bluuebunny/crossref_metadata_2025 huggingface dataset:
Acetylcholinesterase of mammalian neuromuscular junctions: presence of tailed asymmetric acetylcholinesterase in synaptic basal lamina and sarcolemma.
Authors: Dreyfus P A , Rieger F , Pinçon-Raymond M
Published: May 2006

A sarcolemma-rich fraction can be isolated after subcellular fractionation of mouse intercostal muscles by sedimentation on a discontinuous sucrose gradient. The quantitative recovery of the acetylcholine receptor in this fraction is about 50%, which indicates the presence of a high proportion of postsynaptic membranes. Acetylcholinesterase (AcChoEase; EC 3.1.1.7) is found mainly in three different layers: the top layer, which contains soluble AcChoEase, the intermediate layer (fraction A), and the last, AcChoR-rich, layer (fraction C). The relative proportions of the molecular forms of AcChoEase are different in the three layers. The "16S" AcChoEase is in a higher proportion in both types of membrane fractions (A and C) compared to soluble AcChoEase. Both total AcChoEase and 16S AcChoEase are enriched in the A and C fractions. In the C fraction, the sequential use of homogenizations in the presence of detergent and high ionic strength allows the "solubilization" of two distinct AcChoEase pools. One is detergent-soluble and mainly composed of slow-sedimenting forms; the other one is detergent-insoluble, high-ionic strength-soluble, and composed mainly of collagen-like, tailed, asymmetric (16S) AcChoEase. Thus, most of the asymmetric AcChoEase is specifically localized in the synaptic extracellular matrix of the mammalian muscle fiber. However, in the A fraction, most of the 16S AcChoEase found is solubilized by detergent alone, suggesting an association with microsomal membranes. It may mean that at least some of the basal lamina-embedded 16S AcChoEase is preassembled intracellularly in the sarcoplasmic reticulum.
Targeted selection of recombinant clones through gene dosage effects.
Authors: Rine J , Hansen W , Hardeman E , Davis R W
Published: May 2006

The availability of multicopy plasmid vectors for the yeast Saccharomyces cerevisiae allows the selective amplification of individual segments of the genome. Increased dosage of particular genes results in overproduction of specific gene products and thereby confers resistance to certain metabolic inhibitors. Advantage was taken of this fact to isolate recombinant clones that increase the activities of the enzymes UDP-N-acetylglucosamine-1-P transferase and 3-hydroxy-3-methylglutaryl-CoA reductase.
Definition of oriR, the minimum DNA segment essential for initiation of R1 plasmid replication in vitro.
Authors: Masai H , Kaziro Y , Arai K
Published: May 2006

The 3.6-kilobase Bgl II-EcoRI fragment from R1 plasmid containing copA, repA, and the replication origin (ori) was inserted into the ColE1-type plasmid pUC8. The resulting hybrid plasmid replicates in extracts prepared from both polA- and polA+ cells, whereas pUC8 replicates only in a polA+ extract. This characteristic provides a method for assaying the repA and ori functions. Hybrid plasmids that were either repA- or ori- were unable to replicate in a polA- cell extract. Replication of the repA- ori+ plasmid was restored by complementation of the repA defect by a repA+ ori- plasmid in vitro. Successful complementation of the repA function in vitro provides a method for assaying the repA protein. In order to define the minimum DNA segment with origin function (oriR), deletions were introduced starting from either side of the insert, and the replication properties of the plasmids carrying these deletions were examined in a polA- cell extract. The right end of oriR was located at position 1,611 in the nucleotide coordinates defined previously [Ryder, T., Rosen, J., Armstrong, K., Davidson, D. & Ohtsubo, E. (1981) in The Initiation of DNA Replication: ICN-UCLA Symposia on Molecular and Cellular Biology, ed. Ray, D.S. (Academic, New York), Vol. 22, 91-111]. By complementing repA- ori+ plasmids with the repA+ ori- plasmid, the left end of oriR was localized at position 1,424. Therefore, the oriR sequence, localized within a region of 188 base pairs, is separate from the repA gene. A hybrid plasmid carrying the 206-base-pair segment between positions 1,406 and 1,611 also replicates in a polA- cell extract when the repA function is supplied in trans. Removal of an additional 66 base pairs (positions 1,406-1,471) inactivates the function of the minimal oriR segment.
Calmodulin-binding proteins that interact with actin filaments in a Ca2+-dependent flip-flop manner: survey in brain and secretory tissues.
Authors: Sobue K , Kanda K , Adachi J , Kakiuchi S
Published: May 2006

