bluuebunny/crossref_metadata_2025 huggingface dataset:
Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains.
Authors: Porter T D , Kasper C B
Published: May 2006

The coding nucleotide sequence of the mRNA for NADPH-cytochrome P-450 oxidoreductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) from rat liver was determined from two overlapping cDNA clones, pOR-7 and pOR-8, which together contain 2401 nucleotides complementary to rat liver oxidoreductase mRNA. The single open reading frame of 2034 nucleotides spanning these cDNAs codes for a 678 amino acid polypeptide with a molecular weight of 76,962. The deduced amino acid composition is in excellent agreement with that determined by direct amino acid analysis of purified rat liver P-450 oxidoreductase, and the amino-terminal region (residues 1-80) largely coincides with the amino-terminal sequence of the oxidoreductase isolated from rabbit liver. Comparison of the amino acid sequence to those of other flavoproteins revealed two separate domains that are likely to be involved in flavin binding: a long segment (residues 77-228) homologous with Desulfovibrio vulgaris flavodoxin, an FMN-containing protein, and a shorter segment (residues 452-477) homologous with the FAD-binding segment of fumarate reductase from Escherichia coli.
Stimulation of glucose uptake by transforming growth factor beta: evidence for the requirement of epidermal growth factor-receptor activation.
Authors: Inman W H , Colowick S P
Published: May 2006

Transforming growth factor beta (TGF-beta), derived from human platelets, stimulates the uptake of 2-deoxy-glucose by cultured cell monolayers 2- to 4-fold. Stimulation can be detected as early as 30 min with as little as 0.1 ng of TGF-beta per ml and maximal effects can be obtained at 2 hr with 1 ng of the growth factor per ml. TGF-beta-induced stimulation of sugar uptake is enhanced by the co-addition of platelet-derived growth factor (10 ng/ml) or epidermal growth factor (EGF, 1 ng/ml). The NR-6 variant of mouse 3T3 cells, which lack EGF receptors, is not stimulated by TGF-beta. Antisera to EGF receptors that block 125I-labeled EGF binding also inhibit TGF-beta stimulation of 2-deoxyglucose uptake, although 125I-labeled TGF-beta binding remains unimpaired. In contrast, anti-sera to the EGF receptor, which do not block EGF binding, have no measurable effect on the TGF-beta-stimulated uptake of 2-deoxyglucose. We confirm that the receptor for TGF-beta is distinct from the receptor for EGF and we conclude that TGF-beta stimulation of 2-deoxyglucose uptake requires the co-activation of the EGF receptor kinase system.
Stereochemical studies of a selenium-containing hydrogenase from Methanococcus vannielii: determination of the absolute configuration of C-5 chirally labeled dihydro-8-hydroxy-5-deazaflavin cofactor.
Authors: Yamazaki S , Tsai L , Stadtman T C , Teshima T , Nakaji A , Shiba T
Published: May 2006

Reduction of 7,8-didemethyl-8-hydroxy-[5-2H]-5-deazariboflavin by the selenium-containing hydrogenase from Methanococcus vannielii gave a C-5 chirally labeled 1,5-dihydro derivative. The absolute configuration of the chiral label was shown to be (R) by comparison of the chemically degraded product with authentic samples of known absolute configurations. Therefore, the steric course of the enzymic reactions involving the 8-hydroxy-5-deazaflavin cofactor can be defined as follows: (a) reduction occurs on the si face of the 5-deazaflavin molecule; (b) oxidation proceeds by the abstraction of the pro-S hydrogen at C-5 of the 1,5-dihydro-5-deazaflavin. Thus, the selenium-containing hydrogenase and 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from M. vannielii are si face specific.
Loss of polymorphic restriction fragments in malignant melanoma: implications for tumor heterogeneity.
Authors: Dracopoli N C , Houghton A N , Old L J
Published: May 2006

Loss of genetic material at certain chromosomal sites is implicated in the etiology of retinoblastoma and Wilms tumor. Whether specific chromosomal deletions are associated with other types of human cancer needs to be explored. We have examined 24 melanoma cell lines, derived from 21 patients with nonfamilial malignant melanoma, for evidence of somatically induced hemizygosity or homozygosity. Twelve DNA probes, recognizing single-copy restriction fragment length polymorphisms (RFLP) determined by loci on 11 different chromosomes, were used to screen autologous combinations of melanoma cells and either B cells or fibroblasts. Loss of heterozygosity in melanoma cells was identified at 27 of 100 informative loci. These losses occurred at loci on 8 different chromosomes, and the frequency of loss at individual loci varied between 8% and 67%. We conclude that somatic mutations resulting in homozygosity or hemizygosity are common in melanoma and evidently not restricted to specific chromosomes.
Dissociation from albumin: a potentially rate-limiting step in the clearance of substances by the liver.
Authors: Weisiger R A
Published: May 2006

