benzodiazepine receptors with agonist properties is present in both rat and bovine brains. This substance has been purified to homogeneity from bovine brain by immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reversed-phase HPLC steps. The purified substance was characterized as the benzodiazepine N-desmethyldiazepam (nordiazepam). The techniques used for the identification were mass spectrometry, HPLC, spectrophotometry, benzodiazepine receptor binding, and immunological techniques. Benzodiazepine-like immunoreactivity was also found in all the human brains tested, including six brains that had been stored in paraffin since 1940, fifteen years before the first synthesis of benzodiazepines. These results show that benzodiazepine-like molecules of natural origin--and possibly benzodiazepines themselves--are present in human and other mammalian brains.
Angiotensin-stimulated production of inositol trisphosphate isomers and rapid metabolism through inositol 4-monophosphate in adrenal glomerulosa cells.
Authors: Balla T , Baukal A J , Guillemette G , Morgan R O , Catt K J
Published: May 2006

The production and metabolism of inositol phosphates in rat adrenal glomerulosa cells prelabeled with [3H]inositol and stimulated with angiotensin II were analyzed by high-performance anion-exchange chromatography. Exposure to angiotensin II was accompanied by a rapid and substantial decrease in the phospholipid precursor, phosphatidylinositol (PtdIns) 4,5-bisphosphate with only a slight and transient increase in the level of the biologically active product, inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), to a peak at about 5 sec. Inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), the putative metabolite of Ins-1,4,5-P3, was also formed rapidly and maintained an elevated steady-state level during stimulation by angiotensin II. Inositol 1,4-bisphosphate (Ins-1,4-P2) exhibited a simultaneous and prominent increase that could not be accounted for solely by direct breakdown of PtdIns 4-phosphate, indicating that large amounts of Ins-1,4,5-P3 must also have been produced and metabolized. The rapid formation of a substantial amount of inositol 4-monophosphate (Ins-4-P), with no significant change in the level of inositol 1-monophosphate (Ins-1-P) during the first minute of stimulation, was a notable feature of the glomerulosa cell response to angiotensin II. These observations indicate that PtdIns-4,5-P2 catabolism in the angiotensin-stimulated glomerulosa cell initially proceeds via Ins-1,4,5-P3 through Ins-1,3,4-P3 and Ins-1,4-P2 to form Ins-4-P rather than Ins-1-P and that direct hydrolysis of PtdIns by phospholipase C does not occur during the initial phase of angiotensin action. In glomerulosa cells stimulated by angiotensin II in the presence of Li+, the progressive accumulation of both Ins-4-P, and after a short lag period, Ins-1-P indicated that dephosphorylation of both isomers was inhibited by Li+. The increase of Ins-P isomers in the presence of Li+ was associated with increased and progressive accumulation of Ins-1,4-P2 and Ins-1,3,4-P3 but not of Ins-1,4,5-P3. These data demonstrate that sustained and massive breakdown of PtdIns phosphates begins within seconds during cell activation by angiotensin II. The Ca2+-mobilizing metabolite, Ins-1,4,5-P3, is rapidly converted to Ins-1,3,4-P3 and degraded through Ins-1,4-P2 and Ins-4-P, in contrast to the previous view that conversion to Ins-1-P is the major route of PtdIns 4,5-bisphosphate metabolism.
Photoaffinity labeling of the porcine brain alpha 2-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine: identification of the hormone-binding subunit.
Authors: Lanier S M , Graham R M , Hess H J , Grodski A , Repaske M G , Nunnari J M , Limbird L E , Homcy C J
Published: May 2006

