bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced DNA adduct formation in replicating liver cells during continuous feeding of a chemical carcinogen.
Authors: Huitfeldt H S , Brandtzaeg P , Poirier M C
Published: May 2006

To investigate early cellular alterations in liver DNA during hepatocarcinogenesis, we have visualized replicating cells and analyzed their DNA adduct content in livers of rats continuously fed a carcinogenic level (0.02%) of 2-acetylaminofluorene for periods up to 4 weeks. One hour prior to sacrifice, cells undergoing DNA synthesis were pulse-labeled with the thymidine analogue 5-bromodeoxyuridine. Replicating cells were visualized immunohistochemically with anti-(5-bromodeoxyuridine), and identification of aminofluorene-DNA adducts in replicating nuclei was achieved by staining with an antiserum specific for N-(deoxyguanosin-8-yl)-2-aminofluorene; both stains were observed simultaneously by two-color immunofluorescence. Data were obtained for all cells, including large hepatocytes (nuclei greater than 6 microns) and small cells (nuclei less than 6 microns), such as hepatocytes sliced asymmetrically, oval cells, Kuppfer cells, and sinusoidal lining cells. Based on the size of their nuclei, the hepatocytes were the only cells that could be identified separately from the total. A distinct increase in the number of cells synthesizing DNA was observed after 25 days of 2-acetylaminofluorene feeding; replicating cells were either scattered randomly throughout the liver or clustered in discrete foci. At times up to 28 days, cells with both large and small nuclei that were synthesizing DNA showed reduced aminofluorene-DNA adduct immunofluorescence compared to nonreplicating cells. The results suggest that liver cells replicating during carcinogen exposure have altered metabolic capacities resulting in reduced aminofluorene-DNA adduct formation. It is possible that such cells constitute the progenitors of preneoplastic foci, which have a replicative advantage as compared to normal liver.
Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly.
Authors: Dabiri G A , Sanger J M , Portnoy D A , Southwick F S
Published: May 2006

Listeria monocytogenes is an intracellular parasite that can readily infect the macrophage-like cell line J774 and the kidney epithelial cell PtK2. After being ingested, the organism escapes from the phagolysosome into the host-cell cytoplasm. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin, a specific stain for actin filaments (F-actin), demonstrates that within 1 hr of initiation of infection, the bacteria become surrounded by host-cell cytoplasmic actin filaments. By 3 hr, long projections of F-actin begin to form at one end of the bacteria. These actin structures colocalize with the actin-bundling protein alpha-actinin as well as with tropomyosin. Microinjection of fluorescently labeled alpha-actinin in living cells demonstrates that the formation of these F-actin projections is associated with bacterial movement, actin filaments rapidly assembling behind the bacteria as they migrate through the cytoplasm. These F-actin tails attain lengths up to 40 microns. The movement of the bacteria through the cytoplasm is rapid, 0.12-1.46 microns/sec. Within 2 min of cytochalasin D (0.5 micrograms/ml) treatment, all bacterial intracellular movement stops, and additional bacteria-associated actin assembly is blocked. A nonmotile Listeria mutant induces comparable actin assembly and moves at speeds similar to the wild type, indicating that the forces required for intracellular bacterial movement are generated by the host cell. L. monocytogenes can dramatically stimulate host-cell actin assembly in a directional manner, which serves to rapidly propel the bacteria through the cytoplasm, allowing the organisms to move to peripheral membranes and spread to uninfected cells.
Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes.
Authors: Fan Y S , Davis L M , Shows T B
Published: May 2006

A procedure for mapping small DNA probes directly on banded human chromosomes by fluorescence in situ hybridization has been developed. This procedure allows for the simultaneous visualization of banded chromosomes and hybridization signal without overlaying two separate photographic images. This method is simple and rapid, requires only a typical fluorescence microscope, has proven successful with DNA probes as small as 1 kilobase, is applicable for larger probes, and will greatly facilitate mapping the vast number of probes being generated to study genetic disease and define the human genome. Human metaphase chromosomes were prepared from phytohemagglutinin-stimulated lymphocyte cultures synchronized with bromodeoxyuridine and thymidine. Probes were labeled with biotin-dUTP, and the hybridization signal was amplified by immunofluorescence. Chromosomes were stained with both propidium iodide and 4',6-diamidino-2-phenylindole (DAPI), producing R- and Q-banding patterns, respectively, allowing unambiguous chromosome and band identification while simultaneously visualizing the hybridization signal. Thirteen unique DNA segments have been localized to the long arm of chromosome 11 by using this technique, and localization of 10 additional probes by using radioactive in situ hybridization provides a comparison between the two procedures. These DNA segments have been mapped to all long-arm bands on chromosome 11 and in regions associated with neoplasias and inherited disorders.
Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis.
Authors: Weickert M J , Chambliss G H
Published: May 2006

