bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the regulatory gene xylS on the TOL plasmid is positively controlled by the xylR gene product.
Authors: Inouye S , Nakazawa A , Nakazawa T
Published: May 2006

The regulatory gene xylS on the TOL plasmid of Pseudomonas putida activates the transcription of the xylDLEGF operon for the m-toluate-degrading pathway in the presence of m-toluate. The gene also activates the transcription of the same operon in the presence of m-xylene or m-methylbenzyl alcohol, but for this activation another regulatory gene, xylR, is required. In this study we examined the xylS expression by determining the mRNA by reverse transcriptase mapping and by monitoring the enzyme activity of the xylE gene product, which was expressed under the control of the xylS promoter. The results of the above experiments provide evidence that xylR positively controls the transcription of xylS in the presence of m-xylene or m-methylbenzyl alcohol. The xylS product thus amplified may in turn activate the xylDLEGF operon. The nucleotide sequence of the xylS promoter resembles that of the promoter of the xylCAB operon for the m-xylene-degrading pathway, which is also activated by xylR in the presence of m-xylene or m-methylbenzyl alcohol. In addition, we have demonstrated that the expression of xylR is negatively controlled by its own product. On the basis of these findings, we propose a revised model for the regulation of expression of xyl genes on the TOL plasmid.
Functional analysis of a retroviral host-range mutant: altered long terminal repeat sequences allow expression in embryonal carcinoma cells.
Authors: Hilberg F , Stocking C , Ostertag W , Grez M
Published: May 2006

A retroviral host-range neomycin-resistant myeloproliferative sarcoma virus mutant, which is expressed in the embryonal carcinoma cell lines F9 and PCC4aza1R, was molecularly cloned and analyzed. This mutant virus, PCMV, differs from myeloproliferative sarcoma virus by two major deletions, one of which spans exactly a 75-base-pair repeat of the long terminal repeat. Functional analysis of recombinant viruses shows that the host-range expansion of PCMV is a property of nucleotide changes within the U3 region of the long terminal repeat. Furthermore, expression assays of chimeric long terminal repeats show that the enhancer region of PCMV joined to the promoter region of Moloney murine leukemia virus is sufficient to direct the synthesis of chloramphenicol acetyltransferase in F9 and PCC4 cells.
Carcinoembryonic antigen family: expression in a mouse L-cell transfectant and characterization of a partial cDNA in bacteriophage lambda gt11.
Authors: Kamarck M E , Elting J J , Hart J T , Goebel S J , Rae P M , Nothdurft M A , Nedwin J J , Barnett T R
Published: May 2006

Genomic DNA and mRNA from the adenocarcinoma cell line LoVo were used to generate L-cell transfectants and a bacteriophage lambda gt11 cDNA clone that express epitopes of carcinoembryonic antigen (CEA). Primary and secondary L-cell transfectants expressing CEA were selected with a fluorescence-activated cell sorter (FACS). These transfectants, including some clones that were selected for high-level CEA expression by multiple rounds of FACS sorting, express a surface protein of 150 kDa that reacts with all anti-CEA antibodies tested. In parallel, a cDNA library of LoVo poly(A)+ RNA was constructed in lambda gt11 and fusion proteins were screened with polyclonal antisera against CEA. One positive clone, lambda cLV7, was identified that hybridized specifically to transfectant DNA. The nucleic acid sequence of the cDNA insert (cLV7) contained two regions of extensive internal homology, with greater than 70% identity at the amino acid level. cLV7 hybridized to three mRNA species of LoVo cells and to a predominant mRNA of the CEA-expressing transfectants. Hybridization of cLV7 to restriction endonuclease-digested genomic DNA of colon carcinoma cells, normal human cells, and human-mouse somatic cell hybrids revealed the presence of multiple hybridizing bands, one of which was present in transfectant cells. These CEA-related sequences are not rearranged in tumors and, by somatic cell hybrid analysis, were mapped to human chromosome 19.
Molecular events in the induction of a nonresponsive state in interleukin 2-producing helper T-lymphocyte clones.
Authors: Jenkins M K , Pardoll D M , Mizuguchi J , Chused T M , Schwartz R H
Published: May 2006

Exposure of normal interleukin 2 (IL-2)-producing helper T-cell clones to antigen and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated antigen-presenting cells results in proliferative unresponsiveness to subsequent stimulation with antigen and normal antigen-presenting cells. In the present study, we have examined the molecular events that accompany the induction of this unresponsive state. T cells stimulated in this manner failed to produce IL-2, but interleukin 3, interferon-gamma, and IL-2 receptors were partially induced and T-cell receptor beta mRNA was fully induced. Although T-cell unresponsiveness correlated with an IL-2 production defect, addition of IL-2 during the induction phase failed to prevent development of the unresponsive state. The critical biochemical event appeared to be an increase in intracellular calcium. Removal of calcium from the medium prevented induction of the unresponsive state, whereas addition of the calcium ionophore ionomycin induced unresponsiveness as well as all of the related partial activation events. Thus, an increase in intracellular calcium under nonmitogenic conditions appears to initiate an alternative activation program that prevents the T cell from producing IL-2 in response to subsequent normal activation signals. The significance of this in vitro model for tolerance induction in vivo is discussed.
Bile acid metabolism in hereditary forms of hypertriglyceridemia: evidence for an increased synthesis rate in monogenic familial hypertriglyceridemia.
Authors: Angelin B , Hershon K S , Brunzell J D
Published: May 2006