The hepatic uptake rate for certain albumin-bound drugs and metabolites correlates poorly with their equilibrium unbound concentration in the plasma, suggesting that binding equilibrium may not always exist within the hepatic sinusoids. Currently available models for the uptake process assume binding equilibrium and, thus, cannot be used to investigate this possibility. This report presents a more general model that treats plasma-bound and free concentrations separately. A solution is provided that specifies the hepatic uptake rate as a function of the total plasma concentrations of the transported substance and of binding protein and the rate constants for influx, efflux, elimination, association, dissociation, and flow. Analysis of this solution indicates that hepatic uptake may be limited by the rate of plasma flow, dissociation from the binding protein, influx into the liver, cellular elimination, or any combination of these processes. The affinity and concentration of the binding protein strongly influence which of these steps are rate-limiting in any given case, and binding equilibrium exists within the hepatic sinusoids only for binding protein concentrations greater than a specified value (the ratio of the uptake and association rate constants). The precise conditions under which each step is rate-limiting and the kinetic behavior expected when two or more steps mutually limit uptake are provided. The results are compatible with previously reported data for the uptake of certain albumin-bound ligands such as bilirubin, and they offer an alternative to attributing these kinetics to the presence of an albumin receptor.
Cloning and nucleotide sequence determination of the Clostridium pasteurianum ferredoxin gene.
Authors: Graves M C , Mullenbach G T , Rabinowitz J C
Published: May 2006

We have constructed a library of Clostridium pasteurianum DNA cloned in the plasmid pBR322. Based on the known amino acid sequence for C. pasteurianum ferredoxin, a 64-fold degenerate heptadecanucleotide pool was synthesized. This mixed probe hybridized to two clones which were shown to contain greater than 6 kilobase pairs of the same genomic DNA. Sequence analysis of a common Sau3A1 0.6-kilobase-pair fragment revealed that it contains the information for the apoferredoxin structural gene. According to the DNA sequence, the only post-translational processing of this small apoprotein is the hydrolysis of the initiator methionine. Putative transcription and translation start and stop signals are present within the sequence.
Molecular genetics of a transposon-induced dominant mutation in the Drosophila locus Glued.
Authors: Swaroop A , Paco-Larson M L , Garen A
Published: May 2006

The organization of the Drosophila locus Glued containing the dominant allele Gl was shown to differ from that of the normal locus by an insertion of a 9-kilobase-pair DNA segment near the 3' end of a transcribed region. The insertion causes the formation of a truncated polyadenylylated transcript of 5.1 kilobases instead of the normal 6.0 kilobases. The inserted DNA segment has the properties of a transposon and was identified by its corresponding restriction map as B104, which is a retrovirus-like transposon with direct terminal repeats. B104 appears to be oriented in Gl with the same polarity of transcription as Gl. The truncated Gl transcript terminates prematurely inside the 5' terminal repeat of B104, in the region of a putative polyadenylylation signal. We discuss the general implications of this finding for transposon- and retrovirus-induced mutagenesis and for the origin of dominant mutations.
Role of the Bp35 cell surface polypeptide in human B-cell activation.
Authors: Clark E A , Shu G , Ledbetter J A
Published: May 2006

A 35-kDa polypeptide, Bp35, expressed on the surface of all B cells, plays a role in B-cell activation. Monoclonal antibodies to Bp35 stimulate human tonsillar B cells to proliferate. The activation induced by anti-Bp35 is similar to anti-Ig-mediated in several ways: the activation does not require T cells but is augmented by T-cell-derived allogeneic factors; monovalent Fab fragments to Bp35 do not trigger proliferation but instead block activation by whole antibody, indicating that cross-linking is required; and induction by anti-Bp35, like the induction by anti-Ig, is inhibited by monoclonal anti-IgM via an Fc domain-dependent mechanism. However, several features of anti-Bp35-mediated proliferation are clearly different from activation by anti-Ig: anti-Bp35 monoclonal antibodies do not require attachment to beads to function, the proliferation induced by anti-Bp35 and anti-Ig is additive, and Fab fragments of anti-Bp35 augment proliferation induced by anti-Ig. Models for the possible function of the Bp35 polypeptide as either a "bridge" or a "second signal" with surface Ig in B-cell activation are discussed.
Protection against gram-negative bacteremia and endotoxemia with human monoclonal IgM antibodies.
Authors: Teng N N , Kaplan H S , Hebert J M , Moore C , Douglas H , Wunderlich A , Braude A I
Published: May 2006

Hybridomas producing human monoclonal IgM antibodies (mAbs) against bacterial lipopolysaccharide (LPS) were generated by fusion of B lymphocytes from sensitized human spleen with heteromyeloma cells. The splenocytes were from patients undergoing splenectomy during staging for Hodgkin disease after vaccination with the J5 mutant of Escherichia coli, which is deficient in O antigenic side chains. This deficiency exposes the core oligosaccharide, common to LPS of all Gram-negative bacteria. The mAbs cross-reacted strongly with endotoxins from a wide range of unrelated species of Gram-negative bacteria. The mAbs also gave strong protection against LPS in the dermal Shwartzman reaction and against lethal Gram-negative bacteremia in mice. These findings indicate that monoclonal IgM against LPS endotoxin can neutralize its toxicity in vivo and might be valuable for treatment of patients with Gram-negative bacteremia. Analysis of one of the hybridoma clones, A6(H4C5), showed that the IgM mAb is directed against the covalently bound lipid A, which represents the most conservative and least variable structural element of LPS.
Abnormal polyunsaturated fatty acid patterns of serum lipids in alcoholism and cirrhosis: arachidonic acid deficiency in cirrhosis.
Authors: Johnson S B , Gordon E , McClain C , Low G , Holman R T
Published: May 2006