Catabolite repression of the Bacillus subtilis alpha-amylase gene (amyE) involves an operator sequence located just downstream of the promoter (amyR), overlapping the transcription start site. Oligonucleotide site-directed mutagenesis of this sequence identified bases required for catabolite repression. Two mutations increased both the 2-fold symmetry of the operator and the repression ratio. Although many mutations reduced the repression ratio 3- to 11-fold, some also caused a 2-fold or greater increase in amylase production. Others caused hyperproduction without affecting catabolite repression. Homologous sequences in other catabolite-repressed B. subtilis promoters suggest a common regulatory site may be involved in catabolite repression.
Escherichia coli helicase II (UvrD) protein initiates DNA unwinding at nicks and blunt ends.
Authors: Runyon G T , Bear D G , Lohman T M
Published: May 2006

The Escherichia coli uvrD gene product, helicase II, is required for both methyl-directed mismatch and uvrABC excision repair and is believed to function by unwinding duplex DNA. Initiation of unwinding may occur specifically at either a mismatch or a nick, although no direct evidence for this has previously been reported. It has recently been shown that helicase II can unwind fully duplex linear and nicked circular DNA with lengths of at least approximately 2700 base pairs in vitro; hence, a flanking region of single-stranded DNA is not required to initiate DNA unwinding. In studies with uniquely nicked duplex DNA, we present EM evidence that helicase II protein initiates DNA unwinding at the nick, with unwinding proceeding bidirectionally. We also show that helicase II protein initiates DNA unwinding at the blunt ends of linear DNA, rather than in internal regions. These data provide direct evidence that helicase II protein can initiate unwinding of duplex DNA at a nick, in the absence of auxiliary proteins. We propose that helicase II may initiate unwinding from a nick in a number of DNA repair processes.
Alternative splicing produces transcripts encoding two forms of the alpha subunit of GTP-binding protein Go.
Authors: Strathmann M , Wilkie T M , Simon M I
Published: May 2006

The alpha subunit of the guanine nucleotide-binding protein Go ("o" for other) is believed to mediate signal transduction between a variety of receptors and effectors. cDNA clones encoding two forms of Go alpha subunit were isolated from a mouse brain library. These two forms, which we call GoA alpha and GoB alpha, appear to be the products of alternative splicing. GoA alpha differs from GoB alpha over the C-terminal third of the deduced protein sequence. Both forms are predicted to be substrates for ADP-ribosylation by pertussis toxin. GoA alpha transcripts are present in a variety of tissues but are most abundant in brain. The GoB alpha transcript is expressed at highest levels in brain and testis. It is possible that GoA alpha and GoB alpha have different functions.
Peripheral tolerance in mice expressing a liver-specific class I molecule: inactivation/deletion of a T-cell subpopulation.
Authors: Wieties K , Hammer R E , Jones-Youngblood S , Forman J
Published: May 2006

We previously demonstrated that C3H/HeJ transgenic (TG) mice that express a laboratory-engineered class I molecule, Q10/L, exclusively on liver parenchymal cells show no evidence of hepatic disease even after deliberate immunization. Nevertheless, these animals demonstrate cytotoxic T-lymphocyte (CTL) activity specific for Q10/L, although it is less than that obtained from non-TG littermates. We now show that this decrease in CTL activity is not a reflection of a decrease in precursors, since both TG and normal animals have similar numbers. When non-TG C3H mice are primed with H-2Ld and H-2Kbm1 antigens, which extensively crossreact with Q10/L, their specific in vitro CTL activity directed against H-2Ld, H-2Kbm1, and Q10/L is increased 10- to 20-fold, as expected. Although primed TG mice show similar increases in in vitro CTL activity directed against H-2Ld and H-2Kbm1, they display no increase in anti-Q10/L activity. Whereas anti-H-2Ld spleen cells from non-TG mice readily generate CTL lines and clones specific for H-2Ld and Q10/L, TG cells give rise to anti-H-2Ld lines or clones only. These data indicate that the tolerance in TG mice is accounted for by the inactivation or deletion of an important CTL subpopulation having the capability of recognizing the peripheral antigen in situ. To determine whether tolerance would persist in the absence of Q10/L, TG cells were transferred into non-TG recipients. Three weeks later Q10/L-specific lytic activity generated in in vitro bulk cultures remained reduced compared to non-TG cells, indicating that the tolerant phenotype was stable during this interval.
Molecular cloning, sequence, and expression of a human GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase cDNA that can form the H blood group antigen.
Authors: Larsen R D , Ernst L K , Nair R P , Lowe J B
Published: May 2006

