bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacteriorhodopsin's L550 intermediate contains a C14-C15 s-trans-retinal chromophore.
Authors: Fodor S P , Pollard W T , Gebhard R , van den Berg E M , Lugtenburg J , Mathies R A
Published: May 2006

Conformational changes of the retinal chromophore about the C14-C15 bond in bacteriorhodopsin (BR) have been proposed in models for the mechanism of light-driven proton transport. To determine the C14-C15 conformation in BR's L550 intermediate, we have examined the resonance Raman spectra of BR derivatives regenerated with retinal deuterated at the 14 and 15 positions. Vibrational calculations show that the C14-2H and C15-2H rocking modes form symmetric (A) and antisymmetric (B) combinations in [14,15-2H]retinal chromophores. When there is a trans conformation about the single bond between C14 and C15 (14-s-trans), a small frequency separation or splitting is observed between the A and B modes, which are found at approximately equal to 970 cm-1. In 14-s-cis molecules, the splitting is large, and the Raman-active symmetric A mode is predicted at approximately equal to 850 cm-1. In addition, the monodeuterium rock should appear at an unusually low frequency (920-930 cm-1) in the 14-2H-labeled 14-s-cis molecules. These patterns are insensitive to computational details: similar results are predicted by a modified Urey-Bradley force field and by MNDO (modified neglect of differential overlap) calculations for twisted chromophores and for highly delocalized protonated Schiff base cations. Time-resolved resonance Raman spectra were obtained of BR's L550 intermediate regenerated with [14-2H]-, [15-2H]- and [14,15-2H]retinal. The symmetric A rock in L550 is found at 968 cm-1, within 4 cm-1 of the frequencies for the monodeuterio derivatives, and no scattering is observed between 800 and 940 cm-1. The rocking frequencies of deuterated L550 are within 5 cm-1 of those observed in BR568, which contains a 14-s-trans chromophore. These results show that L550 contains a 14-s-trans chromophore and suggest that only 14-s-trans structures are involved in the proton pumping photocycle of BR.
Mutational analysis of insertion sequence 50 (IS50) and transposon 5 (Tn5) ends.
Authors: Makris J C , Nordmann P L , Reznikoff W S
Published: May 2006

Insertion sequence 50 (IS50) transposition utilizes a 19-base-pair "outside" end and a 19-base-pair "inside" end in inverted orientation relative to each other, whereas transposon 5 (Tn5) transposition utilizes two inverted outside ends. The frequency of transposition events that involve an inside end is regulated 1000-fold by the host dam methylase system. The end sequence requirements for transposition and its regulation by dam methylase were analyzed in Escherichia coli by generating random single base pair mutations in either an IS50 inside end or outside end placed in inverted orientation with respect to an unmutagenized outside end. The mutations were then isolated, assayed for transposition phenotype, and sequenced. Mutations were isolated at 15 of the 19 sites in the outside end. All of these mutations except those at position 4 decreased transposition. Mutations at position 4 (which is the only nonidentical base pair in a region of homology between the outside and inside ends) had no effect on transposition. Mutations were isolated at 11 of the 19 sites in the inside end. All of these mutations, including one at position 4, decreased transposition in dam- cells. Mutations at position 10 (within a dam recognition sequence) and 2 (not within a dam recognition sequence) reduced the magnitude of dam regulation. A mutation within a dam recognition sequence adjacent to the required 19 base pairs of the inside end did not reduce the magnitude of dam regulation.
Thiazide diuretic drug receptors in rat kidney: identification with [3H]metolazone.
Authors: Beaumont K , Vaughn D A , Fanestil D D
Published: May 2006

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. We report here that [3H]metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. [3H]Metolazone bound with high affinity (Kd = 4.27 nM) to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or no binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated with their clinical potency. Halide anions (Cl-, Br-, and I-) specifically inhibited high-affinity binding of [3H]metolazone to this site. [3H]Metolazone also bound with lower affinity (Kd = 289 nM) to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had Ki values of 0.6-10 microM for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity [3H]metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.
Some rules for predicting the base-sequence dependence of DNA conformation.
Authors: Peticolas W L , Wang Y , Thomas G A
Published: May 2006

Two tables have been constructed showing the crystal and solution conformations of short sequences of DNA. Each of these DNAs has been found to be in one of three different conformations--the A, B, or Z form--depending upon the base sequence and the environmental conditions. A set of rules is presented showing the tendency of certain base pairs to direct the DNA conformation into the A, B, or Z genus in saturated salt solutions and in crystals. These rules are based on a consideration of nearest-neighbor interactions that are interpreted in terms of 10 different two-letter code words made from the letters denoting the bases guanine (G), cytosine (C), adenine (A), and thymine (T). One table discusses the effect on DNA conformation of 3 strong words that tend to direct a DNA oligomer into either the A, B, or Z genus in crystals or in aqueous solutions containing a high salt concentration (6 M). The second table discusses the remaining 7 code words that appear to have a much weaker effect on conformation. The sequences that are most likely to lead to A-Z, B-Z, and A-B junctions are discussed, as is the possible biological significance of these rules.
Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia-inhibitory factor.
Authors: Gough N M , Gearing D P , King J A , Willson T A , Hilton D J , Nicola N A , Metcalf D
Published: May 2006