Patterns of polyunsaturated fatty acids of serum phospholipids were measured for groups of alcoholics without cirrhosis, alcoholics with cirrhosis, cirrhotics without alcoholism, and a control population. Alcoholics without cirrhosis showed increased polyunsaturated fatty acids derived from linoleic and linolenic acids, but in cirrhotics these products were decreased. Alcoholism accentuated the abnormal polyunsaturated fatty acid pattern of cirrhosis. In alcohol abuse without cirrhosis, the level of 20:3 omega 9 (20 acyl carbon atoms:3 double bonds, omega, 9 carbon atoms beyond last double bond) was significantly increased, despite adequate levels of linoleic and arachidonic acids. Liver involvement appears necessary for development of deficiencies of polyunsaturated fatty acids in serum phospholipids, of which arachidonic acid deficiency is of the largest magnitude.
Mu and kappa opioids inhibit transmitter release by different mechanisms.
Authors: Cherubini E , North R A
Published: May 2006

The actions of various opioids were examined on calcium action potentials in the cell somata of guinea pig myenteric neurones and on the release of acetylcholine at synapses onto these cells. The opioids morphine, normorphine, and [D-Ala2, MePhe4, Met5(O)]enkephalin-ol caused membrane hyperpolarizations resulting from an increase in potassium conductance; opioids that are more selective agonists for the kappa receptor subtype (dynorphin, tifluadom, U50488H) did not. Conversely, calcium action potentials were depressed or abolished by the kappa opioids but were not affected by morphine and [D-Ala2, MePhe4, Met(O)5]enkephalin-ol. Both groups of opioids caused presynaptic inhibition of acetylcholine release in the myenteric plexus, depressing the amplitude of the fast excitatory postsynaptic potential. The presynaptic inhibition caused by [D-Ala2, MePhe4, Met(O)5]enkephalin-ol, morphine, and normorphine, but not that caused by the kappa opioids, was prevented by pretreatment with the selective mu site-directed irreversible antagonist beta-funaltrexamine. Furthermore, the presynaptic inhibitory action of morphine and [D-Ala2, MePhe4, Met(O)5]enkephalin-ol, but not that of the kappa-receptor agonists, was reversibly blocked by barium. The results suggest that presynaptic inhibition caused by mu receptor activation probably results from an increase in potassium conductance, whereas kappa-receptor agonists may depress the release of acetylcholine by directly reducing calcium entry into the nerve terminals.
Epidermal growth factor precursor is related to the translation product of the Moloney sarcoma virus oncogene mos.
Authors: Baldwin G S
Published: May 2006

Murine epidermal growth factor (EGF) is synthesized as part of a large precursor (pro-EGF), which is thought to span the cell membrane. Comparison of the published pro-EGF sequence with the sequences of the translation products of viral oncogenes reveals that pro-EGF is related to the translation product of mos, the oncogene of Moloney murine sarcoma virus. Similarity is greatest between the COOH-terminal region of v-mos (residues 317-360) and part of the cytoplasmic domain of pro-EGF (residues 1127-1174). Statistical comparison of these sequences indicates that the probability of the similarity arising by chance is less than 2 X 10(-8). This similarity extends to the corresponding regions of the translation products of the cellular homologues (c-mos) of the v-mos gene present in normal murine and human DNA. Similarities are also observed between two other regions of the murine c-mos sequence (residues 48-134 and 196-275) and parts of the extracellular domain of pro-EGF (residues 565-651 and 741-817, respectively). All three mos genes are members of the tyrosine kinase family of oncogenes, as is erbB, the oncogene of avian erythroblastosis virus. Since the sequences of the erbB translation product and the EGF receptor are closely related, the relationship between mos and pro-EGF suggests that pro-EGF and the EGF receptor have evolved from a common ancestor.
Chromatin structure around the c-myc gene in Burkitt lymphomas with upstream and downstream translocation points.
Authors: Dyson P J , Rabbitts T H
Published: May 2006

Burkitt lymphoma cells seem to have abnormal c-myc gene activity resulting from chromosomal translocation. We have examined the consequences of translocation on putative control sequences near to the c-myc gene by DNase I hypersensitivity mapping of chromatin. There is no detectable difference in the pattern of hypersensitivity (compared with the actively transcribed c-myc gene of lymphoblastoid cells) in Burkitt lymphoma cells where the translocation point occurs at a considerable distance upstream or downstream of c-myc. When the translocation occurs near the 5' end of the c-myc gene, resulting in loss of hypersensitive sites, those that remain show the same sensitivity as in lymphoblastoid cell lines. We conclude that translocation has little general effect on the usual pattern of hypersensitive sites near to the c-myc gene but new sites can be observed in some cases in the immunoglobulin region near to the breakpoint. These may be sites normally involved in immunoglobulin gene transcription and may exert a subtle effect on the translocated c-myc gene.
One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription.
Authors: Bertholet C , Drillien R , Wittek R
Published: May 2006

A vaccinia virus late gene coding for a major structural polypeptide of 11 kDa was sequenced. Although the 5' flanking gene region is very A+T rich, it shows little homology either to the corresponding region of vaccinia early genes or to consensus sequences characteristic of most eukaryotic genes. Three DNA fragments (100, 200, and 500 base pairs, respectively), derived from the flanking region and including the late gene mRNA start site, were inserted into the coding sequence of the vaccinia virus thymidine kinase (TK) early gene by homologous in vivo recombination. Recombinants were selected on the basis of their TK- phenotype. Cells were infected with the recombinant viruses and RNA was isolated at 1-hr intervals. Transcripts initiating either from the TK early promoter, or from the late gene promoter at its authentic position, or from the translocated late gene promoters within the early gene were detected by nuclease S1 mapping. Early after infection, only transcripts from the TK early promoter were detected. Later in infection, however, transcripts were also initiated from the translocated late promoters. This RNA appeared at the same time and in similar quantities as the RNA from the late promoter at its authentic position. No quantitative differences in promoter efficiency between the 100-, 200-, and 500-base-pair insertions were observed. We conclude that all necessary signals for correct regulation of late-gene expression reside within only 100 base pairs of 5' flanking sequence.
Isolation and analysis of cDNA clones expressing human lupus La antigen.
Authors: Chambers J C , Keene J D
Published: May 2006