We have previously used a gene-transfer scheme to isolate a human genomic DNA fragment that determines expression of a GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.69]. Although this fragment determined expression of an alpha(1,2)FT whose kinetic properties mirror those of the human H blood group alpha(1,2)FT, their precise nature remained undefined. We describe here the molecular cloning, sequence, and expression of a human cDNA corresponding to these human genomic sequences. When expressed in COS-1 cells, this cDNA directs expression of cell surface H structures and a cognate alpha(1,2)FT activity with properties analogous to the human H blood group alpha(1,2)FT. The cDNA sequence predicts a 365-amino acid polypeptide characteristic of a type II transmembrane glycoprotein with a domain structure analogous to that of other glycosyltransferases but without significant primary sequence similarity to these or other known proteins. To directly demonstrate that the cDNA encodes an alpha(1,2)FT, the COOH-terminal domain predicted to be Golgi-resident was expressed in COS-1 cells as a catalytically active, secreted, and soluble protein A fusion peptide. Southern blot analysis showed that this cDNA identifies DNA sequences syntenic to the human H locus on chromosome 19. These results strongly suggest that this cloned alpha(1,2)FT cDNA represents the product of the human H blood group locus.
Biophysical model of a Hebbian synapse.
Authors: Zador A , Koch C , Brown T H
Published: May 2006

We present a biophysical model of electrical and Ca(2+) dynamics following activation of N-methyl-D-aspartate (NMDA) receptors located on a dendritic spine. The model accounts for much of the phenomenology of the induction of long-term potentiation at a Hebbian synapse in hippocampal region CA1. Computer simulations suggested four important functions of spines in this Ca(2+)-dependent synaptic modification: (i) compartmentalizing transient changes in [Ca(2+)] to just those synapses that satisfy the conjunctive requirement for synaptic modification; (ii) isolating the spine head from changes in the [Ca(2+)] at the dendritic shaft; (iii) amplifying the concentration changes at those synapses; and (iv) increasing the voltage dependence of the processes underlying long term potentiation induction. This proposed role of spines in the regulation of Ca(2+) dynamics contrasts with traditional approaches to spine function that have stressed electronic properties. This model can be used to explore the computational implications of Hebbian synapses.
Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing two distinct domains.
Authors: Bhargava A K , Woitach J T , Davidson E A , Bhavanandan V P
Published: May 2006

A lambda gt11 cDNA library prepared from bovine submaxillary gland mRNA was screened with polyclonal anti-apo-bovine submaxillary mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. One of the positive clones had a 1.8 kilobase (kb) cDNA insert and coded for an incomplete protein. A 2.0-kb cDNA clone was isolated by rescreening the library with the 1.8-kb cDNA. Nucleotide sequencing of the full-length 2.0-kb cDNA revealed an open reading frame that coded for a 563-amino acid protein. A striking feature of the cloned protein is the skewed distribution of the amino acids, most notably that of the hydroxy amino acids and cysteine. The amino-terminal domain of 339 residues is very rich in threonine, serine, and glycine and poor in cysteine, aspartic acid, tyrosine, phenylalanine, and tryptophan. In contrast, the carboxyl-terminal domain of 224 residues is rich in cysteine, aspartic acid, tyrosine, lysine, and asparagine and relatively poor in threonine, serine, and glycine. A search of the protein data bank for homologies to the deduced amino acid sequence revealed statistically significant matches to several proteins, including the porcine submaxillary apomucin fragment. The cysteine-rich domain by itself was not statistically homologous with any of the registered polypeptide sequences. RNA blot analysis using DNA probes corresponding to the mucin-like and cysteine-rich regions detected a nearly identical pattern of transcripts, demonstrating that the characterized clones are not artifacts of cDNA library construction. The blots also showed the presence of polydisperse transcripts in bovine submaxillary gland but no detectable hybridization signals in liver or brain RNA.
Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A.
Authors: Carroll M C , Fathallah D M , Bergamaschini L , Alicot E M , Isenman D E
Published: May 2006

The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)--namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the alpha chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed (ester vs. amide). Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not "catalytic" as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to "select" binding sites on potential acceptor molecules.
Molecular structure of leucine aminopeptidase at 2.7-A resolution.
Authors: Burley S K , David P R , Taylor A , Lipscomb W N
Published: May 2006

The three-dimensional structure of bovine lens leucine aminopeptidase (EC 3.4.11.1) complexed with bestatin, a slow-binding inhibitor, has been solved to 3.0-A resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, the structure of the isomorphous native enzyme has been refined at 2.7-A resolution, and the current crystallographic R factor is 0.169 for a model that includes the two zinc ions and all 487 amino acid residues comprising the asymmetric unit. The enzyme is physiologically active as a hexamer, which has 32 symmetry and is triangular in shape with a triangle edge length of 115 A and maximal thickness of 90 A. The monomers are crystallographically equivalent and each is folded into two unequal alpha/beta domains connected by an alpha-helix to give a comma-like shape with approximate maximal dimensions of 90 x 55 x 55 A3. The secondary structural composition is 40% alpha-helix and 19% beta-strand. The N-terminal domain (160 amino acids) mediates trimer-trimer interactions and does not appear to participate directly in catalysis. The C-terminal domain (327 amino acids) is responsible for catalysis and binds the two zinc ions, which are 2.88 A apart. The pair of metal ions is located near the edge of an eight-stranded, saddle-shaped beta-sheet. One zinc ion is coordinated by carboxylate oxygen atoms of Asp-255, Asp-332, and Glu-334 and the carbonyl oxygen of Asp-332. The other zinc ion is coordinated by the carboxylate oxygen atoms of Asp-255, Asp-273, and Glu-334. The active site also contains two positively charged residues, Lys-250 and Arg-336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 A and thickness 10 A. Access to this cavity is provided by solvent channels that run along the twofold symmetry axes.
Pvt-1 transcripts are found in normal tissues and are altered by reciprocal(6;15) translocations in mouse plasmacytomas.
Authors: Huppi K , Siwarski D , Skurla R , Klinman D , Mushinski J F
Published: May 2006