Regulatory actions of calmodulin on the contractile apparatus and cytoskeleton of smooth muscle and nonmuscle tissue are mediated by a number of specific calmodulin-binding proteins that bind to F-actin in a flip-flop manner--i.e., they bind to calmodulin or F-actin depending on the presence or absence, respectively, of Ca2+. A survey for such proteins in brain, adrenal gland, and pituitary gland identified six polypeptides on polyacrylamide gels--Mr 340,000 (band 1), Mr 240,000/235,000 doublet (band 2), Mr 150,000 (band 3), Mr 129,000 (band 4), Mr 105,000 (band 5), and Mr 94,000 (band 6)--as flip-flop-regulated calmodulin- and F-actin-binding polypeptides. In addition to these polypeptides, a Mr 58,000 non-flip-flop calmodulin-binding actin-binding polypeptide (band 7) was found in all tissues examined. Band 2 was identified as calspectin (spectrin-related protein; fodrin). The flip-flop regulation of calspectin required the presence of a heat-labile nondialyzable factor contained in a supernatant fraction of brain homogenates. Band 1 was distinct from microtubule-associated proteins (MAPs) 1 and 2. However, when band 1 polypeptide was kept on ice 3 days, it converted to a lower molecular weight doublet that migrated with MAP2 on NaDodSO4 gel electrophoresis. Bands 1 and 2 were found in all tissues examined.
Specific DNA sequences associated with the nuclear matrix in synchronized mouse 3T3 cells.
Authors: Goldberg G I , Collier I , Cassel A
Published: May 2006

Eukaryotic chromatin appears to be organized into arrays of supercoiled loops anchored to the scaffolding structure of the mitotic chromosome core or to the nuclear matrix of interphase nuclei. To reveal whether specific DNA sequences are involved in this level of chromatin organization, we isolated and cloned a population of DNA molecules [average length of 150 base pairs (bp)] closely associated with the nuclear matrix after exhaustive DNase digestion and subsequent extensive protease digestion. The nuclear matrix was obtained from murine BALB/c 3T3 cells synchronized at the G1/S border of the cell cycle. We report the structure of two sequences, designated G4 and G5, which are highly enriched in the matrix DNA. Sequence G4, of 152 bp, contains three 31-bp direct head-to-tail repeats. An 11-bp sequence at the end of each repeat is homologous to the first large tumor antigen recognition site of human papova virus. Sequence G5, of 135 bp, consists of two well-defined domains, in which the first domain is a fragment of the B1 repetitive sequence. The results suggest the possibility that the loops of histone-depleted chromatin are connected to the scaffold of the nuclear matrix, with specific DNA sequences at the anchorage sites.
Electrophoretic analysis of human HLA-DR antigens from HLA-DR4 homozygous cell lines: correlation between beta-chain diversity and HLA-D.
Authors: Nepom B S , Nepom G T , Mickelson E , Antonelli P , Hansen J A
Published: May 2006

Two-dimensional gel electrophoresis of immunoprecipitated human HLA-DR antigens from cells expressing the HLA-DR4 haplotype shows distinct clustering of beta-chain patterns. Six unique electrophoretic variants were observed among 17 HLA-DR4 homozygous cell lines (HCL) analyzed. These patterns correlate precisely with the HLA-D phenotype of the HCL donor as determined by reactivity in mixed lymphocyte culture. All DR4 HCL that belong to one of the well-defined HLA-D antigen groups (Dw4, Dw10, LD "40", LD "DYT", LD "KT2", or LD "TAS") have identical DR beta-chain patterns; DR4 HCL belonging to different HLA-D antigen groups do not. The concordance of the functional expression in mixed lymphocyte culture of a specific D phenotype with a distinct DR beta-chain pattern on gel analysis provides a direct structural basis for understanding the genetic control of HLA-D polymorphisms; HLA-D specificities as revealed by T-cell recognition in mixed lymphocyte culture thus might be accounted for by DR beta-chain polymorphisms. The extent of this beta-chain diversity within a single DR haplotype may aid in understanding variations in Ia-regulated functions, such as Ir gene control and certain disease susceptibilities.
Crystallographic studies on apocarboxypeptidase A and the complex with glycyl-L-tyrosine.
Authors: Rees D C , Lipscomb W N
Published: May 2006