This study was undertaken to characterize bile acid metabolism in hereditary forms of hypertriglyceridemia. Ten hypertriglyceridemic patients (type IV phenotype) with familial combined hyperlipidemia and 7 patients with monogenic familial hypertriglyceridemia (FHTG) were compared with 18 healthy controls; all subjects were males. Pool size, synthesis rate, and fractional catabolic rate of cholic and chenodeoxycholic acids were determined with an isotope dilution technique. Patients with FHTG had synthesis rates of cholic acid, chenodeoxycholic acid, and total bile acids above those seen in normal controls (P less than 0.001); also the fractional catabolic rates of both bile acids were increased (P less than 0.001). In contrast, bile acid kinetic parameters were--with one exception--within normal limits in patients with familial combined hyperlipidemia. The abnormality of bile acid metabolism could also be identified in a normolipidemic individual presumed to carry the gene for FHTG. The postprandial rise of serum bile acids was blunted in FHTG, indicating that the intestinal uptake of bile acids may be deficient in this condition. We conclude that FHTG, but not familial combined hyperlipidemia, is frequently associated with a defective regulation of bile acid synthesis, resulting in abnormally high production rate of bile acids. It is hypothesized that this abnormality is important for the subsequent development of hypertriglyceridemia.
Full-length cDNA for rabbit tryptophan hydroxylase: functional domains and evolution of aromatic amino acid hydroxylases.
Authors: Grenett H E , Ledley F D , Reed L L , Woo S L
Published: May 2006

A full-length cDNA for tryptophan hydroxylase was cloned from rabbit pineal body by screening an expression library with antibody against rat phenylalanine hydroxylase, which crossreacts with rabbit tryptophan hydroxylase. Clones producing immunoreactive material contain sequences homologous to, yet distinct from, phenylalanine hydroxylase. The rabbit cDNA hybridizes to mRNA in pineal body and brainstem but not in liver. Comparison of the rabbit tryptophan hydroxylase sequence with the sequences of phenylalanine hydroxylase and tyrosine hydroxylase demonstrates that these three biopterin-dependent aromatic amino acid hydroxylases are highly homologous, reflecting a common evolutionary origin from a single primordial genetic locus. The pattern of sequence homology supports the hypothesis that the carboxyl-terminal two-thirds of the molecules constitute the enzymatic activity cores, and the amino-terminal thirds of the molecules constitute domains for substrate specificity.
Multiple repressor binding sites in the genome of bacteriophage P1.
Authors: Velleman M , Dreiseikelmann B , Schuster H
Published: May 2006

After digestion of bacteriophage P1 DNA with EcoRI in the presence of P1 repressor, 6 repressor binding sites were identified in 5 of 26 EcoRI fragments. Binding sites were localized by the decreased mobility of DNA fragment-repressor complexes during electrophoresis and by DNase protection ("footprinting") analysis. The repressor binding sites, or operators, comprise a 17-base-pair-long consensus sequence lacking symmetrical elements. Three operators can be related to known genes, whereas the function of the others is still unknown. The mutant P1 bac, rendering ban expression constitutive, is identified as an operator-constitutive mutation of the ban operon.
Adenosine triphosphate stimulates inositol phospholipid metabolism and prostacyclin formation in adrenal medullary endothelial cells by means of P2-purinergic receptors.
Authors: Forsberg E J , Feuerstein G , Shohami E , Pollard H B
Published: May 2006

In the adrenal medulla, chromaffin cells secrete high concentrations of catecholamines, ATP, peptides and other factors that must pass through an endothelial cell barrier to enter the bloodstream. We have measured the effect of several of these chromaffin cell secretory products on cultured bovine adrenal medullary endothelial cells and have found that only ATP stimulates prostacyclin formation. The stimulation of prostacyclin formation by ATP coincides with the metabolism of inositol phospholipids and the accumulation of the putative second messenger inositol trisphosphate. The time course, concentration dependence, and P2-purinergic receptor specificity were similar for ATP-stimulated prostacyclin formation and ATP-stimulated inositol phospholipid metabolism. Thus, the increase in prostacyclin formation may be secondary to mobilization of intracellular Ca2+ by inositol trisphosphate, leading to activation of phospholipase A2, liberation of arachidonic acid, and the conversion of arachidonic acid to prostacyclin. We propose that the function of ATP, which is often colocalized with cell-specific hormones in secretory cells, may be to regulate blood flow in the adrenal medulla and other endocrine tissues by interacting with adjacent endothelial cells.
A cryptic proofreading 3'----5' exonuclease associated with the polymerase subunit of the DNA polymerase-primase from Drosophila melanogaster.
Authors: Cotterill S M , Reyland M E , Loeb L A , Lehman I R
Published: May 2006