Several cDNA clones of the La antigen recognized by certain lupus autoantibodies were isolated from lambda gt11 expression libraries made from human liver. Recombinant clones were used to hybrid-select HeLa cell mRNA that was subsequently translated in vitro into a single protein species that comigrated with HeLa cell La protein. The in vitro translated protein was reactive with anti-La patient sera and was identical to the authentic La protein by peptide mapping. By analyzing overlapping cDNA clones, we mapped an antigenic site of La protein at the terminal 12% of the carboxyl end of the molecule. Within this region we identified a unique decapeptide of high hydrophilicity that may constitute a La antigenic determinant. We further demonstrated that the La antigen expressed from the recombinant clones can be used in a definitive enzyme-linked assay (ELISA) for the classification of sera from patients with systemic lupus erythematosus.
Solution of the embedding problem and decomposition of symmetric matrices.
Authors: Sippl M J , Scheraga H A
Published: May 2006

A solution of the problem of calculating cartesian coordinates from a matrix of interpoint distances (the embedding problem) is reported. An efficient and numerically stable algorithm for the transformation of distances to coordinates is then obtained. It is shown that the embedding problem is intimately related to the theory of symmetric matrices, since every symmetric matrix is related to a general distance matrix by a one-to-one transformation. Embedding of a distance matrix yields a decomposition of the associated symmetric matrix in the form of a sum over outer products of a linear independent system of coordinate vectors. It is shown that such a decomposition exists for every symmetric matrix and that it is numerically stable. From this decomposition, the rank and the numbers of positive, negative, and zero eigenvalues of the symmetric matrix are obtained directly.
Correlation between catalytic activity and bonding and coordination number of atoms and molecules on transition metal surfaces: Theory and experimental evidence
Authors: Falicov L. M. , Somorjai G. A.
Published: May 2006

Correlation between catalytic activity and low-energy local electronic fluctuations in transition metals is proposed. A theory and calculations are presented which indicate that maximum electronic fluctuations take place at high-coordination metal sites. Either (
i
) atomically rough surfaces that expose to the reactant molecules atoms with large numbers of nonmagnetic or weakly magnetic neighbors in the first or second layer at the surface or (
ii
) stepped and kinked surfaces are the most active in carrying out structure-sensitive catalytic reactions. The synthesis of ammonia from N
2
and H
2
over iron and rhenium surfaces,
1
H
2
/
2
H
2
exchange over stepped platinum crystal surfaces at low pressures, and the hydrogenolysis (C—C bond breaking) of isobutane at kinked platinum crystal surfaces are presented as experimental evidence in support of the theory.
UGA is read as tryptophan in Mycoplasma capricolum.
Authors: Yamao F , Muto A , Kawauchi Y , Iwami M , Iwagami S , Azumi Y , Osawa S
Published: May 2006

UGA is a nonsense or termination (opal) codon throughout prokaryotes and eukaryotes. However, mitochondria use not only UGG but also UGA as a tryptophan codon. Here, we show that UGA also codes for tryptophan in Mycoplasma capricolum, a wall-less bacterium having a genome only 20-25% the size of the Escherichia coli genome. This conclusion is based on the following evidence. First, the nucleotide sequence of the S3 and L16 ribosomal protein genes from M. capricolum includes UGA codons in the reading frames; they appear at positions corresponding to tryptophan in E. coli S3 and L16. Second, a tRNATrp gene and its product tRNA found in M. capricolum have the anticodon sequence 5' U-C-A 3', which can form a complementary base-pairing interaction with UGA.
Two tissue-specific isozymes of creatine kinase have closely matched amino acid sequences.
Authors: Pickering L , Pang H , Biemann K , Munro H , Schimmel P
Published: May 2006

Creatine kinase activity is associated with different isozyme species. We have examined two of these: the cytoplasmic brain (B) isozyme that is expressed in many tissues and is reported to be induced by estrogen and the developmentally regulated cytoplasmic muscle (M) isozyme that is found predominantly in differentiated muscle tissue. Recently, we cloned and sequenced the cDNA for the M isoenzyme of rabbit creatine kinase. We now report the isolation of B-isozyme cDNAs and the deduced primary structure of the polypeptide. The translated cDNA nucleotide sequence was cross-checked by fast-atom bombardment/mass spectrometry of tryptic fragments from the protein. The sequence is exactly colinear with the rabbit M isozyme and the two isozymes have 80% nucleotide and amino acid sequence identity. There are blocks of 36 and 41 amino acids where the amino acid sequence is conserved exactly. The colinearity of the two sequences and the extent of their identity makes it unlikely that either isozyme has unique polypeptide domains that account for specialized functions. The rationale for the existence of these creatine kinase isozymes, with distinct biological features, evidently is at the level of regulation of individual isozyme expression.
Metabolic activation of the nucleoside analog 9-[( 2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human diploid fibroblasts infected with human cytomegalovirus.
Authors: Biron K K , Stanat S C , Sorrell J B , Fyfe J A , Keller P M , Lambe C U , Nelson D J
Published: May 2006