A human homologue of the recently cloned murine leukemia-inhibitory factor (LIF) gene was isolated from a genomic library by using the murine cDNA as a hybridization probe. The nucleotide sequence of the human gene indicated that human LIF has 78% amino acid sequence identity with murine LIF, with no insertions or deletions, and that the region of the human gene encoding the mature protein has one intervening sequence. After oligonucleotide-mediated mutagenesis, the mature protein-coding region of the LIF gene was introduced into the yeast expression vector YEpsec1. Yeast cells transformed with the resulting recombinant could be induced with galactose to produce high levels of a factor that induced the differentiation of murine M1 leukemic cells in a manner analogous to murine LIF. This factor competed with 125I-labeled native murine LIF for binding to specific cellular receptors on murine cells, compatible with a high degree of structural similarity between the murine and human factors.
Learning and memory: regional changes in N-methyl-D-aspartate receptors in the chick brain after imprinting.
Authors: McCabe B J , Horn G
Published: May 2006

An extensive series of experiments has implicated a restricted region of the chick forebrain in the learning process of imprinting. The region is the intermediate and medial part of the hyperstriatum ventrale (IMHV). Previous studies have shown that training is associated with an increase in the area of the postsynaptic density of axospinous synapses in the left but not the right IMHV. The postsynaptic density is a site of high receptor density, and at least some axospinous synapses are excitatory. We found that imprinting is associated with a 59% increase in N-methyl-D-aspartate-sensitive binding of the excitatory amino acid L-[3H]glutamic acid in the left IMHV. The increase is probably due to an increased number of binding sites. The profile of sensitivity of the sites to a series of amino-, phosphono-substituted carboxylic acids (2-amino-3-phosphonopropionate to 2-amino-8-phosphonooctanoate) is characteristic of N-methyl-D-aspartate-type receptors. There were no significant effects of training on binding in the right IMHV. The effect of training on left IMHV binding could not be attributed to light exposure, arousal, or motor activity per se but was a function of how much the chicks learned. The changes in the left IMHV could increase the effectiveness of synaptic transmission in a region crucial for information storage and so form a neural basis for recognition memory.
Accurate and efficient pre-mRNA splicing in Drosophila cell-free extracts.
Authors: Rio D C
Published: May 2006

Synthetic mRNA precursors from the Drosophila fushi tarazu (ftz) gene were shown to be accurately and efficiently spliced in Drosophila nuclear extracts derived from Kc tissue culture cells or 0- to 12-hr embryos. Splicing the ftz pre-mRNA requires ATP and low levels of Mg2+. The reaction proceeds with a lag of 20-30 min prior to appearance of spliced mRNA and appears to proceed in two steps. The first step is cleavage at the 5' splice site to generate a 5' exon (E1) fragment and an intron-3' exon (IVS-E2) species. The second step involves cleavage at the 3' splice site, ligation of the two exons (E1-E2), and intron (IVS) release. The excised intron (IVS) and intron-3' exon (IVS-E2) exhibit anomalous electrophoretic mobility, suggesting that they contain branched structures. Nuclease analysis using two-dimensional thin-layer chromatography indicates that both the IVS and IVS-E2 species possess branched trinucleotides in which a guanosine residue at the 5' end of the intron is linked in a 2'-5' phosphodiester bond to the 2' hydroxyl group of an adenosine residue in the intron. The site of branchpoint formation was localized by debranching the Drosophila lariat with mammalian (HeLa) cell debranching enzyme and by P1 and T2 nuclease analysis. These findings indicate that nuclear extracts derived from Drosophila cultured cells or embryos can accurately splice mRNA precursors and that the reaction mechanism is the same as has been observed in yeast and mammalian cells. This system provides an initial step toward the biochemical analysis of developmentally regulated pre-mRNA splicing events in Drosophila.
Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid).
Authors: Bolander M E , Young M F , Fisher L W , Yamada Y , Termine J D
Published: May 2006

Osteonectin is a prominent noncollagenous protein of developing bone. A 2150-base-pair cDNA coding for osteonectin, isolated from a bovine bone cell lambda gt11 expression library, was sequenced and identified by comparison with protein sequence data. The nucleotide sequence predicts that osteonectin contains 304 amino acids, including a 17-residue signal peptide. Analysis of the deduced protein sequence suggests that the secreted protein contains at least four distinct structural domains. An acidic region at the amino terminus of the protein appears to be a potential hydroxyapatite-binding site. This is followed by a second domain, rich in cysteine, that shows sequence homology with cysteine-rich domains in turkey ovomucoid and other serine proteinase inhibitors. Two sequences homologous with central calcium-binding loops of "EF hands" and thus having potential to be high-affinity calcium-binding sites are located in two other domains within the carboxyl-terminal half of the protein. Finally, the osteonectin sequence shows near identity (greater than 90%) with another protein, SPARC (secreted protein, acidic and rich in cysteine), secreted by mouse parietal endoderm. These data suggest that osteonectin, a protein present in bone and other selected tissues, is a multifunctional protein.
Chimeric cytotoxin IL2-PE40 delays and mitigates adjuvant-induced arthritis in rats.
Authors: Case J P , Lorberboum-Galski H , Lafyatis R , FitzGerald D , Wilder R L , Pastan I
Published: May 2006