The mouse Pvt-1 (for plasmacytoma variant translocation) region maps to a chromosome 15 breakpoint region that is frequently interrupted by "variant" reciprocal chromosome translocations, rcpt(6;15), in plasmacytomas. This region lies several hundred kilobases (kb) 3' of the mouse c-myc gene (Myc) which is deregulated in both rcpt(6;15) and rcpt(12;15) plasmacytomas. rcpt(12;15) translocations apparently activate c-myc directly by interrupting the gene itself, but the mechanism causing c-myc deregulation in tumors bearing rcpt(6;15) translocations remains unknown. The indirect activation of c-myc by Pvt-1 interruption has remained an appealing possibility, but heretofore it has not been possible to establish such a connection. Furthermore, no genes from the Pvt-1 locus have been shown to be transcribed in normal tissues or in tumors with rcpt(6;15) translocations. We report the isolation of a cDNA clone, Pvt-1-1, from mouse spleen mRNA that is specific to the Pvt-1 region. This cDNA probe detects low levels of large (ca. 14 kb) RNA transcripts in normal mouse tissues. In plasmacytomas with rcpt(6;15) translocations, the Pvt-1 transcripts are elevated in abundance and truncated in size. Both changes are apparently induced by the chromosomal translocation. Expression of 14-kb Pvt-1 RNA is elevated in B-cell tumor lines that express immunoglobulin light chain genes; thus, we postulate that these translocations are facilitated by the increased DNA accessibility of immunoglobulin kappa light chain and Pvt-1 genes when they are simultaneously expressed at certain times during B-cell ontogeny.
Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase.
Authors: Volarević S , Burns C M , Sussman J J , Ashwell J D
Published: May 2006

Immunoprecipitation of Thy-1 from Triton X-100 detergent lysates of surface-iodinated and chemically cross-linked T cells precipitated at least five major and discrete bands. Four of these bands were identified as Thy-1, CD45 (a transmembrane tyrosine phosphatase), a major histocompatibility complex-encoded class I molecule, and beta 2-microglobulin. Similar analyses revealed that CD45 was coprecipitated from lysates of cross-linker-treated cells by antibodies to the T-cell antigen receptor (TCR). The same pattern of coprecipitated bands was observed when digitonin was used to lyse untreated cells. Immunoprecipitation of Thy-1 or the TCR from lysates of cross-linked T cells precipitated CD45 tyrosine phosphatase activity. Calculations based upon the amounts of coprecipitated enzymatic activity or TCR zeta chain indicate that a substantial fraction of Thy-1 and TCR complexes can be cross-linked to CD45. These data support a model in which the dependence of Thy-1 signaling on TCR coexpression is due to their common interaction with a tyrosine phosphatase and provide a possible structural basis for the influence of CD45 on TCR-mediated signaling.
Uptake of a nido-carboranylporphyrin by human glioma xenografts in athymic nude mice and by syngeneic ovarian carcinomas in immunocompetent mice.
Authors: Kahl S B , Joel D D , Nawrocky M M , Micca P L , Tran K P , Finkel G C , Slatkin D N
Published: May 2006

A tetraphenylporphyrin bearing four dicarbollide ([B9C2H11]-) cages linked to the o-phenyl ring positions by anilide bonds, known as boronated tetraphenylporphyrin (BTPP), has been synthesized in excellent yield from tetra-(o-aminophenyl) porphyrin and carborane carbonyl chloride followed by base-assisted cage opening and ion exchange to give the highly water-soluble potassium salt. Preliminary studies showed that BTPP accumulates in liver and in a syngeneic ovarian carcinoma, but not in normal brain parenchyma, of mice infused with BTPP subcutaneously for 6 or 7 days via surgically implanted osmotic minipumps. In this study, the uptake of boron was measured in human gliomas xenografted subcutaneously to athymic nude mice in which BTPP was infused intraperitoneally or subcutaneously or both for 3 or 7 days by using similar minipumps. Immunocompetent mice bearing a syngeneic ovarian carcinoma were similarly infused to provide comparative data. Bulk concentrations of boron up to 18 micrograms/g of glioma and up to 45 micrograms/g of carcinoma were observed when up to 102 micrograms/g of tissue was present in the liver after 7 days of BTPP infusion. Glioma boron concentrations were increased by approximately 80% on the average (up to 33 micrograms/g) when correspondingly greater amounts of BTPP were infused in only 3 days. Cell counts and chemical tests on blood samples from individual mice indicate that BTPP causes moderate hepatotoxicity and thrombocytopenia. This hepatohematic toxicity syndrome should be taken into account if BTPP or a similar agent is used for boron neutron-capture therapy (BNCT) of human malignancies.
Genetic analysis of membrane protein topology by a sandwich gene fusion approach.
Authors: Ehrmann M , Boyd D , Beckwith J
Published: May 2006