The DNA polymerase-primase from Drosophila lacks 3'----5' exonuclease activity. However, a potent exonuclease can be detected after separating the 182-kDa polymerase subunit from the other three subunits of the enzyme (73, 60, and 50 kDa) by glycerol gradient sedimentation in the presence of 50% ethylene glycol. The exonuclease activity cosediments with the polymerase subunit, suggesting that the two activities reside in the same polypeptide. The 3'----5' exonuclease excises mismatched bases at the 3' termini of primed synthetic and natural DNA templates. Excision of a mispaired base at the 3' terminus occurs at a 10-fold greater rate than excision of the correctly paired base. When replication fidelity is measured by the bacteriophage phi X174 am3 reversion assay, the isolated polymerase subunit is at least 100-fold more accurate than either the intact polymerase-primase or a complex of the 182- and 73-kDa subunits. These results suggest that the 3'----5' exonuclease functions as a proofreading enzyme during Drosophila DNA replication in vitro and very likely in vivo.
Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA.
Authors: Indik Z , Yeh H , Ornstein-Goldstein N , Sheppard P , Anderson N , Rosenbloom J C , Peltonen L , Rosenbloom J
Published: May 2006

Poly(A)+ RNA, isolated from a single 7-mo fetal human aorta, was used to synthesize cDNA by the RNase H method, and the cDNA was inserted into lambda gt10. Recombinant phage containing elastin sequences were identified by hybridization with cloned, exon-containing fragments of the human elastin gene. Three clones containing inserts of 3.3, 2.7, and 2.3 kilobases were selected for further analysis. Three overlapping clones containing 17.8 kilobases of the human elastin gene were also isolated from genomic libraries. Complete sequence analysis of the six clones demonstrated that: the cDNA encompassed the entire translated portion of the mRNA encoding 786 amino acids, including several unusual hydrophilic amino acid sequences not previously identified in porcine tropoelastin, exons encoding either hydrophobic or crosslinking domains in the protein alternated in the gene, and a great abundance of Alu repetitive sequences occurred throughout the introns. The data also indicated substantial alternative splicing of the mRNA. These results suggest the potential for significant variation in the precise molecular structure of the elastic fiber in the human population.
Drosophila Krüppel gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA.
Authors: Ollo R , Maniatis T
Published: May 2006

The product of the Drosophila segmentation gene Krüppel was produced in cultured insect cells using the baculovirus expression system. When a cloned Krüppel cDNA sequence was inserted into the viral genome downstream from the promoter of the polyhedrin gene, a polypeptide with an apparent molecular weight of approximately equal to 72,000 was observed in the nuclei of infected cells. Antibodies were raised against this protein and used to detect Krüppel in Drosophila embryos. Characterization of the Krüppel protein extracted from infected cells showed that it is tightly bound to the nucleus, it binds to calf thymus DNA-cellulose, and it is phosphorylated. These results support the hypothesis that Krüppel is a regulatory protein that acts by binding DNA.
Activation of transforming potential of the human insulin receptor gene.
Authors: Wang L H , Lin B , Jong S M , Dixon D , Ellis L , Roth R A , Rutter W J
Published: May 2006

A retrovirus containing part of the human insulin receptor (hIR) gene was constructed by replacing ros sequences in the avian sarcoma virus UR2 with hIR cDNA sequences coding for 46 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains of the beta subunit of hIR. The resulting virus, named UIR, contains the hIR sequence fused to the 5' portion of the UR2 gag gene coding for p19. UIR is capable of transforming chicken embryo fibroblasts and promoting formation of colonies in soft agar; however, it does not form tumors in vivo. A variant that arose from the parental UIR is capable of efficiently inducing sarcomas in vivo. UIR-transformed cells exhibit higher rates of glucose uptake and growth than normal cells. The 4-kilobase UIR genome codes for a membrane-associated, glycosylated gag-hIR fusion protein of 75 kDa designated P75gag-hir. P75gag-hir contains a protein tyrosine kinase activity that is capable of undergoing autophosphorylation and of phosphorylating foreign substrates in vitro; it is phosphorylated at both serine and tyrosine residues in vivo.
Expression of a complete soybean leghemoglobin gene in root nodules of transgenic
Lotus corniculatus
Authors: Stougaard Jens , Petersen Torben E. , Marcker Kjeld A.
Published: May 2006

The complete soybean leghemoglobin
lbc
3
gene was transferred into the legume
Lotus corniculatus
using an
Agrobacterium rhizogenes
vector system. Organ-specific expression of the soybean gene was observed in root nodules formed on regenerated transgenic plants after infection with
Rhizobium loti
. The primary transcript was processed in the same way as in soybean nodules and the resulting mRNA was translated into Lbc
3
protein. Quantitative determination of the Lbc
3
protein in nodules of transgenic plants indicated that the steady-state level of the soybean protein is comparable to that of endogenous
Lotus
leghemoglobin.
Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer disease.
Authors: van Duinen S G , Castaño E M , Prelli F , Bots G T , Luyendijk W , Frangione B
Published: May 2006