9-[( 2-Hydroxy-1-(hydroxymethyl)ethoxy]-methyl)guanine (BW B759U) is a more potent inhibitor of human cytomegalovirus (HCMV) in vitro than is the related nucleoside analog acyclovir (ACV). BW B759U was selectively activated to the 5'-triphosphate (BW B759U-triphosphate) in cells infected with HCMV to levels at least 10-fold higher than those measured for ACV-triphosphate and up to as much as 100-fold higher than the levels found in uninfected cells. BW B759U-triphosphate accumulated in HCMV-infected cells with time; the rate of this increase was dependent upon the drug dose and virus multiplicity of infection. Enzyme activities that catalyzed the phosphorylation of thymidine and 2'-deoxycytidine increased 3- to 7-fold in extracts of cells early after HCMV infection but thereafter declined. No concomitant increase in the rate of BW B759U phosphorylation was detected under these assay conditions. Maximal rate of accumulation of both BW B759U-triphosphate and ACV-triphosphate after a short exposure to drug occurred in the late phase of the infective cycle, as the titer of extracellular virus reached a peak in untreated cultures, but after the decline of stimulated host deoxypyrimidine kinase activities. Once formed, the BW B759U-triphosphate pool decreased very slowly and thus it persisted for several days in both HCMV-infected and uninfected cells.
An endothelial cell-dependent pathway of coagulation.
Authors: Stern D , Nawroth P , Handley D , Kisiel W
Published: May 2006

Although the endothelial cell is considered antithrombogenic, endothelium has recently been shown to participate in procoagulant reactions. In this report cultured bovine aortic endothelial cells are shown to propagate a procoagulant pathway starting with factor XIa, leading to activation of factors IX, VIII, X, and prothrombin, culminating in fibrinopeptide A cleavage from fibrinogen and formation of a fibrin clot. Electron microscopic studies demonstrated that fibrin strands are closely associated with the endothelial cells. Endotoxin-treated endothelial cells, having acquired tissue factor activity, generated fibrinopeptide A in the presence of factors VIIa, IX, VIII, X, prothrombin, and fibrinogen. Factor X activation by factor VIIa and tissue factor expressed by endothelial cells is 10 times greater in the presence of factors IX and VIII than in their absence. This indicates that on the perturbed endothelial cell surface, factors IX and VIII do have an important role in the activation of factor X. Addition of platelets (10(8) per ml) augmented thrombin formation seen in the presence of endothelium alone by about 15-fold. Anti-human factor V IgG decreased this enhanced thrombin formation in the presence of platelets, indicating that factor V from platelets was playing an important role in thrombin formation. These data lead us to propose that endothelial cells can actively participate in procoagulant reactions. Although platelets can augment thrombin formation by these endothelial cell-dependent reactions, endothelial cells alone can lead to formation of a cell-associated fibrin clot. The endotoxin-treated endothelial cell provides a model of the thrombotic state supplying tissue factor to initiate coagulation and propagating the reactions leading to fibrin formation. This endothelial cell-dependent pathway suggests a central role for factors VIII and IX consistent with their importance in hemostasis.
Primary structure and gene organization of human hepatitis A virus.
Authors: Najarian R , Caput D , Gee W , Potter S J , Renard A , Merryweather J , Van Nest G , Dina D
Published: May 2006

The RNA genome of human hepatitis A virus (HAV) was molecularly cloned. Recombinant DNA clones representing the entire HAV RNA were used to determine the primary structure of the viral genome. The length of the viral genome is 7478 nucleotides. An open reading frame starting at nucleotide 734 and terminating at nucleotide 7415 encodes a polyprotein of Mr 251,940. Comparison of the HAV nucleotide sequence with that of other picornaviruses has failed to reveal detectable areas of homology. However, a computer analysis of the putative amino acid sequence of HAV and poliovirus demonstrated the existence of short areas of homology in virion protein 3 (VP3) and throughout the carboxyl-terminal portion of the polyproteins. In addition, extensive protein structural homologies with poliovirus were detected.
Characterization of the spo0A locus and its deduced product.
Authors: Ferrari F A , Trach K , LeCoq D , Spence J , Ferrari E , Hoch J A
Published: May 2006

The highly pleiotropic stage 0 sporulation locus of Bacillus subtilis, spo0A, has been cloned in bacteriophage lambda, subcloned in plasmids, and sequenced. The locus was found to code for a protein of 29,691 Da. Analysis of the in vivo transcripts from this region by nuclease S1 protection experiments located the start and stop of transcription of the locus. The transcription start site was preceded by a promoter resembling sigma 37-dependent promoters. Two mutations originally assigned to a second locus, spo0C, in this region because of their weakly pleiotropic phenotypes were cloned and sequenced. The mutations were found to be different missense alterations in the same base of the 10th codon preceding the carboxyl end of the Spo0A protein. These results, along with the finding that mutations in the spo0A gene product [Hoch, J. A., Trach, K., Kawamura, F. & Saito, H. (1985) J. Bacteriol. 161, 552-555] suppress the requirement for spo0B, spo0E, and spo0F gene products in transcription from sigma 28-dependent promoters, suggest that the Spo0A protein interacts directly with the transcription machinery to effect the initiation of sporulation. The deduced amino acid sequence of the Spo0A protein was highly related to that of the OmpR regulatory protein of Escherichia coli.
Preferential binding of benzo[a]pyrene diol epoxide to the linker DNA of human foreskin fibroblasts in S phase in the presence of benzamide.
Authors: Kurian P , Jeffrey A M , Milo G E
Published: May 2006