We describe a cloning vector that allows the construction of phoA sandwich fusions in which mature alkaline phosphatase is inserted into target proteins. In contrast to previous fusions obtained using the TnphoA transposon, the entire amino acid sequence of the target protein is present in the fusion product. We have constructed a series of sandwich fusions of alkaline phosphatase to the multispanning cytoplasmic membrane protein MalF. Despite the fact that the alkaline phosphatase was tethered to MalF at both its N and its C terminus, the enzyme exhibited high activity when it was fused to a periplasmic domain of the membrane protein. Cells harboring an alkaline phosphatase sandwich fusion to the end of the first membrane-spanning segment of MalF exhibited both MalF and alkaline phosphatase activity. When alkaline phosphatase was inserted into a cytoplasmic domain of MalF, its specific activity was very low. Our results suggest that the alkaline phosphatase activity of phoA sandwich fusions provides a more sensitive monitor than previous methods of the cellular localization of the domain of the target protein to which the enzyme is fused. Thus, the sandwich fusion approach can give a more accurate picture of membrane protein topology.
Detection of heteroduplex DNA molecules among the products of Saccharomyces cerevisiae meiosis.
Authors: Lichten M , Goyon C , Schultes N P , Treco D , Szostak J W , Haber J E , Nicolas A
Published: May 2006

We have used denaturant-gel electrophoresis to provide a physical demonstration of heteroduplex DNA in the products of yeast meiosis. We examined heteroduplex formation at arg4-nsp, a G.C----C.G transversion that displays a moderately high level of postmeiotic segregation. Of the two possible arg4-nsp/ARG4 mismatches (G.G and C.C), only C.C was detected in spores from mismatch repair-competent (Pms1+) diploids. In contrast, C.C and G.G were present at nearly equal levels in spores from Pms1- diploids. These results confirm previous suggestions that postmeiotic segregation spores contain heteroduplex DNA at the site of the marker in question, that C.C is repaired less frequently than is G.G, and that the PMS1 gene product plays a role in mismatch correction. Combined with the observation that Pms1+ ARG4/arg4-nsp diploids produce 3 times more 3+:5m (wildtype:mutant) tetrads (+, +, +/m, m) than 5+:3m tetrads (+, +/m, m, m), these results indicate that, during meiosis, formation of heteroduplex DNA at ARG4 involves preferential transfer of the sense (nontranscribed) strand of the DNA duplex.
Immunodominant regions for T helper-cell sensitization on the human nicotinic receptor alpha subunit in myasthenia gravis.
Authors: Protti M P , Manfredi A A , Straub C , Howard J F , Conti-Tronconi B M
Published: May 2006

In myasthenia gravis an autoimmune response against the nicotinic acetylcholine receptor (AChR) occurs. The alpha subunit of the AChR contains both the epitope(s) that dominates the antibody response (main immunogenic region) and epitopes involved in T helper cell sensitization. In this study, overlapping synthetic peptides corresponding to the complete AChR alpha-subunit sequence were used to propagate polyclonal AChR-specific T helper cell lines from four myasthenic patients of different HLA types. Response of the T helper lines to the individual peptides was studied. Four immunodominant sequence segments were identified--i.e., residues 48-67, 101-120, 304-322, and 419-437. These regions did not include residues known to form the main immunogenic region or the cholinergic binding site, and they frequently contained sequence motifs that have been proposed to be related to T-epitope formation.
Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment.
Authors: Jones T L , Simonds W F , Merendino J J , Brann M R , Spiegel A M
Published: May 2006

We transfected COS cells with cDNAs for the alpha subunits of stimulatory and inhibitory GTP-binding proteins, alpha s and alpha i1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled alpha s and alpha i were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected alpha i but could not be detected in alpha s even when it was overexpressed. We converted the second residue, glycine, of alpha i1 into alanine by site-directed mutagenesis. Upon transfection of the mutant alpha i1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal alpha i1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant alpha i1 could still interact with the beta-gamma complex, since purified beta gamma subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant alpha i1 subunits. These results indicate that myristoylation is critical for membrane attachment of alpha i but not alpha s subunits.
Localizing multiple X chromosome-linked retinitis pigmentosa loci using multilocus homogeneity tests.
Authors: Ott J , Bhattacharya S , Chen J D , Denton M J , Donald J , Dubay C , Farrar G J , Fishman G A , Frey D , Gal A
Published: May 2006