Hereditary cerebral hemorrhage with amyloidosis in Dutch patients is an autosomal dominant form of vascular amyloidosis restricted to the leptomeninges and cerebral cortex. Clinically the disease is characterized by cerebral hemorrhages leading to an early death. Immunohistochemical studies of five patients revealed that the vascular amyloid deposits reacted intensely with an antiserum raised against a synthetic peptide homologous to the Alzheimer disease-related beta-protein. Silver stain-positive, "senile plaque-like" structures were also labeled by the antiserum, yet these lesions lacked the dense amyloid cores present in typical plaques of Alzheimer disease. No neurofibrillary tangles were present. Amyloid fibrils were purified from the leptomeningeal vessels of one patient who clinically had no signs of dementia. The protein had a molecular weight of approximately equal to 4000 and its partial amino acid sequence to position 21 showed homology to the beta-protein of Alzheimer disease and Down syndrome. These results suggest that hereditary cerebral hemorrhage with amyloidosis of Dutch origin is pathogenetically related to Alzheimer disease and support the concept that the initial amyloid deposition in this disorder occurs in the vessel walls before damaging the brain parenchyma. Thus, deposition of beta-protein in brain tissue seems to be related to a spectrum of diseases involving vascular syndromes, progressive dementia, or both.
Type XII collagen: distinct extracellular matrix component discovered by cDNA cloning.
Authors: Gordon M K , Gerecke D R , Olsen B R
Published: May 2006

We have screened a cDNA library constructed from tendon fibroblast mRNA for the presence of collagenous coding sequences. Nucleotide sequence analysis of one isolated clone, pMG377, reveals that the clone encodes a polypeptide that is homologous to, yet distinctly different from, type IX short-chain collagen polypeptides. The structure of the conceptual translation product of the cDNA is also different from that of all other collagen types. Therefore, we have given the type IX-like collagen chain encoded by pMG377 the designation alpha 1(XII). Ribonuclease protection assays with single-stranded cRNA probes demonstrate that alpha 1(XII) mRNA is present in several tissues such as calvaria, tendon, and sternal cartilage of 17-day-old chicken embryo and in cornea from 6-day-old embryos. Using pMG377 as the hybridization probe, we isolated a fragment of the corresponding gene from a chicken genomic library. Partial nucleotide sequence analysis of the genomic clone DG12 shows that the exon/intron structure of the alpha 1(XII) collagen gene appears to be homologous to that of the alpha 1(IX) and alpha 2(IX) collagen genes. Our data demonstrate that types IX and XII collagen are two homologous members of a family of unique collagenous proteins that show tissue-specific patterns of expression. Based on their structure and the properties of their genes, we conclude that this family of collagens is distinctly different from that of fibrillar collagens.
Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of [Val181]CAP and [Leu181]CAP.
Authors: Ebright R H , Kolb A , Buc H , Kunkel T A , Krakow J S , Beckwith J
Published: May 2006

It has been proposed that Glu-181 of the catabolite gene activator protein (CAP) makes direct contact with certain base pairs of the specific DNA site. We have purified wild-type CAP and two substituted CAP variants, [Val181]CAP and [Leu181]CAP, and have assessed the DNA-sequence-recognition properties in vitro with respect to positions 5, 6, 7, 8, and 16 of the DNA site. The data indicate that [Val181]CAP and [Leu181]CAP fail to discriminate between the consensus DNA base pair and the three non-consensus-DNA base pairs at 2-fold-related positions 7 and 16 of the DNA site. In contrast, [Val181]CAP and [Leu181]CAP retain the ability to discriminate between different base pairs at positions 5 and 8 of the DNA site. We conclude that Glu-181 of CAP makes a direct contact with 2-fold-related positions 7 and 16 of the DNA site, as proposed previously based on in vivo results. We propose that upon replacement of Glu-181 by valine or leucine, this contact is eliminated and is replaced by no other functional contact. We estimate that the contact by Glu-181 with each position contributes -0.7 kcal/mol to the total CAP-DNA binding free energy.
Molecular dynamics simulations of fluorescence polarization of tryptophans in myoglobin.
Authors: Henry E R , Hochstrasser R M
Published: May 2006

The fluorescence of heme proteins is influenced by energy transfer from the excited tryptophan to the heme. Molecular dynamics simulations of the tryptophan and heme motions in sperm whale myoglobin were used to calculate the fluorescence intensity and anisotropy decays. The side chains underwent both small rapid orientational fluctuations and large infrequent transitions between conformations. The predicted motions of the tryptophans and the heme produce large fluctuations in the instantaneous rate of energy transfer, but no stable conformations in which energy transfer is suppressed were found. The calculated fluorescence anisotropies exhibited a large subpicosecond decay, corresponding to nondiffusive side-chain motions. The calculations adequately predict the observed fluorescence decay curve for myoglobin and the total anisotropy decay at 16-ps time resolution. The subnanosecond decays of anisotropy for tryptophan-14 in tuna myoglobin are not reproduced by the calculation.
Cotranslational assembly of myosin heavy chain in developing cultured skeletal muscle.
Authors: Isaacs W B , Fulton A B
Published: May 2006

To examine how nascent myosin heavy chains associate with the cytoskeletons of developing muscle cells, we used pulse labeling, cell fractionation, and immunoprecipitation. More than 80% of nascent myosin heavy chains associate with the cytoskeleton. More than one-third of these nascent chains are not released by puromycin and/or RNase. The fraction of nascent heavy chains that resists release increases during development of muscle cells in culture. Treatment with cytochalasin D but not nocodazole decreases myosin heavy chain cotranslational assembly. These results indicate that (i) cotranslational assembly of myosin heavy chains is developmentally regulated, (ii) structures containing actin and not microtubules may mediate initial association of the heavy chains with the cytoskeleton, and (iii) the site of translation dictates where a significant fraction of the heavy chains will be inserted into the cytoskeleton.
Reconstituted voltage-sensitive sodium channel from Electrophorus electricus: chemical modifications that alter regulation of ion permeability.
Authors: Cooper E C , Tomiko S A , Agnew W S
Published: May 2006