Adjuvant arthritis in rats is a T-cell dependent "autoimmune" disease with close similarities to several forms of human arthritis. Injection of mycobacterial adjuvant leads to T-cell activation and proliferation, processes in which the de novo expression of the interleukin 2 (IL-2) receptor plays a pivotal role. The subsequent massive mononuclear cell infiltration of the joints ultimately results in complete joint destruction. Because activation of the helper/inducer subset of T lymphocytes is critical to the establishment of disease, we reasoned that IL2-PE40, a cytotoxic IL-2-Pseudomonas exotoxin fusion protein that targets the membrane-penetration and ADP-ribosylation domains of the toxin to cells bearing the IL-2 receptor, would be an effective and specific therapy. Adjuvant-injected rats were randomized to treatment with IL2-PE40, phosphate-buffered saline, or either of two control proteins related to IL2-PE40 but lacking either the receptor-binding moiety or an enzymatically active toxin domain and previously demonstrated to lack cytotoxicity in vitro. Intraperitoneal IL2-PE40 given before the establishment of overt clinical disease proved an effective and specific modifier of adjuvant arthritis by clinical, histological, and radiographic criteria. Our data suggest that IL2-PE40 may be effective in those diseases in which activated T-cells play an important role.
Protection of rhesus monkeys from fatal Lassa fever by vaccination with a recombinant vaccinia virus containing the Lassa virus glycoprotein gene.
Authors: Fisher-Hoch S P , McCormick J B , Auperin D , Brown B G , Castor M , Perez G , Ruo S , Conaty A , Brammer L , Bauer S
Published: May 2006

Lassa fever is an acute febrile disease of West Africa, where there are as many as 300,000 infections a year and an estimated 3000 deaths. As control of the rodent host is impracticable at present, the best immediate prospect is vaccination. We tested as potential vaccines in rhesus monkeys a closely related virus, Mopeia virus (two monkeys), and a recombinant vaccinia virus containing the Lassa virus glycoprotein gene, V-LSGPC (four monkeys). Two monkeys vaccinated with the New York Board of Health strain of vaccinia virus as controls died after challenge with Lassa virus. The two monkeys vaccinated with Mopeia virus developed antibodies measurable by radioimmunoprecipitation prior to challenge, and they survived challenge by Lassa virus with minimal physical or physiologic disturbances. However, both showed a transient, low-titer Lassa viremia. Two of the four animals vaccinated with V-LSGPC had antibodies to both Lassa glycoproteins, as determined by radioimmunoprecipitation. All four animals survived a challenge of Lassa virus but experienced a transient febrile illness and moderate physiologic changes following challenge. Virus was recoverable from each of these animals, but at low titer and only during a brief period, as observed for the Mopeia-protected animals. We conclude that V-LSGPC can protect rhesus monkeys against death from Lassa fever.
Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor.
Authors: Yamagata H , Nakahama K , Suzuki Y , Kakinuma A , Tsukagoshi N , Udaka S
Published: May 2006

Using previously isolated Bacillus brevis strains that secrete large amounts of proteins but little protease into the medium, we have developed a host-vector system for very efficient synthesis and secretion of heterologous proteins. The multiple promoters and the signal-peptide-coding region of the MWP gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, were used to construct expression-secretion vectors. With this system, a synthetic gene for human epidermal growth factor (hEGF) was expressed efficiently and a large amount (0.24 g per liter of culture) of mature hEGF was secreted into the medium. hEGF purified from the culture supernatant had the same NH2-terminal amino acid sequence, COOH-terminal amino acid, and amino acid composition as natural hEGF, and it was fully active in biological assays. These results, in combination with previous results, showed that mammalian proteins can be produced in active form 10-100 times more efficiently in B. brevis than has been reported in other systems.
Heat shock protein hsp70 cognate gene expression in vegetative and reproductive organs of
Lycopersicon esculentum
Authors: Duck Nicholas , McCormick Sheila , Winter Jill
Published: May 2006

We have detected hsc70 gene expression (heat shock protein hsp70 cognate) during vegetative growth and reproductive development in tomato (
Lycopersicon esculentum
). Using RNA from a tomato hsc70 cDNA as a probe in
in situ
hybridizations, we have determined expression patterns of hsc70 in nonstressed tomato roots, stems, leaves, flowers, and developing fruits. We have localized high levels of hsc70 transcript to the vascular system of the ovary, dividing cells of the lateral root tips, and the inner integument of developing seeds. We also see expression in the transmitting tissue, in immature anthers, and in embryos. We cannot detect expression in mature pollen, xylem, or ovules. These data indicate that the expression of at least some tomato hsp70 family members is developmentally regulated.
Plasmin catalyzes binding of lipoprotein (a) to immobilized fibrinogen and fibrin.
Authors: Harpel P C , Gordon B R , Parker T S
Published: May 2006