Addition of benzamide (BZ) at the onset of S phase inhibited expression of the neoplastic phenotype in human foreskin fibroblasts treated in vitro with (+/-)-7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P diol epoxide) in early S phase. Analysis of the specific B[a]P diol epoxide-DNA adducts revealed that ca. 65% of the total adducts in BZ and non-BZ carcinogen-treated cells was the B[a]P diol epoxide-deoxyguanine adduct. Limited micrococcal nuclease digestion of the early S phase nuclei from cells treated with B[a]P diol epoxide indicated that the carcinogen binds equally to linker and core DNA. However, when the cells were predominantly in S phase, in the presence of BZ, there was ca. three times more binding of B[a]P diol epoxide to the linker DNA compared to the core region. The confluent cells in G1 cell arrest treated only with B[a]P diol epoxide also bound the carcinogen preferentially to the linker region. These data indicate that pretreatment of the cells with BZ at the onset of S phase established a preferential binding pattern in the linker DNA similar to that observed in the cells treated with B[alpha]P diol epoxide in G1 arrest.
Phorbol esters induce transient internalization without degradation of unoccupied epidermal growth factor receptors.
Authors: Beguinot L , Hanover J A , Ito S , Richert N D , Willingham M C , Pastan I
Published: May 2006

4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment of KB cells at 37 degrees C rapidly induces a 50% reduction in epidermal growth factor (EGF) binding that is maximal by 30 min. EGF binding activity returns to the original value by 1 hr and remains constant for 2 hr thereafter. Using a polyclonal antibody directed against the cytoplasmic domain of the EGF receptor (EGF-R), we examined the fate of the receptor after PMA treatment. Immunofluorescent and electron microscopic localization of the EGF-R after PMA treatment demonstrated that about 50% of the receptor became internalized into endocytic vesicles (receptosomes) and Golgi-associated structures. Unlike EGF-induced internalization, PMA-induced internalization did not cause delivery of EGF-R to lysosomes or receptor degradation. Rather, receptor reappeared on the cell surface. No stimulation of EGF-R synthesis was observed after 1 hr of PMA treatment. Loss of cell surface binding correlated with the internalization of the EGF-R observed morphologically. A possible explanation for these observations is that PMA, an activator of protein kinase C, confers a signal sufficient for EGF-R clustering and internalization but not for transport to lysosomes.
Insertion of long interspersed repeated elements at the Igh (immunoglobulin heavy chain) and Mlvi-2 (Moloney leukemia virus integration 2) loci of rats.
Authors: Economou-Pachnis A , Lohse M A , Furano A V , Tsichlis P N
Published: May 2006

Restriction enzyme analysis of normal DNA derived from individual rats of the National Institutes of Health outbred Osborn-Mendel colony revealed that two independent single-copy loci, the Igh (immunoglobulin heavy chain) locus and the Mlvi-2 (Moloney leukemia virus integration 2) locus, a putative oncogene, are polymorphic (i.e., exhibit allelic variation). The polymorphism at both loci was due to the presence or absence of a long interspersed repeated DNA element (LINE). The LINE insertion in the Igh locus occurred in the joining (J) region, which is involved in the physiological rearrangement of this locus. The LINE insertion in the Mlvi-2 locus has occurred approximately 6 kilobases from the region of provirus integration in Moloney murine leukemia virus-induced rat thymomas. The two inserts are colinear with each other and with other randomly selected cloned copies of the rat LINE family, the general characteristics of which we also present. LINE insertion in the Mlvi-2 locus was observed in several rat strains, established from independent rat colonies, suggesting that LINE-containing Mlvi-2 alleles may be widespread in the rat population. LINE insertion in the Igh locus was observed in 1 of 27 rats. The detection of a LINE-related polymorphism at two nonselected loci indicates that LINEs are transposable. The presence or absence of these long (greater than 5 kilobases), highly transcribed elements at single-copy loci could have profound effects on gene activity. Furthermore, LINE-containing single-copy loci could be affected by homologous interaction between the resident LINE and any of the other 50,000 or so copies of these elements in the rat genome.
Propagation of some human DNA sequences in bacteriophage lambda vectors requires mutant Escherichia coli hosts.
Authors: Wyman A R , Wolfe L B , Botstein D
Published: May 2006

The growth of clones of human genomic DNA fragments in a bacteriophage lambda vector has been examined in a number of different Escherichia coli hosts. A large proportion (8.9%) of the phages carrying different fragments of the human genome fail to grow on standard rec+ hosts but will grow on hosts carrying mutations in the recB, recC, and sbcB genes. Heteroduplex analysis in the electron microscope of DNA from four of these phages revealed substantial secondary structure, including snap-back regions 200-500 base pairs in length. Such structures were not found in phages from the same DNA library that grow in rec+ hosts. These results are interpreted in the light of prior observations [Leach, D.R.F. & Stahl, F. (1983) Nature (London) 305, 448-451] showing that inverted repetitions cloned in phage lambda can be propagated in recB recC sbcB hosts but not in rec+ hosts.
Assignment of the gene coding for the T3-delta subunit of the T3-T-cell receptor complex to the long arm of human chromosome 11 and to mouse chromosome 9.
Authors: van den Elsen P , Bruns G , Gerhard D S , Pravtcheva D , Jones C , Housman D , Ruddle F A , Orkin S , Terhorst C
Published: May 2006