The crystal structures of zinc-free carboxypeptidase A (apocarboxypeptidase A) and the complex of glycyl-L-tyrosine with apocarboxypeptidase A are described and compared to the corresponding structures of the zinc-containing enzyme. Only small conformational changes in the zinc ligands accompany removal of the metal. Interactions between the tyrosine residue of glycyl-L-tyrosine and apocarboxypeptidase A are similar to those observed in the complex with the holoenzyme. However, in the absence of zinc, the carbonyl oxygen of the glycyl moiety now receives a hydrogen bond from the side chain of arginine-127. Although not as yet observed, a similar shift of the carbonyl oxygen of a susceptible bond from the zinc to arginine-127 could stabilize tetrahedral intermediates generated during the hydrolysis of substrates by carboxypeptidase.
3-Hydroxy-3-methylglutaryl-CoA reductase: a transmembrane glycoprotein of the endoplasmic reticulum with N-linked "high-mannose" oligosaccharides.
Authors: Liscum L , Cummings R D , Anderson R G , DeMartino G N , Goldstein J L , Brown M S
Published: May 2006

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) is an abundant protein of the crystalloid endoplasmic reticulum of UT-1 cells, a line of cultured hamster cells that over-produces the reductase as a result of gene amplification. In the current studies, we show that reductase in UT-1 cells is a glycoprotein. The solubilized enzyme (Mr = 97,000) from UT-1 cells, Chinese hamster ovary cells, and rat liver was adsorbed quantitatively and specifically to concanavalin A-Sepharose. UT-1 cells incorporated [1,6-3H]glucosamine into the reductase; after release with endo-N-acetylglucosaminidase H most of the radioactivity was found in N-linked "high-mannose" chains, including Man6(GlcNAc)2, Man7(GlcNAc)2, and Man8(GlcNAc)2. The carbohydrate of the reductase was localized to a 30- to 35-kilodalton fragment that was separable proteolytically from a cytoplasmic 53-kilodalton fragment that contained the active site of the enzyme. We conclude that 3-hydroxy-3-methylglutaryl-CoA reductase is a transmembrane glycoprotein with an active site facing the cytoplasm and a carbohydrate-bearing site oriented toward the lumen of the endoplasmic reticulum.
Laser-induced multiphoton processes in living cells.
Authors: Calmettes P P , Berns M W
Published: May 2006

It is shown that specific light-induced subcellular alterations, usually referred to as phase "paling," result from multiphoton absorption processes. For green light (532 nm), four photons are required to induce paling in chromosomes and nucleoli; a two-photon process is observed for UV light (266 nm).
Weight gain and adjustment of feeding territory size in migrant hummingbirds
Authors: Carpenter F. Lynn , Paton David C. , Hixon Mark A.
Published: May 2006

Rufous hummingbirds periodically establish and defend territories along their summer southward migration route. Using artificial perches attached to spring or electronic balances in the field, we were able to measure daily weight changes in undistributed, individually marked birds. The territory size (number of flowers) of individual birds varied from day to day. Four of five intensively studied birds adjusted their territories to that size which was associated with the fastest sustained rate of weight gain attained at any stable territory size. The one exception was explicable on the basis of its unusually high weight. These results are consistent with the assumption of optimization theory that animals are capable of assessing when their behavior (e.g., territory size) is suboptimal and then making adjustments toward an optimum. The results also suggest, although not conclusively, that these birds are selected to maximize their rate of weight gain on each stopover prior to resuming migration.
Inducible repair of phosphotriesters in Escherichia coli.
Authors: McCarthy J G , Edington B V , Schendel P F
Published: May 2006

Extracts from Escherichia coli cells induced for the adaptive response have been prepared that are capable of repairing O6-methylguanine, O4-methylthymine, and the phosphotriesters produced on the DNA backbone by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The phosphotriesters are repaired by a methyltransferase distinct from the one that demethylates O6-methylguanine. We propose that this increased capacity to repair phosphotriesters accounts for much of the increased resistance to MNNG toxicity seen in cultures induced for the adaptive response.
Regulation of proline and glucose transport in mouse intestine by dietary substrate levels.
Authors: Karasov W H , Pond R S , Solberg D H , Diamond J M
Published: May 2006