Lipoprotein (a) [Lp(a)] is a plasma component whose concentration is related to the development of atherosclerosis, although the underlying mechanisms are not known. Lp(a) contains a unique structure, apolipoprotein (a), that shares partial homology with plasminogen. We now report that plasmin catalyzes the binding of Lp(a) to both immobilized fibrinogen and fibrin in a manner analogous to our previously reported studies with plasminogen. Plasmin treatment of immobilized fibrinogen induces a 3.7-fold increase in Lp(a) binding. Low density lipoprotein, molecules similar to Lp(a) but lacking apolipoprotein (a), bind poorly to immobilized fibrinogen and binding is not increased by plasmin. Trypsin but not neutrophil elastase also increases the binding of Lp(a) to fibrinogen. Lp(a) also complexes to plasmin-fibrinogen digests, and binding increases in proportion to the time of plasmin-induced fibrinogen degradation. Lp(a) binding is lysine-binding site dependent as it is inhibited by epsilon-aminocaproic acid. Lp(a) inhibits the binding of plasminogen to plasmin-modified immobilized fibrinogen, indicating that both molecules compete for similar lysine-binding sites. These findings demonstrate an affinity between Lp(a) and protease-modified fibrinogen or fibrin and thereby provide a potential mechanism to explain the association between thrombosis, coronary atherosclerosis, and increased blood concentrations of Lp(a).
Activity of ion channels during volume regulation by clonal N1E115 neuroblastoma cells.
Authors: Falke L C , Misler S
Published: May 2006

When exposed to a hypotonic bathing solution, clonal N1E115 neuroblastoma cells initially swell and then undergo a regulatory volume decrease (RVD). Using cell-attached patch-clamp recording, we have found that the activity of a stretch-sensitive, nonselective cation [C+(SA)] channel increases shortly after the onset of osmotically induced cell swelling; this depolarizes the cells as much as 30 mV. Shortly thereafter, and roughly coincident with the onset of RVD, two types of voltage-dependent channels open at the new resting potential; these are (i) a delayed-rectifier type K+ [K+(DR)] channel and (ii) a large-conductance anion channel. We suggest that opening of the C+(SA) channel may contribute to the volume "sensor" mechanism, while the depolarization-induced opening of the K+(DR) and anion channels may constitute a significant K+ salt exit pathway, operating in RVD.
Translational repression in bacteriophage f1: characterization of the gene V protein target on the gene II mRNA.
Authors: Michel B , Zinder N D
Published: May 2006

Previous studies have shown that the single-stranded DNA binding protein of bacteriophage f1 (gene V protein) represses the translation of the mRNA of the phage-encoded replication protein (gene II protein). We have characterized phage mutations in the repressor and in its target. Using a gene II-lacZ translational fusion, we have defined a 16-nucleotide-long region in the gene II mRNA sequence that is required in vivo for repression by the gene V protein. We have shown that in vitro the binding affinity of the gene V protein is at least 10-fold higher to an RNA carrying this sequence than to an RNA lacking it. We propose that this sequence constitutes the gene II mRNA operator.
Structural features of the acetyl-CoA carboxylase gene: mechanisms for the generation of mRNAs with 5' end heterogeneity.
Authors: Luo X C , Park K , Lopez-Casillas F , Kim K H
Published: May 2006

Acetyl-CoA carboxylase [acetyl-CoA:carbondioxide ligase (ADP-forming), EC 6.4.1.2] is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. We have previously characterized five acetyl-CoA carboxylase mRNA species that differ in their 5' untranslated regions but not in the coding region. We have now characterized the exon-intron structure of the genomic DNA that encodes the 5' untranslated region of the mRNA. Generation of different forms of the mRNA is the result of the selective use of two promoters and differential splicing of five different exons. These five exons contain a total of 645 nucleotides and they are scattered over a 50-kilobase-pair genomic DNA region that we have characterized.
An Escherichia coli chemoreceptor gene is temporally controlled in Caulobacter.
Authors: Frederikse P H , Shapiro L
Published: May 2006

Flagellar and chemotaxis genes are transcribed at a discrete time in the Caulobacter cell cycle. We demonstrate here that the expression of the Escherichia coli chemoreceptor gene tsr, with 2.6 kilobases of its upstream sequence, is temporally controlled in Caulobacter crescentus. The tsr gene was placed on the chromosome in single copy or on a low-copy-number plasmid. It was found that the Tsr protein appeared at the same point in the cell cycle as an endogenous C. crescentus methyl-accepting chemotaxis protein. Nuclease S1 mapping experiments showed that the tsr transcript was also controlled by the cell cycle, suggesting that the E. coli tsr gene is regulated by C. crescentus factors that mediate the timing of transcription initiation. The apparent transcription start site of the E. coli tsr gene was determined in both E. coli and C. crescentus, and we found that in both backgrounds the promoter used conforms to the consensus sequence for the promoters of the flagellar and chemosensory genes of Bacillus subtilis and E. coli. The use of this promoter suggests that C. crescentus has a cognate sigma factor and predicts that other C. crescentus genes are expressed from this consensus promoter.
Limited diversity of T-cell receptor gamma-chain expression of murine Thy-1+ dendritic epidermal cells revealed by V gamma 3-specific monoclonal antibody.
Authors: Havran W L , Grell S , Duwe G , Kimura J , Wilson A , Kruisbeek A M , O'Brien R L , Born W , Tigelaar R E , Allison J P
Published: May 2006