Multilocus linkage analysis of 62 family pedigrees with X chromosome-linked retinitis pigmentosa (XLRP) was undertaken to determine the presence of possible multiple disease loci and to reliably estimate their map location. Multilocus homogeneity tests furnished convincing evidence for the presence of two XLRP loci, the likelihood ratio being 6.4 x 10(9):1 in favor of two versus a single XLRP locus and gave accurate estimates for their map location. In 60-75% of the families, location of an XLRP gene was estimated at 1 centimorgan distal to OTC, and in 25-40% of the families, an XLRP locus was located halfway between DXS14 (p58-1) and DXZ1 (Xcen), with an estimated recombination fraction of 25% between the two XLRP loci. There is also good evidence for a third XLRP locus, midway between DXS28 (C7) and DXS164 (pERT87), supported by a likelihood ratio of 293:1 for three versus two XLRP loci.
Specific patterns of oncogene activation in transplacentally induced tumors.
Authors: Sukumar S , Barbacid M
Published: May 2006

Transplacental exposure of rats to a single dose of the direct acting carcinogen methylnitrosourea (MNU) results in the induction of a variety of neoplasias of neuroectodermal, epithelial, and mesenchymal origin. Molecular analysis of the oncogenes present in these tumors revealed a striking degree of tissue specificity. neu oncogenes were found to be reproducibly activated in tumors derived from the peripheral nervous system (PNS), but not in those arising from the central nervous system (CNS). No ras oncogenes were found in either PNS- or CNS-derived tumors. However, Ha-ras oncogenes were detected in each of three mammary carcinomas and Ki-ras oncogenes were present in each of five kidney mesenchymal tumors. These results illustrate that phenotypic expression of activated oncogenes in vivo is not a random process and suggest that normal developmental programs may play an important role in modulating the activation of specific oncogenes by chemical carcinogens. PCR analysis revealed that each of the ras oncogenes detected in these transplacentally induced tumors became activated by the same G----A transition in the second base of codon 12. Since G----A transitions are the preferred mutations induced by MNU, it is likely that these ras oncogenes may have been directly targeted by MNU during embryonic development.
Origin of the Metazoa.
Authors: Lake J A
Published: May 2006

The origin of the multicellular animals has been investigated by rate invariant analysis of 18S rRNA sequences. These analyses indicate that (i) the Metazoa is a monophyletic taxon; (ii) the Deuterostomia is a monophyletic taxon; (iii) the Annelida-Mollusca lineage is the sister group of an arthropod subgroup; and (iv) the last common ancestor of the Annelida-Mollusca lineage is most parsimoniously derived from a segmented, hemocoelic ancestor with an open circulatory system.
Stimulation of casein kinase II by epidermal growth factor: relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit.
Authors: Ackerman P , Glover C V , Osheroff N
Published: May 2006

To determine relationships between the hormonal activation of casein kinase II and its phosphorylation state, epidermal growth factor (EGF)-treated and EGF-naive human A-431 carcinoma cells were cultured in the presence of [32P]orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated [32P]phosphate than did its counterpart in untreated cells. Levels of kinase phosphorylation paralleled levels of kinase activity over a wide range of EGF concentrations as well as over a time course of hormone action. Approximately 97% of the incorporated [32P]phosphate was found in the beta subunit of casein kinase II. Both activated and hormone-naive kinase contained radioactive phosphoserine and phosphothreonine but no phosphotyrosine. On the basis of proteolytic mapping experiments, EGF treatment of A-431 cells led to an increase in the average [32P]phosphate content (i.e., hyperphosphorylation) of casein kinase II beta subunit peptides which were modified prior to hormone treatment. Finally, the effect of alkaline phosphatase on the reaction kinetics of activated casein kinase II indicated that hormonal stimulation of the kinase resulted from the increase in its phosphorylation state.
Finding sequence motifs in groups of functionally related proteins.
Authors: Smith H O , Annau T M , Chandrasegaran S
Published: May 2006

We have developed a method for rapidly finding patterns of conserved amino acid residues (motifs) in groups of functionally related proteins. All 3-amino acid patterns in a group of proteins of the type aa1 d1 aa2 d2 aa3, where d1 and d2 are distances that can be varied in a range up to 24 residues, are accumulated into an array. Segments of the proteins containing those patterns that occur most frequently are aligned on each other by a scoring method that obtains an average relatedness value for all the amino acids in each column of the aligned sequence block based on the Dayhoff relatedness odds matrix. The automated method successfully finds and displays nearly all of the sequence motifs that have been previously reported to occur in 33 reverse transcriptases, 18 DNA integrases, and 30 DNA methyltransferases.
Saccharomyces cerevisiae U1 small nuclear RNA secondary structure contains both universal and yeast-specific domains.
Authors: Kretzner L , Krol A , Rosbash M
Published: May 2006