At equilibrium, voltage-sensitive sodium channels normally are closed at all potentials. They open transiently in response to changes in membrane voltage or chronically under the influence of certain neurotoxins. Covalent modifications that result in chronic opening may help identify molecular domains involved in conductance regulation. Here, the purified sodium channel from electric eel electroplax, reconstituted in artificial liposomes, has been used to screen for such modifications. When the liposomes were treated with the alkaloid neurotoxin batrachotoxin, sodium-selective ion fluxes were produced, with permeability ratios PNa greater than PTl greater than PK greater than PRb greater than PCs. When the liposomes were treated with either of two oxidizing reagents (N-bromoacetamide or N-bromosuccinimide), or with Pronase or trypsin, ion-selective fluxes also were stimulated. These were blocked by tetrodotoxin and the anesthetic QX-314 in a manner suggesting that only modification of the cytoplasmic protein surface resulted in stimulation. Limited exposure to trypsin resulted in strong flux activation, with the concomitant appearance of peptide fragments with masses of approximately equal to 130, 70, and 38 kDa and fragments with masses of 45 and 24 kDa appearing later. We propose that characterization of these fragments may allow identification of channel domains important for inactivation gating.
Extensive amino acid sequence homologies between animal lectins.
Authors: Paroutaud P , Levi G , Teichberg V I , Strosberg A D
Published: May 2006

We have established the amino acid sequence of the beta-D-galactoside binding lectin from the electric eel and the sequences of several peptides from a similar lectin isolated from human placenta. These sequences were compared with the published sequences of peptides derived from the beta-D-galactoside binding lectin from human lung and with sequences deduced from cDNAs assigned to the beta-D-galactoside binding lectins from chicken embryo skin and human hepatomas. Significant homologies were observed. One of the highly conserved regions that contains a tryptophan residue and two glutamic acid residues is probably part of the beta-D-galactoside binding site, which, on the basis of spectroscopic studies of the electric eel lectin, is expected to contain such residues. The similarity of the hydropathy profiles and the predicted secondary structure of the lectins from chicken skin and electric eel, in spite of differences in their amino acid sequences, strongly suggests that these proteins have maintained structural homologies during evolution and together with the other beta-D-galactoside binding lectins were derived from a common ancestor gene.
Glucose transport and antilipolysis are differentially regulated by the polar head group of an insulin-sensitive glycophospholipid.
Authors: Kelly K L , Mato J M , Merida I , Jarett L
Published: May 2006

A glycophospholipid has been purified from rat liver membranes, which copurified with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid, which is a phosphooligosaccharide, was generated by treatment with a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. There was an "insulin-like" inhibitory effect of this phosphooligosaccharide on isoproterenol-stimulated lipolysis in adipocytes, whereas there was no effect on glucose oxidation under conditions that measure glucose transport. The antilipolytic effect of this phosphooligosaccharide was demonstrated in intact adipocytes. There was a linear correlation between the concentration of phosphooligosaccharide and its antilipolytic effect, the magnitude and time course of which were similar to that obtained with physiological concentrations of insulin. Submaximal concentrations of insulin and phosphooligosaccharide produced an additive antilipolytic effect. The antilipolytic effect of the phosphooligosaccharide was demonstrated only after release of this compound from the precursor glycophospholipid with phosphatidylinositol-specific phospholipase C, and the activity of the phosphooligosaccharide was sensitive to alkali. It is proposed that this phosphooligosaccharide plays a role in mediating certain insulin actions.
Promoter trans-activation of protooncogenes c-fos and c-myc, but not c-Ha-ras, by products of adenovirus early region 1A.
Authors: Sassone-Corsi P , Borrelli E
Published: May 2006

The E1A (early region 1A) oncogene products of adenovirus type 2 trans-activate the other early viral transcription units, as well as some cellular promoters. Using a short-term cotransfection assay in murine NIH 3T3 fibroblasts, we show that c-fos and c-myc promoter activities are stimulated by the E1A proteins, whereas c-Ha-ras transcription is not affected. The product of E1A 13S mRNA is responsible for the trans-activation, whereas the 12S mRNA product has no effect. Analysis of the c-fos promoter sequences required for the E1A stimulation shows that responsive sequences are located between positions -402 and -240 upstream of the transcription initiation site. This same region also contains the c-fos serum-responsive element. Furthermore, transcription of the endogenous c-fos gene in HeLa cells is increased after E1A transfection.
Molecular cloning of complementary DNAs encoding the heavy chain of the human 4F2 cell-surface antigen: a type II membrane glycoprotein involved in normal and neoplastic cell growth.
Authors: Quackenbush E , Clabby M , Gottesdiener K M , Barbosa J , Jones N H , Strominger J L , Speck S , Leiden J M
Published: May 2006

Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a lambda gt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggests that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution.
Treatment of type 1 diabetes mellitus in non-obese diabetic mice by transplantation of allogeneic bone marrow and pancreatic tissue.
Authors: Yasumizu R , Sugiura K , Iwai H , Inaba M , Makino S , Ida T , Imura H , Hamashima Y , Good R A , Ikehara S
Published: May 2006

Non-obese diabetic (NOD) mice provide a model for type 1 diabetes mellitus. We previously showed that allogeneic bone marrow transplantation (ABMT) can prevent and treat insulitis and overt diabetes in NOD mice. However, ABMT alone could not be used to treat overt diabetes in NOD mice whose islets had been completely destroyed. To provide insulin-producing cells, pancreatic tissue from newborn mice was grafted under the renal capsules in combination with ABMT. The aims of concomitant ABMT are as follows. (i) It induces immunological tolerance to the donor-type major histocompatibility complex determinants and permits the host to accept subsequent pancreatic allografts from the bone marrow donor. (ii) ABMT replaces abnormal stem cells with normal stem cells. After transplantation of bone marrow plus newborn pancreas, NOD mice showed reduction of the glycosuria and a normal response in the glucose-tolerance test. Immunohistological study revealed the presence of clustered insulin-containing beta cells in the grafted pancreatic transplants. ABMT may become a viable treatment of established type 1 diabetes mellitus in humans.
In vitro transformation of immature hematopoietic cells by the P210 BCR/ABL oncogene product of the Philadelphia chromosome.
Authors: McLaughlin J , Chianese E , Witte O N
Published: May 2006

The Philadelphia chromosome [t(9;22)-(q34;q11)] is the cytogenetic hallmark of human chronic myelogenous leukemia. RNA splicing joins sequences from a gene on chromosome 22 (BCR) across the translocation breakpoint to a portion of the ABL oncogene from chromosome 9, resulting in a chimeric protein (P210) that is an active tyrosine kinase. Although strongly correlated with this specific human neoplasm, and implicated as an oncogene by analogy to the gene product of the Abelson murine leukemia virus, the P210 gene had not been tested directly for oncogenic potential in hematopoietic cells. We have used a retroviral gene-transfer system to express P210 in mouse bone marrow cells. When infected bone marrow is plated under conditions for long-term culture of cells of the B-lymphoid lineage, cells expressing high amounts of P210 tyrosine kinase dominate the culture and rapidly lead to clonal outgrowths of immature lymphoid cells. Expression of P210 is growth-stimulatory but not sufficient for full oncogenic behavior. Some clonal lines progress toward a fully malignant phenotype as judged by increased cloning efficiency in agar suspension and frequency and rapidity of tumor induction in syngeneic mice. Such in vitro systems should be useful in evaluating the sequential and perhaps synergistic involvement of the P210 gene and other oncogenes as models for the progressive changes observed in human chronic myelogenous leukemia.
Both barium and calcium activate neuronal potassium currents.
Authors: Ribera A B , Spitzer N C
Published: May 2006

Amphibian spinal neurons in culture possess both rapidly inactivating and sustained calcium-dependent potassium current components, similar to those described for other cells. Divalent cation-dependent whole-cell outward currents were isolated by subtracting the voltage-dependent potassium currents recorded from Xenopus laevis neurons in the presence of impermeant cadmium (100-500 microM) from the currents produced without cadmium but in the presence of permeant divalent cations (50-100 microM). These concentrations of permeant ions were low enough to avoid contamination by macroscopic inward currents through calcium channels. Calcium-dependent potassium currents were reduced by 1 microM tetraethylammonium. These currents can also be activated by barium or strontium. Barium as well as calcium activated outward currents in young neurons (6-8 hr) and in relatively mature neurons (19-26 hr in vitro). However, barium influx appeared to suppress the sustained voltage-dependent potassium current in most cells. Barium also activated at least one class of potassium channels observed in excised membrane patches, while blocking others. The blocking action may have masked and hindered detection of the stimulatory action of barium in other systems.
Isolation and characterization of a cDNA clone for the Cat2 gene in maize and its homology with other catalases.
Authors: Bethards L A , Skadsen R W , Scandalios J G
Published: May 2006

A 230-base-pair cDNA representing the 3' end of the Cat2 gene in maize was isolated and used to screen a lambda gt11 cDNA library made from scutellar poly(A)+ RNA. Several clones were subcloned into a pUC12 vector; one of the subclones, pCat2.1c, appears to represent a near-complete sequence of the Cat2 coding region. Immunological screening, hybridization-selection translation, and RNA blot analysis confirmed that pCat2.1c was derived from the Cat2 transcript. The DNA sequence of pCat2.1c was determined. The deduced amino acid sequence shows homology with catalase amino acid sequences from rat liver, bovine liver, human kidney, and yeast.
The t(8;14) chromosome translocation of the Burkitt lymphoma cell line Daudi occurred during immunoglobulin gene rearrangement and involved the heavy chain diversity region.
Authors: Haluska F G , Tsujimoto Y , Croce C M
Published: May 2006