To study the origin of and the degree of T-cell antigen receptor (TCR) diversity of Thy-1+ dendritic epidermal cells (Thy-1+ dECs) in mice, we have developed a monoclonal antibody (mAb 536) to the gamma delta TCR. mAb 536 binds to and stimulates interleukin 2 secretion from Thy-1+ dEC but not cells that express TCR composed of alpha and beta chains. mAb 536 precipitates CD3-associated gamma and delta chains from lysates of radioiodinated Thy-1+ dECs. Analysis of a panel of hybridomas that express gamma delta TCR indicated that mAb 536 defines an epitope of the variable region (V gamma 3) gene product. Flow cytometric analysis revealed that expression of V gamma 3 in the adult mouse is restricted to cells in the epidermis, where essentially all Thy-1+ cells are V gamma 3+. The majority of CD3+ cells in the 14-day fetal thymus also express V gamma 3. These results indicate that the T-cell complement in epidermis are cells that express gamma delta TCR and that the diversity of antigens recognized by the cells might be restricted by the use of a single V gamma gene segment. Finally, the data raise the intriguing possibility that Thy-1+ dECs may arise from precursors that are among the first to emerge from the developing thymus. This suggests that V gene usage during thymocyte development is highly regulated and has important consequences on the tissue localization and function of the emerging cells. As in other developing tissues, it appears that programmed and transient gene expression determines the fate of the emerging cells.
Allorecognition of DR1 by T cells from a DR4/DRw13 responder mimics self-restricted recognition of endogenous peptides.
Authors: Lombardi G , Sidhu S , Batchelor J R , Lechler R I
Published: May 2006

Coculture of a series of anti-DR1Dw1 alloreactive human T-cell clones with autologous (DR4Dw4/DRw13Dw19) antigen-presenting cells and a series of recall antigens revealed that two of four clones tested proliferated in response to Candida albicans. One was restricted by DR4Dw4 and the other was restricted by DRw13Dw19. These results provide further evidence that many alloreactive T cells have a primary self-restricted specificity and cross-react on allogeneic major histocompatibility complex products. Structural comparison of the responder and stimulator DR molecules for these clones revealed that the regions predicted to contact the T cells' receptor, and thereby to determine self-restriction, are identical in sequence for DR4Dw4 and DR1Dw1 and differ by one residue between DRw13Dw19 and DR1Dw1. The DR beta residues that differ between responder, DR4Dw4 and DR13Dw19, and stimulator, DR1Dw1, are predicted to contribute to antigen binding. This implies that these anti-DR1 T cells may be specific for endogenous peptides that are bound by DR1 and not by the responder DR products, seen in a self-restricted manner. These T-cell clones also cross-reacted on DR4Dw13 and DRw14Dw16 molecules and on a human/murine hybrid class II dimer DR1 beta/I-E alpha. These reactions are discussed in terms of self-restricted peptide recognition. Thus these data suggest that in certain responder/stimulator combinations allorecognition may resemble self-restricted recognition of fragments of endogenous antigens that are bound by stimulator but not by responder major histocompatibility complex products.
T5 DNA polymerase: structural--functional relationships to other DNA polymerases.
Authors: Leavitt M C , Ito J
Published: May 2006

T5 DNA polymerase, a highly processive single-polypeptide enzyme, has been analyzed for its primary structural features. The amino acid sequence of T5 DNA polymerase has a high degree of homology with that of DNA polymerase I from Escherichia coli and retains many of the amino acid residues that have been implicated in the 3'----5' exonuclease and DNA polymerase activities of that enzyme. Alignment with sequences of polymerase I and T7 DNA polymerase was used to identify regions possibly involved in the high processivity of this enzyme. Further, amino acid sequence comparisons of T5 DNA polymerase with a large group of DNA polymerases previously shown to exhibit little similarity to polymerase I indicate certain sequence segments are shared among distantly related DNA polymerases. These shared regions have been implicated in the 3'----5' exonuclease function of polymerase I, which suggests that the proofreading domains of all these enzymes may be evolutionarily related.
Structural homology between lymphocyte receptors for high endothelium and class III extracellular matrix receptor.
Authors: Gallatin W M , Wayner E A , Hoffman P A , St John T , Butcher E C , Carter W G
Published: May 2006

We have identified extensive structural homology between one type of heterotypic adhesion receptor (HAR) involved in lymphocyte interactions with high endothelium in lymphoid organs and a collagen-binding protein, termed class III extracellular matrix receptor (ECMRIII), expressed on most nucleated cell types. Both receptors have been described as heterogeneous 90-kDa transmembrane glycoproteins, referred to here as gp90. Monoclonal anti-HAR antibodies, Hermes-1 and Hutch-1, and monoclonal anti-ECMRIII antibodies, P1G12 and P3H9, were utilized to compare the two receptors. (i) All these monoclonal antibodies immunoprecipitated major gp90 components as well as uncharacterized additional higher molecular mass antigens of 120-200 kDa in human and macaque fibroblasts and peripheral blood mononuclear cells. (ii) Competitive binding analyses with the antibodies identified distinct epitopes present on gp90. (iii) Enzymatic and chemical digestions generated identical peptide fragments from all the antigens in human and macaque fibroblasts and peripheral blood mononuclear cells. (iv) Sequential immunoprecipitation with P1G12 followed by the other monoclonal antibodies indicated that all gp90 species reactive with Hermes-1 and Hutch-1 also expressed the P1G12 defined epitope. In reciprocal experiments, Hermes-1 and Hutch-1 immunoprecipitation did not completely remove all P1G12-reactive gp90 from cellular extracts. One inference from these data would be that gp90 is serologically heterogeneous, encompassing HARs as a major subset of this broadly expressed class of molecules.
Antibody-dependent enhancement of human immunodeficiency virus type 1 (HIV-1) infection in vitro by serum from HIV-1-infected and passively immunized chimpanzees.
Authors: Robinson W E , Montefiori D C , Mitchell W M , Prince A M , Alter H J , Dreesman G R , Eichberg J W
Published: May 2006