The five small nuclear RNAs (snRNAs) involved in mammalian pre-mRNA splicing (U1, U2, U4, U5, and U6) are well conserved in length, sequence, and especially secondary structure. These five snRNAs from Saccharomyces cerevisiae show notable size and sequence differences from their metazoan counterparts. This is most striking for the large S. cerevisiae U1 and U2 snRNAs, for which no secondary structure models currently exist. Because of the importance of U1 snRNA in the early steps of "spliceosome" assembly, we wanted to compare the highly conserved secondary structure of metazoan U1 snRNA (approximately 165 nucleotides) with that of S. cerevisiae U1 snRNA (568 nucleotides). To this end, we have cloned and sequenced the U1 gene from two other yeast species possessing large U1 RNAs. Using computer-derived structure predictions, phylogenetic comparisons, and structure probing, we have arrived at a secondary structure model for S. cerevisiae U1 snRNA. The results show that most elements of higher eukaryotic U1 snRNA secondary structure are conserved in S. cerevisiae. The hundreds of "extra" nucleotides of yeast U1 RNA, also highly structured, suggest that large insertions and/or deletions have occurred during the evolution of the U1 gene.
A somatic gene rearrangement contributing to genetic diversity in maize.
Authors: Das O P , Levi-Minzi S , Koury M , Benner M , Messing J
Published: May 2006

We have discovered a somatic genomic rearrangement that occurs at high frequency at a duplicated zein locus in certain cultures of the maize inbred line A188. The rearranged allele arises from the duplication by a two-step process involving a homologous recombination and a second event, which may be a deletion, inversion, or insertion; both steps always occur together. The frequency of rearrangement is lower in homozygous states of the parental allele than in heterozygotes. In both cases, the rearrangement is shown to be mitotic. The rearranged product can be transmitted through meiosis, providing another mechanism for genome evolution in higher eukaryotes.
Protective effect of eicosapentaenoic acid on ouabain toxicity in neonatal rat cardiac myocytes.
Authors: Hallaq H , Sellmayer A , Smith T W , Leaf A
Published: May 2006

Isolated neonatal cardiac myocytes have been utilized as a model for the study of cardiac arrhythmogenic factors. The myocytes respond to the toxic effects of a potent cardiac glycoside, ouabain at 0.1 mM, by an increase in their spontaneous beating rate and a reduction in amplitude of contractions resulting within minutes in a lethal state of contracture. Incubating the isolated myocytes for 3-5 days in culture medium enriched with 5 microM arachidonic acid [20:4 (n-6)] had no effect on the development of lethal contracture after subsequent exposure to 0.1 mM ouabain. By contrast, incubating the myocytes for 3-5 days with 5 microM eicosapentaenoic acid [20:5 (n-3)] completely prevented the toxic effects of ouabain at 0.1 mM. There were no measurable differences in the degree to which ouabain inhibited Na,K-ATPase activity by comparing the control with the arachidonic acid- or the eicosapentaenoic acid-enriched myocytes. No differences in bumetanide-inhibitable 86Rb flux were observed between the three preparations. However, measurements with fura-2 of cytosolic free calcium levels indicated that control and arachidonic acid-enriched myocytes developed toxic cytosolic calcium concentrations of 845 +/- 29 and 757 +/- 64 nM, respectively, on exposure to 0.1 mM ouabain, whereas in eicosapentaenoic acid-enriched myocytes, physiologic calcium levels (214 +/- 29 nM) were preserved. Incubating the myocytes with eicosapentaenoic acid (5 microM) for 3-5 days resulted in a small reduction of arachidonic acid and a small but significant increase of eicosapentaenoic acid in membrane phospholipids of the myocytes.
Cloning of the cDNA encoding an RNA regulatory protein--the human iron-responsive element-binding protein.
Authors: Rouault T A , Tang C K , Kaptain S , Burgess W H , Haile D J , Samaniego F , McBride O W , Harford J B , Klausner R D
Published: May 2006

Iron-responsive elements (IREs) are stemloop structures found in the mRNAs encoding ferritin and the transferrin receptor. These elements participate in the iron-induced regulation of the translation of ferritin and the stability of the transferrin receptor mRNA. Regulation in both instances is mediated by binding of a cytosolic protein to the IREs. High-affinity binding is seen when cells are starved of iron and results in repression of ferritin translation and inhibition of transferrin receptor mRNA degradation. The IRE-binding protein (IRE-BP) has been identified as an approximately 90-kDa protein that has been purified by both affinity and conventional chromatography. In this report we use RNA affinity chromatography and two-dimensional gel electrophoresis to isolate the IRE-BP for protein sequencing. A degenerate oligonucleotide probe derived from a single peptide sequence was used to isolate a cDNA clone that encodes a protein containing 13 other sequenced peptides obtained from the IRE-BP. Consistent with previous characterization of the IRE-BP, the cDNA encodes a protein of 87 kDa with a slightly acidic pI, and the corresponding mRNA of approximately 3.6 kilobases is found in a variety of cell types. The encoded protein contains a nucleotide-binding consensus sequence and regions of cysteine and histidine clusters. This mRNA is encoded by a single gene on human chromosome 9, a finding consistent with previous localization by functional mapping. The protein contains no previously defined consensus motifs for either RNA or DNA binding. The simultaneous cloning of a different, but highly homologous, cDNA suggests that the IRE-BP is a member of a distinct gene family.
Diastrophic dysplasia gene maps to the distal long arm of chromosome 5.
Authors: Hästbacka J , Kaitila I , Sistonen P , de la Chapelle A
Published: May 2006