The gene encoding the 20-kDa glycoprotein of the T3-T-cell receptor complex (T3-delta chain) has been mapped to human chromosome 11 by hybridization of a T3-delta cDNA clone (pPGBC#9) to DNA from a panel of human-rodent somatic cell hybrids. In Southern blotting experiments with DNAs of somatic cell hybrids that contained segments of chromosome 11, we were able to assign the T3-delta gene to the distal portion of the long arm of human chromosome 11 (11q23-11qter). By use of a newly developed cDNA clone (pPEM-T3 delta) that codes for the murine T3-delta chain, the mouse T3-delta gene was mapped on chromosome 9. The importance of the T3-delta map position and its relationship to the other genes on the long arm of human chromosome 11 and to those on mouse chromosome 9 is discussed.
Amelioration of lytic abnormalities of paroxysmal nocturnal hemoglobinuria with decay-accelerating factor.
Authors: Medof M E , Kinoshita T , Silber R , Nussenzweig V
Published: May 2006

Purified decay-accelerating factor (DAF), from the stroma of normal human erythrocytes, was incorporated into the membranes of erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH), and its effect on the complement sensitivity of the cells was investigated. Reconstitution with exogenous DAF restored the ability of the affected PNH cells to resist assembly of the homologous C3 convertase, C4b2a, on their surfaces, and decreased the susceptibility of the cells to lysis in acidified serum. Conversely, treatment of normal erythrocytes with monoclonal or polyclonal anti-DAF antibodies abrogated the capacity of the normal cells to circumvent C4b2a assembly and rendered the cells sensitive to acid lysis. These findings show that the previously reported association of DAF deficiency with PNH is causally related to the lytic abnormalities of the cells and clarify the molecular basis for restriction of autologous convertase formation on normal human erythrocytes.
Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat.
Authors: Adelman J P , Mason A J , Hayflick J S , Seeburg P H
Published: May 2006

Cloned cDNAs encoding the precursor protein for gonadotropin-releasing hormone (Gn-RH) and prolactin release-inhibiting factor (PIF) were isolated from libraries derived from human and rat hypothalamic mRNA. Nucleotide sequence analyses predict precursor proteins of 92 amino acids for both species and show identity between the human placental and human hypothalamic precursor proteins. Whereas the Gn-RH peptide structure is completely conserved in human and rat, the PIF domain of the precursor displays 70% interspecies homology. Genomic analyses revealed the presence of a single Gn-RH-PIF gene in human and rat containing sequences corresponding to the cDNA distributed across four exons.
Agreement with the disulfide stretching frequency-conformation correlation of Sugeta, Go, and Miyazawa.
Authors: Van Wart H E , Scheraga H A
Published: May 2006

Two hypotheses have been advanced to explain the conformational dependence of the disulfide stretching frequency of the CCSSCC moiety in unstrained disulfides. Such compounds can adopt conformations that exhibit disulfide stretching bands near 510, 525, and 540 cm-1. Sugeta et al. [Sugeta, H., Go, A. & Miyazawa, T. (1973) Bull. Chem. Soc. Jpn. 46, 3407-3411] have attributed these three bands to conformations having none, one, and two trans conformations, respectively, about the C-S bonds. In apparent contradiction to this correlation, Van Wart and Scheraga [Van Wart, H. E. & Scheraga, H. A. (1976) J. Phys. Chem. 80, 1812-1823] found that dithioglycolic acid crystals, believed at the time to exist only in the trans conformation about both C-S bonds, exhibited the S-S stretching band near 510 cm-1. On the basis of this observation, it was suggested instead that the 510, 525, and 540 cm-1 bands arose from CCSSCC moieties having none, one, and two A conformations (i.e., those with CC-SS dihedral angles close to 30 degrees), respectively, about the C-S bonds. It has recently been shown by Nash et al. [Nash, C. P., Olmstead, M. M., Weiss-Lopez, B., Musker, W. K., Ramasubbu, M. & Parthasarathy, R. (1985) J. Am. Chem. Soc. 107, 7194-7195] that dithioglycolic acid can crystallize in two different forms, one with trans and the other with gauche conformations about both C-S bonds, and that these have S-S stretching bands at 536 and 510 cm-1, respectively. These results are confirmed here and it is shown that our earlier data were collected on the all-gauche (rather than the all-trans) form. Thus, the correlation proposed by Sugeta et al. is correct.
Biphasic effects of estrogen on apolipoprotein synthesis in human hepatoma cells: mechanism of antagonism by testosterone.
Authors: Tam S P , Archer T K , Deeley R G
Published: May 2006

Treatment of HepG2 cells with various concentrations of 17 beta-estradiol has revealed two distinct thresholds for induction of different apolipoproteins. Maximal increases in apolipoprotein AI and CII (apoAI and apoCII) secretion can be obtained with initial concentrations of hormone of 20 nM or greater, while a similar induction of apoB and apoE requires in excess of 500 nM. Both responses involve alterations in the concentrations of apolipoprotein mRNAs. Analyses of the kinetics of accumulation of the apolipoproteins in response to high concentrations of hormone indicate that induction of apoB and apoE occurs coordinately, but it lags behind that of apoAI and apoCII by 5-6 hr. This lag can be eliminated by preexposing the cells to low concentrations of hormone. The ability to induce apoAI and apoCII and the kinetics with which they respond to low levels of estrogen correlate with levels of nuclear type I estrogen binding sites, while increases in apoE and apoB synthesis in response to high concentrations of hormone correlate with the induction of type II sites. Testosterone alone has no effect on the rates of apolipoprotein secretion, but it does increase the concentration of estrogen required to maximally induce apoCII and apoAI by a mechanism that involves high-affinity androgen receptors. This effect may be attributable to the testosterone-dependent induction of a cytoplasmic moderate-affinity estrogen-binding component.
RETRACTED: Binding of thymopoietin to the acetylcholine receptor.
Authors: Venkatasubramanian K , Audhya T , Goldstein G
Published: May 2006