Based on recent reports of antibody-dependent enhancement of human immunodeficiency virus type 1 (HIV-1) infection in vitro by serum from HIV-1-infected humans, sera from HIV-1 antibody-positive chimpanzees (Pan troglodytes) was evaluated for enhancing activity in an in vitro infection assay that uses MT-2 cells (a human lymphoblastoid cell line). Although fresh chimpanzee serum was found to have pronounced infection-enhancing properties in the absence of antibody to HIV-1, this effect was abolished by heat inactivation (57 degrees C, 1 hr) or treatment with cobra venom anticomplementary protein. Heat-inactivated, HIV-1 antibody-positive chimpanzee serum could enhance HIV-1 infection of MT-2 cells in vitro when combined with fresh, normal human serum. By serial serum samples from three HIV-1-infected chimpanzees, HIV-1 antibody-positive chimpanzees are shown to develop enhancing antibodies early in infection (2 mo postchallenge), whereas neutralizing antibodies develop later. Over the course of HIV-1 infection, this enhancing activity decreases while neutralizing activity increases, suggesting a possible role for enhancing and neutralizing activities in HIV-1 pathogenesis. The enhancing activity of an IgG fraction used to passively immunize chimpanzees against HIV-1 infection is shown to be present at dilutions as high as 1:65,000, offering an interesting possible reason for the failure of passive immunization to protect chimpanzees from HIV infection. These results suggest that serum from HIV-1-immunized chimpanzees might be tested to determine whether current HIV-1 candidate vaccines induce production of antibodies that mediate antibody-dependent enhancement of HIV-1 infection in this in vitro assay.
Role of aspartate-96 in proton translocation by bacteriorhodopsin.
Authors: Gerwert K , Hess B , Soppa J , Oesterhelt D
Published: May 2006

Proton transfer reactions in bacteriorhodopsin were investigated by Fourier transform infrared spectroscopy, using a mutant protein in which Asp-96 was replaced by Asn-96. By comparison of the BR - K, BR - L, and BR - M difference spectra (BR indicating bacteriorhodopsin ground state and K, L, and M indicating photo-intermediates) of the wild-type protein with the corresponding difference spectra of the mutant protein, detailed insight into the functional role of this residue in the proton pump mechanism is obtained. Asp-96 is protonated in BR, as well as another aspartic residue, which is tentatively assigned to be Asp-115. Asp-96 is not affected in the primary photoreaction. During formation of the L intermediate it is subjected to a change in the H-bonding character of its carboxylic group, but no deprotonation occurs at this reaction step. Also, in the mutant protein a light-induced structural change of the protein interior near the Asn-96 residue is probed. The BR - M difference spectrum of the mutant protein lacks the negative carbonyl band at 1742 cm-1 of Asp-96 and in addition a positive band at about 1378 cm-1, which is most likely to be caused by the carboxylate vibration of Asp-96. This argues for a deprotonation of Asp-96 in the time range of the M intermediate during its photostationary accumulation. On the basis of these results, it is suggested that the point mutation does not induce a gross change of the protein structure, but a proton-binding site in the proton pathway from the cytoplasmic side to the Schiff base is lost.
Transcriptional bias: a non-Lamarckian mechanism for substrate-induced mutations.
Authors: Davis B D
Published: May 2006

In bacterial cultures in the stationary phase, substrates can selectively stimulate mutations that lead to their own utilization, but because of apparent conflict with the neo-Darwinian view of evolution the phenomenon has encountered widespread resistance. Building on further evidence for this process, Cairns et al. [Cairns, J., Overbaugh, J. & Miller, S. (1988) Nature (London) 335, 142-145] have suggested a Lamarckian mechanism of directed mutation. This paper proposes an alternative mechanism: transcription induced by the substrate introduces a bias in the random process of mutation, because the resulting single-stranded regions of DNA are more mutable. This stimulation of adaptive mutations by the environment has implications for evolution similar to those of directed mutation, but without contradicting the central "dogma" of molecular genetics. In addition, in eukaryotic cells a mutagenic effect of induction on protooncogenes could contribute to the stimulatory effect of proliferation on carcinogenesis.
Identification of a monocyte specific pre-interleukin 1 beta convertase activity.
Authors: Kostura M J , Tocci M J , Limjuco G , Chin J , Cameron P , Hillman A G , Chartrain N A , Schmidt J A
Published: May 2006

Interleukin 1 (IL-1) is a lymphokine secreted by monocytes in response to a variety of inflammatory stimuli. IL-1 beta, the predominant form of IL-1 produced by human monocytes, is synthesized as an inactive precursor of 31 kDa and is cleaved at Asp116-Ala117 to yield a 17.5-kDa extracellular form. The exact cellular site of cleavage and mechanism of secretion is at present unknown. We have prepared cell-free postnuclear extracts from freshly isolated human monocytes as well as THP.1 cells, a human monocyte-like cell line, and various blood lymphocytes and fibroblast cell lines. Using pre-IL-1 beta synthesized by in vitro transcription and translation, we have shown that only extracts derived from human monocytes and THP.1 cells were capable of cleaving precursor IL-1 beta to authentic mature IL-1 beta. Subcellular fractionation of the extracts suggested that the processing activity is found in the cytosol of monocytes or monocyte-like cell lines. The cleavage product of this protease is identical to authentic IL-1 beta as shown by mobility on SDS/PAGE and amino acid sequence analysis of the [3H]leucine-labeled product. The cleavage product is also capable of binding to the IL-1 receptor found on fibroblast membranes. Finally, mutation of Asp116----Ala116 rendered the IL-1 beta precursor resistant to cleavage by the processing activity. We conclude that a protease activity found only in monocytes will specifically process IL-1 beta to an active form.
Structural characterization of a 2:1 distamycin A.d(CGCAAATTGGC) complex by two-dimensional NMR.
Authors: Pelton J G , Wemmer D E
Published: May 2006