A functionalized derivative of the alpha 2-selective antagonist rauwolscine formed the basis for a photoaffinity adduct that has allowed identification of the hormone-binding subunit of the brain alpha 2-adrenergic receptor protein. Rauwolscine carboxylate underwent reaction with 4-N-t-butyloxycarbonyl-aminoaniline, leading to the synthesis of rauwolscine 4-aminophenyl carboxamide (Rau-AmPC). Rau-AmPC was radioiodinated and converted to the arylazide derivative, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-(4-azido-3-[125I]iodo)phenyl] carboxamide (125I-Rau-AzPC), via a diazonium salt intermediate. The characterization of 125I-Rau-AzPC as a photolabile probe employed alpha 2-adrenergic receptors, which were first solubilized from porcine brain membranes and partially purified by affinity chromatography utilizing a yohimbine-agarose affinity matrix. In the partially purified receptor preparation incubated with 125I-Rau-AzPC, photolysis resulted in covalent labeling of a major (Mr, 62,000) peptide as determined by NaDodSO4/PAGE and autoradiography. Labeling of this peptide was inhibited by the alpha 2-selective antagonist, yohimbine, and the non-subtype-selective alpha-antagonist, phentolamine, but not by the alpha 1-antagonist, prazosin, or the beta-receptor antagonist, (-)-alprenolol. The alpha-adrenergic agonist epinephrine also inhibited labeling in a stereoselective manner. These data indicate that the photolabeled Mr 62,000 peptide is the hormone-binding subunit of the alpha 2-adrenergic receptor protein. The availability of this radioiodinated photoaffinity probe for the alpha 2-adrenergic receptor should facilitate further structural and biophysical characterization of the receptor protein.
Dynamic exchange of myosin molecules between thick filaments.
Authors: Saad A D , Pardee J D , Fischman D A
Published: May 2006

To examine thick filament assembly and myosin exchange, a fluorescence energy transfer assay has been established. Assembly-competent myosin molecules labeled with the sulfhydryl-specific fluorochromes 5-(2-[(iodoacetyl)-amino]ethyl)aminonaphthalene-1-sulfonic acids (IAEDANS) or 5-iodoacetamidofluorescein (IAF) were prepared. Using IAEDANS-labeled myosin as fluorescence donor and IAF-labeled myosin as acceptor, thick filament formation was followed by the decrease in donor fluorescence at 0.1 M KCl/10 mM potassium phosphate, pH 6.9. The critical concentration of myosin--i.e., that concentration that remained unassembled at equilibrium with fully formed filaments--was 40 nM. In FET and 125I-labeled myosin incorporation assays, extensive exchange of myosin between thick filaments was observed. The presence of a critical concentration and the measurements of extensive exchange suggest a dynamic equilibrium between fully polymerized myosin and a small pool of soluble myosin.
Phosphorylation of the secreted, free alpha subunit of human chorionic gonadotropin.
Authors: Saccuzzo J E , Krzesicki R F , Perini F , Ruddon R W
Published: May 2006

Phosphorylation of secretory proteins is an uncommon event. In this manuscript, the phosphorylation of human chorionic gonadotropin, a glycoprotein hormone secreted by the JAR choriocarcinoma cell line, is described. Labeling of JAR cells with 32PO4 indicates that both the intracellular and the secreted forms of the free alpha subunit are phosphorylated. Although the secreted alpha beta dimer also incorporates 32PO4, there is little detectable phosphorylation of the intracellular precursors of alpha beta dimer, suggesting that dimer phosphorylation occurs as a late event in post-translational processing. In addition, phorbol 12-myristate 13-acetate markedly stimulates the phosphorylation of both intracellular and secreted forms of free alpha subunit and to a lesser extent of secreted alpha beta dimer. In vitro assays, using homogenates of JAR cells as a source of protein kinase activity, indicate that the uncombined alpha subunit is preferentially phosphorylated. The phosphorylation sites are on serine and threonine residues in the alpha subunit.
High-affinity fluorescent peptide binding to I-Ad in lipid membranes.
Authors: Watts T H , McConnell H M
Published: May 2006

Membranes isolated from antigen-pulsed, antigen-presenting cells were extensively dialyzed and shown to retain the ability to present antigen to an I-Ad-restricted, antigen-specific T cell, 3DO-54.8. This ability to retain antigen on the membrane was duplicated in lysates of antigen-presenting cells as well as with pure I-A molecules in phospholipid vesicles. Measurement of the concentration of surface-associated fluorescent peptide on planar membranes prepared from antigen-pulsed phospholipid vesicles showed that about 1 peptide molecule was retained per 100 I-A molecules. We calculate that about 1000 I-A-associated peptide molecules are sufficient to stimulate the response of a single 3DO-54.8 cell. The association of fluorescent peptide with the surface depended on the presence of I-A and was blocked by unlabeled ovalbumin peptide or by a digest of hen egg lysozyme, added at the time of the pulse, but not after pulsing.