Thymopoietin is a polypeptide hormone of the thymus with physiological effects on the immune system and on acetylcholine-mediated transmission at the neuromuscular synapse. Elucidation of the structure and function of the nicotinic acetylcholine receptor has been facilitated by the use of the electric organs of Torpedo ray or Electrophorus eel as rich sources of the receptor and by the use of snake polypeptide toxins such as alpha-bungarotoxin as highly selective labels of the acetylcholine binding site. We now show that thymopoietin binds with high affinity (Ka approximately equal to 2.5 X 10(9) M-1) to the acetylcholine binding region of the acetylcholine receptor of Torpedo californica, as evidenced by similar and complete inhibition of the binding of radiolabeled thymopoietin or alpha-bungarotoxin by either of these polypeptides. These findings raise intriguing questions concerning the mechanisms whereby alpha-bungarotoxin and the thymopoietin affect acetylcholine receptor function, since these two polypeptides with such similar binding properties have very different functional effects.
Transcriptional activation encoded by the v-fos gene.
Authors: Setoyama C , Frunzio R , Liau G , Mudryj M , de Crombrugghe B
Published: May 2006

We present evidence that the fos oncogene encodes a transcriptional trans-activation function. trans-activation was assayed by cotransfection into NIH 3T3 mouse fibroblasts of v-fos DNA containing plasmids together with a plasmid containing a test promoter. Three v-fos DNAs were used: (i) pFBR-1, a plasmid containing the FBR proviral sequences; (ii) pFBJ-2, a plasmid harboring the FBJ proviral sequences; (iii) pMF-J, a plasmid containing the FBJ fos sequences linked to a mouse metallothionein promoter. Each of the three v-fos DNA plasmids stimulated the expression of a cotransfected chimeric gene consisting of a promoter segment of the mouse alpha 1(III) collagen gene linked to the gene for chloramphenicol transacetylase. In similar experiments the v-fos gene also stimulated the long terminal repeat promoter of Rous sarcoma virus (RSV) but neither the early promoter of simian virus 40 nor the beta-actin promoter. Evidence that the trans-activation function is specified by the v-fos coding sequences comes from the fact that a frameshift mutation in the v-fos coding sequence inhibits the trans-activation. Two mutations that map around nucleotide -100 in the RSV promoter do not respond to cotransfection with v-fos, whereas other mutations respond like the wild-type RSV promoter. These experiments suggest that the v-fos gene either encodes or induces an activator of transcription that recognizes specific sequences in promoters.
Structure and developmentally regulated expression of a Strongylocentrotus purpuratus collagen gene.
Authors: Venkatesan M , de Pablo F , Vogeli G , Simpson R T
Published: May 2006

We have isolated and partially characterized an ca. 20-kilobase-pair Strongylocentrotus purpuratus genomic clone, using a mouse alpha 1 (type IV) collagen cDNA probe. A 1-kilobase-pair HindIII fragment of the clone hybridizes strongly to the probe; this has been subcloned and sequenced. It contains 212 base pairs of sequence coding for (Gly-Xaa-Yaa)n (where Xaa and Yaa are different unspecified amino acids), characteristic of all known collagen genes. There is a single point of discontinuity within the repeating pattern in this exon, similar to the genomic structure of mouse type IV collagen. The (Gly-Xaa-Yaa)n-encoding element is flanked by consensus splicing sequences, and the intervening sequences on either side of it have multiple in-phase termination codons. Electron microscopy of R loops between the phage lambda recombinant clone and poly(A)+ RNA reveals multiple short exons, a feature also seen in vertebrate collagen genes. The (Gly-Xaa-Yaa)n protein-encoding sequence hybridizes to a developmentally regulated 9-kilobase mRNA; the message appears during the morula stage, rises sharply in abundance at the blastula stage, and decreases in proportion to total RNA later in development.
Mitotic recombination in germ cells generated two major histocompatibility complex mutant genes shown to be identical by RNA sequence analysis: Kbm9 and Kbm6.
Authors: Geliebter J , Zeff R A , Melvold R W , Nathenson S G
Published: May 2006

RNA sequencing represents a major procedural simplification for nucleotide sequence analysis of a transcribed gene. Using newly adapted mRNA and cDNA sequencing procedures, we have sequenced 855 nucleotides of Kbm9 mRNA, corresponding to the codons for the aminoterminal 285 amino acids. The inferred DNA sequence of the Kbm9 gene differs from the parental Kb sequence by single nucleotide alterations in each of codons 116 and 121, resulting in Tyr----Phe and Cys----Arg substitutions, respectively. The Kbm9 sequence is identical to that of another independently arising MHC mutant gene, Kbm6. As both the Kbm9 and Kbm6 genes were generated by recombination between the Kb and Q4 genes, our data indicate that the identical genetic interactions have occurred at least twice. The relatively large extent of identity between Q4 and Kb may be responsible for frequent recombination between the two genes. The parents of the original bm9 mutant mice had five identical mutant offspring, which can be explained by mitotic recombination in the germ cells, producing gonadal mosaicism in the C57BL/6 mother. Thus, mitotic recombination, and not meiotic recombination, appears to be responsible for the formation of at least some of the Kb mutants. Such a mechanism probably plays a major role in the generation of diversity in the major histocompatibility complex.