We have used polymorphic DNA markers to map the gene for a clinically well-characterized form of osteochondrodysplasia, diastrophic dysplasia (DD), an autosomal recessive disorder of unknown pathogenesis. Linkage was analyzed in 13 families with two or three affected sibs comprising a total of 84 individuals. Positive two-point logarithm-of-odds (lod) scores were obtained between the DD locus and three polymorphic markers on chromosome 5. The highest pairwise lod score estimate of 7.37 with zero recombination to locus D5S72 suggests very tight linkage. There was no evidence of heterogeneity. Multipoint linkage analysis against the published order of the three loci gave the result centromere-D5S84-(DD, D5S72)-D5S61-terminus with a four-point lod score of 9.11. The present findings place the DD locus distal to the gene for adenomatous polyposis coli on the distal part of the long arm of chromosome 5. Our results provide a basis for refining the map position of the DD locus followed by physical localization, isolation, and characterization of the gene.
Genetic organization of the cellulose synthase operon in Acetobacter xylinum.
Authors: Wong H C , Fear A L , Calhoon R D , Eichinger G H , Mayer R , Amikam D , Benziman M , Gelfand D H , Meade J H , Emerick A W
Published: May 2006

An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum.
Direct measurement of oligonucleotide substrate binding to wild-type and mutant ribozymes from Tetrahymena.
Authors: Pyle A M , McSwiggen J A , Cech T R
Published: May 2006

Like protein enzymes, RNA enzymes (ribozymes) provide specific binding sites for their substrates. We now show that equilibrium dissociation constants for complexes between the Tetrahymena ribozyme and its RNA substrates and products can be directly measured by electrophoresis in polyacrylamide gels containing divalent cations. Binding is 10(3)- to 10(4)-fold tighter (4-5 kcal/mol at 42 degrees C) than expected from base-pairing interactions alone, implying that tertiary interactions also contribute to energetic stabilization. Binding decreases with single base changes in the substrate, substitution of deoxyribose sugars, and lower Mg2+ concentration. Ca2+, which enables the ribozyme to fold but is unable to mediate efficient RNA cleavage, promotes weaker substrate binding than Mg2+. This indicates that Mg2+ has special roles in both substrate binding and catalysis. Mutagenesis of a region near the internal guide sequence disrupts substrate binding, whereas binding is not significantly affected by a mutation of the guanosine-binding site. This approach should be generally useful for analysis of ribozyme variants independent of their catalytic activities.
Fructose-induced aberration of metabolism in familial gout identified by 31P magnetic resonance spectroscopy.
Authors: Seegmiller J E , Dixon R M , Kemp G J , Angus P W , McAlindon T E , Dieppe P , Rajagopalan B , Radda G K
Published: May 2006

The hyperuricemia responsible for the development of gouty arthritis results from a wide range of environmental factors and underlying genetically determined aberrations of metabolism. 31P magnetic resonance spectroscopy studies of children with hereditary fructose intolerance revealed a readily detectable rise in phosphomonoesters with a marked fall in inorganic phosphate in their liver in vivo and a rise in serum urate in response to very low doses of oral fructose. Parents and some family members heterozygous for this enzyme deficiency showed a similar pattern when given a substantially larger dose of fructose. Three of the nine heterozygotes thus identified also had clinical gout, suggesting the possibility of this defect being a fairly common cause of gout. In the present study this same noninvasive technology was used to identify the same spectral pattern in 2 of the 11 families studied with hereditary gout. In one family, the index patient's three brothers and his mother all showed the fructose-induced abnormality of metabolism, in agreement with the maternal inheritance of the gout in this family group. The test dose of fructose used produced a significantly larger increment in the concentration of serum urate in the patients showing the changes in 31P magnetic resonance spectra than in the other patients with familial gout or in nonaffected members, thus suggesting a simpler method for initial screening for the defect.
Hypomutability in Fanconi anemia cells is associated with increased deletion frequency at the HPRT locus.
Authors: Papadopoulo D , Guillouf C , Mohrenweiser H , Moustacchi E
Published: May 2006

Fanconi anemia (FA) is an inherited human disorder associated with a predisposition to cancer and characterized by anomalies in the processing of DNA cross-links and certain monoadducts. We reported previously that the frequency of psoralen-photoinduced mutations at the HPRT locus is lower in FA cells than in normal cells. This hypomutability is shown here to be associated with an increased frequency of deletions in the HPRT gene when either a mixture of cross-links and monoadducts or monoadducts alone are induced. Molecular analysis of mutants in the HPRT gene was carried out. In normal cells the majority of spontaneous and induced mutants are point mutations whereas in FA deletion mutations predominate. In that case a majority of mutants were found to lack individual exons or small clusters of exons whereas in normal cells large (complete or major gene loss) and small deletions are almost equally represented. Thus we propose that the FA defect lies in a mutagenic pathway that, in normal cells, involves bypassing lesions and subsequent gap filling by a recombinational process during replication.