Recent molecular analyses of Burkitt lymphomas carrying the t(8;14) chromosome translocation have indicated that a dichotomy exists regarding the molecular mechanisms by which the translocations occur. Most sporadic Burkitt tumors carry translocations that apparently arise due to mistakes in the immunoglobulin isotype-switching process. In contrast, there is evidence that the translocations of most endemic Burkitt lymphomas occur as a consequence of aberrant V-D-J recombination of variable, diversity, and joining gene segments, catalyzed by the recombinase enzymes. This phenomenon was first noted in follicular lymphomas and chronic lymphocytic leukemias of the B-cell lineage and has been described in T-cell malignancies as well. In each of these cases, analysis of the nucleotide sequence at chromosome breakpoints demonstrated the involvement of immunoglobulin heavy chain JH or T-cell-receptor alpha-chain J alpha gene segments in the translocation. We now have cloned and sequenced both the 8q- and 14q+ translocation breakpoints deriving from the t(8;14) translocation of the endemic Burkitt lymphoma line Daudi. Our data show that the translocation resulted from a reciprocal exchange between the DH region on chromosome 14 and sequences far 5' of the MYC protooncogene on chromosome 8. Features of the nucleotide sequences surrounding the breakpoint further implicate the V-D-J joining machinery in the genesis of chromosome translocations in endemic Burkitt lymphomas and, more generally, in other lymphoid malignancies as well.
Effective tumor immunotherapy directed against an oncogene-encoded product using a vaccinia virus vector.
Authors: Bernards R , Destree A , McKenzie S , Gordon E , Weinberg R A , Panicali D
Published: May 2006

We have constructed a vaccinia virus recombinant that expresses the extracellular domain of the rat neu oncogene-encoded protein, a 185-kDa transmembrane glycoprotein termed p185. Strain NFS mice immunized with this recombinant virus developed a strong antibody response against the neu oncogene product and were fully protected against subsequent tumor challenge with neu-transformed NIH 3T3 cells. No tumor immunoprotection was found when recombinant virus-immunized mice were challenged with Ha-ras-transformed NIH 3T3 cells. These data indicate that immunization with a single oncogene-encoded antigen can fully and specifically protect animals against tumor cells bearing this antigen.
An IgE-binding protein with a distinctive repetitive sequence and homology with an IgG receptor.
Authors: Albrandt K , Orida N K , Liu F T
Published: May 2006

Proteins that bind IgE play important roles in both the synthesis and function of IgE are therefore intimately involved in IgE-mediated human allergic disorders. This report describes the structure of an IgE-binding protein, as predicted from sequencing a cDNA cloned from rat basophilic leukemia cells. This protein contains two domains: the amino-terminal domain (140 amino acids) consists of a highly conserved repetitive amino acid sequence, Tyr-Pro-Gly-Pro/Gln-Ala/Thr-Pro/Ala-Pro-Gly-Ala, whereas the carboxyl-terminal domain (122 amino acids) shares significant sequence homology with a domain of lymphocyte/macrophage receptor for the Fc portion of IgG. Other proteins with this type of structure but with affinity for other immunoglobulin isotypes may exist and may represent a heretofore unidentified component of the immune system.
Interleukin 4 (B-cell stimulatory factor 1) can enhance or antagonize the factor-dependent growth of hemopoietic progenitor cells.
Authors: Rennick D , Yang G , Muller-Sieburg C , Smith C , Arai N , Takabe Y , Gemmell L
Published: May 2006

Our studies show that although interleukin 4 (IL-4) fails to stimulate significant colony formation by bone marrow progenitor cells, it enhances erythroid, granulocyte, macrophage, and mast-cell colony formation when used as a costimulant with erythropoietin, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and interleukin 3 (IL-3), respectively. In contrast, IL-4 suppresses IL-3-dependent colony formation by granulocyte and macrophage progenitor cells and by multipotential progenitor cells. Furthermore, it appears to inhibit the in vitro generation of colony-forming progenitor cells from immature IL-3-dependent stem cells. We also found that IL-4 inhibits stromal cell-dependent growth of bone marrow-derived pre-B cells. The ability of IL-4 to directly or indirectly regulate both positive and negative aspects of progenitor cell growth is discussed.
Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells.
Authors: Rusche J R , Lynn D L , Robert-Guroff M , Langlois A J , Lyerly H K , Carson H , Krohn K , Ranki A , Gallo R C , Bolognesi D P
Published: May 2006

The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120.
Target-controlled differentiation of axon terminals and synaptic organization.
Authors: Campbell G , Frost D O
Published: May 2006

These experiments investigate the processes regulating the morphological differentiation of synaptic connections. Electron microscopy showed that the terminal boutons and synaptic complexes of retinal afferent axons in the main thalamic visual nucleus, the dorsal lateral geniculate nucleus, differ in their morphology from those of ascending afferent axons in the main thalamic somatosensory (ventrobasal) nucleus. Developing retinal ganglion cell axons in hamsters were made to project permanently to the ventrobasal nucleus, rather than to the lateral geniculate nucleus. With respect to most of the ultrastructural features examined, the terminals and synaptic complexes of mature, anterogradely labeled retino-ventrobasal axons more closely resembled those of normal somatosensory afferents to the ventrobasal nucleus than they did those of normal retinofugal axons within the lateral geniculate nucleus. These results suggest that the ultrastructural differentiation of axon terminals and synaptic complexes is regulated largely by the target environment, although some features appear to be intrinsic to the afferent axons themselves.
Direct neurotrophic action of glycyl-L-glutamine in the maintenance of acetylcholinesterase and butyrylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.
Authors: Koelle G B , Sanville U J , Thampi N S
Published: May 2006