Two-dimensional NMR has been used to study the 2:1 distamycin A.d(CGCAAATTGGC).d(GCCAATTTGCG) complex. The nuclear Overhauser effect spectroscopy (NOESY) experiment was used to assign the aromatic and C1'H DNA protons and to identify drug-DNA contacts. These data indicate that two drug molecules bind simultaneously in the minor groove of the central 5'-AAATT-3' segment and are in close contact with both the DNA and one another. One drug binds with the formyl end close to the second adenine base of the A-rich strand, while the other drug binds with the formyl end close to the second adenine of the complementary strand. With this binding orientation, the positively charged propylamidinium groups are directed toward opposite ends of the helix. Molecular modeling shows that the minor groove must expand relative to the 1:1 complex to accommodate both drugs. Energy calculations suggest that electrostatic interactions, hydrogen bonds, and van der Waals forces contribute to the stability of the complex.
Scanning detection of mutations in human ornithine transcarbamoylase by chemical mismatch cleavage.
Authors: Grompe M , Muzny D M , Caskey C T
Published: May 2006

The mutations in X chromosome-linked diseases are frequently heterogeneous, and yet only a small fraction can be detected by Southern analysis. We therefore adapted the chemical cleavage method of Cotton et al. [Cotton, R. G. H., Rodrigues, N. R. & Campbell, R. D. (1988) Proc. Natl. Acad. Sci. USA 85, 4397-4401] and the polymerase chain reaction to rapidly scan for point mutations in X chromosome-linked ornithine transcarbamoylase (carbamoyl-phosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3.) deficiency. This simple heteroduplex mapping method identified different mismatch sites in polymerase chain reaction-amplified liver cDNA from five unrelated ornithine transcarbamoylase-deficient patients. The predicted sequence alteration was confirmed by DNA sequencing in all five patients and indicated a likely disease-causing mutation in four of these patients. In one atypical ornithine transcarbamoylase-deficient patient a sequence alteration compatible with a cDNA polymorphism was found. One family was studied in detail. Female-carrier detection was performed by chemical cleavage of amplified genomic DNA and verified by allele-specific oligonucleotide hybridization. This mutation scanning approach is simple, sensitive, and applicable to many genetic loci.
Interleukin 7 is a T-cell growth factor.
Authors: Chazen G D , Pereira G M , LeGros G , Gillis S , Shevach E M
Published: May 2006

Interleukin 7 (IL-7) is a 25-kDa cytokine which was purified and its corresponding cDNA was cloned based upon its ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also function as a costimulator with Con A for the proliferation of T lymphocytes by inducing the production of interleukin 2 (IL-2). We demonstrate here that IL-7 in combination with phorbol 12-myristate 13-acetate can directly drive the proliferation of purified T cells and that this response is not inhibited by cyclosporine A or by antibodies to IL-2 and IL-4. Stimulation of T cells with phorbol myristate acetate and IL-2, IL-4, or IL-7 prepared T cells to respond to any of the three lymphokines. Although T cells activated in vitro by anti-CD3 or allogeneic cells failed to proliferate when challenged with IL-7, T cells primed in vivo to the same stimuli demonstrated a significant proliferative response when restimulated in vitro with IL-7. IL-7 can, therefore, function both as a growth factor for T cells in an IL-2-independent manner and as a competence factor for the induction of lymphokine responsiveness. The ability to induce IL-7 responsiveness via stimulation of the T-cell receptor complex in vivo, but not in vitro, raises the possibility that IL-7 may play a role in T-cell growth and differentiation in vivo.
Cationic liposome-mediated RNA transfection.
Authors: Malone R W , Felgner P L , Verma I M
Published: May 2006

We have developed an efficient and reproducible method for RNA transfection, using a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), incorporated into a liposome (lipofectin). Transfection of 10 ng to 5 micrograms of Photinus pyralis luciferase mRNA synthesized in vitro into NIH 3T3 mouse cells yields a linear response of luciferase activity. The procedure can be used to efficiently transfect RNA into human, rat, mouse, Xenopus, and Drosophila cells. Using the RNA/lipofectin transfection procedure, we have analyzed the role of capping and beta-globin 5' and 3' untranslated sequences on the translation efficiency of luciferase RNA synthesized in vitro. Following transfection of NIH 3T3 cells, capped mRNAs with beta-globin untranslated sequences produced at least 1000-fold more luciferase protein than mRNAs lacking these elements.
Absence of unsaturated fatty acid synthesis in murine T lymphocytes.
Authors: Buttke T M , Van Cleave S , Steelman L , McCubrey J A
Published: May 2006