Intracarotid infusion of glycyl-L-glutamine (Gly-Gln) was shown previously to oppose the fall in the acetylcholinesterase and butyrylcholinesterase contents of the cat superior cervical ganglion (SCG) that otherwise follows preganglionic denervation. However, its effect was demonstrable only on the vascularly remote left SCG but not on the directly infused right SCG. Accordingly, it was concluded that a metabolite of Gly-Gln, formed in the blood, is an active neurotrophic factor. Glycyl-L-glutamic acid and L-glutamic acid were subsequently found to have a similar but less marked effect on both SCG. In the present study an alternative explanation has been tested: that Gly-Gln must combine slowly with some component of plasma to enable it to penetrate the ganglion cells and exert its neurotrophic effect. Findings are consistent with the latter proposal.
Covalent structure of biodegradative threonine dehydratase of Escherichia coli: homology with other dehydratases.
Authors: Datta P , Goss T J , Omnaas J R , Patil R V
Published: May 2006

The 987-base-pair coding region of the tdc gene of Escherichia coli K-12 encoding biodegradative threonine dehydratase [Tdc; L-threonine hydro-lyase (deaminating), EC 4.2.1.16], previously cloned in this laboratory, was sequenced. The deduced polypeptide consists of 329 amino acid residues with a calculated Mr of 35,238. Although the purified enzyme was shown to contain tryptophan, no tryptophan codon was found in the tdc reading frame. Incubation of purified Tdc with [14C]tryptophan revealed apparent "covalent" binding of tryptophan, indicating posttranslational modification of the enzyme. A heptapeptide, 54Thr-55Gly-56Ser-57Phe-58Lys-59Ile- 60Arg, was found to contain Lys-58, which binds pyridoxal phosphate coenzyme. A comparison of amino acid sequences between the Tdc polypeptide and the biosynthetic threonine dehydratases of yeast (encoded by ILV1) and E. coli (encoded by ilvA) and the E. coli D-serine dehydratase (DsdA, encoded by dsdA) revealed various extents of homology: five domains of the Tdc polypeptide were 63-93% homologous with the yeast enzyme, and three of these same regions were 80% homologous with the biosynthetic E. coli dehydratase; two different domains showed 67% and 83% homology with DsdA. In addition, two other sequences were highly conserved in all four proteins, one of which was shown to contain the conserved lysine residue that binds pyridoxal phosphate in the Tdc and DsdA polypeptides. These observations suggest that, despite their diverse origin and metabolic significance, these enzymes may have evolved from a common ancestral protein.
Tumor necrosis factor inhibits MYC expression in HL-60 cells at the level of mRNA transcription.
Authors: Krönke M , Schlüter C , Pfizenmaier K
Published: May 2006

Highly purified, recombinant human tumor necrosis factor type alpha (rTNF-alpha) exerts cytostatic activity on the human promyelocytic leukemic cell line HL-60. Between 4 and 10 hr after treatment with rTNF-alpha, HL-60 cells start to exhibit phagocytic activity, indicating the induction of a differentiation process, and show a marked decline in steady-state levels of MYC mRNA. In vitro transcription assays reveal that rTNF-alpha inhibits MYC gene expression at the level of transcription. rTNF-alpha has little or no effect on the half-life of MYC mRNA, indicating that the decreased MYC mRNA level is primarily due to reduced transcription, and the protein synthesis inhibitor cycloheximide does not prevent rTNF-alpha-mediated inhibition of MYC gene expression, suggesting that rTNF-alpha action is independent of de novo protein synthesis. In contrast to MYC, HLA-A, -B, -C mRNA levels are slightly increased in rTNF-alpha-treated HL-60 cells. Recombinant interferon-gamma, which also enhances HLA gene transcription in HL-60 cells, fails to reduce MYC mRNA levels and lacks cytostatic activity. Attenuation of HL-60 cell proliferation occurs as a late response to treatment with rTNF-alpha and thus may be secondary to both MYC inhibition and induction of cell differentiation.
A locus for a human hereditary cataract is closely linked to the gamma-crystallin gene family.
Authors: Lubsen N H , Renwick J H , Tsui L C , Breitman M L , Schoenmakers J G
Published: May 2006

Within the human gamma-crystallin gene cluster polymorphic Taq I sites are present. These give rise to three sets of allelic fragments from the gamma-crystallin genes. Together these restriction fragment length polymorphisms define eight possible haplotypes, three of which (Q, R, and S) were found in the Dutch and English population. A fourth haplotype (P) was detected within a family in which a hereditary Coppock-like cataract of the embryonic lens nucleus occurs in heterozygotes. Haplotype P was found only in family members who suffered from cataract, and all family members who suffered from cataract had haplotype P. The absolute correlation between the presence of haplotype P and cataract within this family shows that the gamma-crystallin gene cluster and the locus for the Coppock-like cataract are closely linked [logarithm of odds (lod) score of 7.58 at its maximum at phi = 0]. This linkage provides genetic evidence that the primary cause of a cataract in humans could possibly be a lesion in a crystallin gene.