Stearic acid is toxic for T lymphocytes in vitro but has little effect on B lymphocytes. To investigate the molecular basis for this difference, purified murine T and B lymphocytes were compared for their abilities to incorporate and metabolize stearic acid. Unstimulated T and B cells incorporated identical amounts of stearic acid into six different phospholipids and four neutral lipids. After mitogen stimulation, fatty acid uptake was increased in both lymphocyte types, but cell-specific differences were seen in the distribution of stearic acid among the various cellular lipids. Doses of stearic acid that selectively inhibited T-cell proliferation resulted in a 5-fold greater accumulation of distearoylphosphatidylcholine in T cells than in B cells. Whereas T cells did not desaturate the exogenously derived stearic acid, up to 25% of the saturated fatty acid was converted to oleic acid in B cells. These findings suggested a deficiency of stearoyl-CoA desaturase (acyl-CoA, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.5) activity in T cells, which was confirmed by subsequent studies. Cell-free extracts from B cells displayed nearly 20-fold more stearoyl-CoA desaturase activity than T-cell extracts, and the level of stearoyl-CoA desaturase mRNA was 30-fold higher in B cells. Collectively, our data indicate that murine T cells are deficient in unsaturated fatty acid synthesis. The deficiency of stearoyl-CoA desaturase in T cells may represent the basis for the differing sensitivities of T and B lymphocytes to inhibition by saturated fatty acids.
A DNA double chain break stimulates triparental recombination in Saccharomyces cerevisiae.
Authors: Ray A , Machin N , Stahl F W
Published: May 2006

Mitotic recombination between his3 heteroalleles on heterologous chromosomes is stimulated by a DNA double chain break delivered in vivo at a site 8.6 kilobase pairs distant from one his3 allele and unlinked to the other. The induced recombination at his3 is accompanied by gap repair at the break site using the uncut homolog as a template. The DNA between the break site and his3 is not deleted in most of the His+ recombinants.
Electrophoretic karyotype of Aspergillus nidulans.
Authors: Brody H , Carbon J
Published: May 2006

An electrophoretic karyotype of Aspergillus nidulans has been obtained using contour-clamped homogeneous electric field gel electrophoresis. Six chromosomal bands were separated, with two of the bands migrating as doublets. Using the Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes as size standards, we estimate the sizes of the chromosomes to be between 2.9 and 5.0 megabase pairs (mb) with a total genome size of approximately 31 mb. Four of the eight genetic linkage groups were assigned to chromosomal bands by hybridization of contour-clamped homogeneous electric field gel blots with various radiolabeled probes each specific to a particular linkage group. Contour-clamped homogeneous electric field gel analysis of reciprocal translocation strains gave chromosomal assignments for the four remaining linkage groups. In order of decreasing size, the A. nidulans chromosomes are: VIII (5.0 mb), VII (4.5 mb), II (4.2 mb), I and V (3.8 mb), III and VI (3.5 mb), and IV (2.9 mb).
Vaccine protection against simian immunodeficiency virus infection.
Authors: Desrosiers R C , Wyand M S , Kodama T , Ringler D J , Arthur L O , Sehgal P K , Letvin N L , King N W , Daniel M D
Published: May 2006

Rhesus monkeys were immunized by multiple inoculations with purified, disrupted, noninfectious simian immunodeficiency virus (SIV) in adjuvant. Immunized monkeys developed anti-SIV antibodies detectable by whole-virus ELISA and by immunoblot reactivity; these antibodies had weak neutralizing activity. One week after the last immunization, monkeys were challenged with 200-1000 animal infectious doses of uncloned, live SIV. The same strain of SIV that was used for vaccination was also used for challenge. Anamnestic antibody responses and SIV recovery from peripheral blood were used to evaluate infection following the live virus challenge; two of six vaccinated monkeys showed no evidence of infection following the live virus challenge. Transfusion of 10 ml of whole blood from these two into uninfected, naive rhesus monkeys did not result infection of the recipients, providing further support for the lack of infection in the two previously vaccinated animals. Four of four unvaccinated control monkeys inoculated with these doses of live SIV became infected and three of these died with AIDS 118-258 days after infection. Only one of the six vaccinated monkeys has died to date. In situ hybridization with lymph node biopsy specimens suggested that the virus load was much higher in control macaques than in vaccinated macaques. These results indicate that vaccination with inactivated whole virus can protect macaques against challenge with live SIV. Furthermore, they provide hope that vaccine protection against human AIDS virus infection may be possible.
Cholesteryl-conjugated oligonucleotides: synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture.
Authors: Letsinger R L , Zhang G R , Sun D K , Ikeuchi T , Sarin P S
Published: May 2006

A family of oligonucleotides and phosphorothioate oligonucleotide analogues was synthesized with a cholesteryl group tethered at the 3'-terminal internucleoside link. This modification, introduced to enhance interaction of the polyanions with cell membranes, significantly increases the antiviral activity of the oligomers, as judged by inhibition of syncytia formation and expression of viral proteins p17, p24, and reverse transcriptase for human immunodeficiency virus 1 in Molt-3 cells. In the most favorable case, with a 20-mer cholesteryl-phosphorothioate derivative, complete inhibition by all assays was obtained with an oligomer concentration of 0.2 microM. Even decamers were active, and some antiviral activity was observed for a heptanucleotide cholesteryl-phosphorothioate derivative, which binds very poorly to complementary oligonucleotides. These facts, and the finding that the activity of the phosphorothioate decamers does not correlate with a specific sequence, suggests that a mechanism other than "antisense inhibition" may be operative in these systems.