Active uptake of D-glucose and L-proline at 50 mM was measured in everted intestinal sleeves of mice whose dietary carbohydrate and protein levels were being varied experimentally. Compared to a nearly carbohydrate-free meat diet, a 50% carbohydrate laboratory chow diet stimulated active glucose uptake in the proximal intestine without affecting proline uptake, passive glucose permeability, or several measures of mucosal mass. Switching from a low-protein high-carbohydrate to a high-protein no-carbohydrate diet reversibly stimulated proline uptake while inhibiting glucose uptake. For each solute and diet switch, the stimulation of transport was complete within 1 day, while the inhibition required several days. The results imply induction and repression of intestinal glucose and proline transport by dietary substrate levels. This mechanism, in conjunction with the normal gradient of nutrient concentrations along the intestine, is probably largely responsible for the gradient in nutrient transport along the intestine.
Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts.
Authors: von Gabain A , Belasco J G , Schottel J L , Chang A C , Cohen S N
Published: May 2006

An assay was developed to investigate the fate of specific segments of beta-lactamase (bla) and ompA gene transcripts in Escherichia coli. DNA probes cloned in bacteriophage M13 were treated with an endonuclease capable of cleaving single-stranded DNA, the fragments produced were annealed with total cellular RNA, and the resulting RNA . DNA hybrids were subjected to S1 nuclease treatment and gel fractionation. By using this assay, direct evidence was obtained for 3'-to-5' directionality in the decay of the long-lived mRNA encoded by the ompA gene, and no preferential stability was observed for translated versus untranslated mRNA segments. In the case of bla mRNA, initial cleavage of the full-length transcript was rate limiting, and no decay intermediates were detected. No difference in degradation rate was seen for bla transcripts having variant 3' or 5' termini.
Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat.
Authors: Byrne B J , Davis M S , Yamaguchi J , Bergsma D J , Subramanian K N
Published: May 2006

The simian virus 40 (SV40) origin region includes the viral replication origin and the early and late promoters and consists of a few palindromes, a 17-base-pair (bp) A + T-rich sequence, three copies of a G + C-rich 21-bp repeat, and two copies of a 72-bp repeat. We have made sequential deletions in the SV40 origin region and determined the early promoter efficiencies of these truncated DNA segments by connecting them in the correct orientation with the coding regions of selectable marker genes and assaying the expression of the chimeric marker genes in vivo in different host cell lines. A truncated SV40 early promoter segment containing only the TATA box and the major in vivo mRNA initiation sites has essentially no promoter efficiency. We have located the major component of the SV40 early promoter within the 21-bp repeated sequences, which consist of an alternating and mutually overlapping array of two C-rich oligonucleotides having the consensus sequences Y-Y-C-C-G-C-C-C (Y = pyrimidine nucleoside) and G-C-C-C-(C)-TA-AT-A(T)-C-T. Between one and two copies of the 21-bp repeat were adequate for gene expression under conditions in which the enhancement effect of the 72-bp repeat was minimal. We also find that the SV40 72-bp repeat exhibits a pronounced host range in its enhancement of gene expression; the enhancement is only 2-fold in the nonpermissive mouse cells but amounts to 10- or 20-fold in the permissive monkey cells or the semipermissive human cells, respectively.
Use of monoclonal antibodies to identify shared idiotypes on human antibodies to native DNA from patients with systemic lupus erythematosus.
Authors: Solomon G , Schiffenbauer J , Keiser H D , Diamond B
Published: May 2006

Antinative DNA antibodies were purified from the serum of a patient with active systemic lupus erythematosus. Using hybridoma technology, we produced several mouse monoclonal antibodies directed against idiotypic determinants on these antinative DNA antibodies. One of these monoclonal anti-idiotypic antibodies, 3I, an IgG2a Kappa, was extensively characterized. 3I is believed to be directed against an idiotypic determinant because (i) its reactivity with antinative DNA antibodies is not inhibited by a large excess of pooled human serum, (ii) it reacts with F(ab')2 fragments of antinative DNA antibodies, and (iii) it reacts with antinative DNA antibodies of all IgG subclasses. 3I is not directed against the antigen binding site in that native DNA and 3I do not compete for binding to antinative DNA antibodies. Eight of nine sera containing antinative DNA activity as determined by the Millipore filter assay reacted with 3I in a solid-phase radioimmunoassay. These findings suggest that antinative DNA antibodies from nonrelated patients with systemic lupus erythematosus share a common idiotype.
Vibrio cholerae
hemagglutinin/lectin/protease hydrolyzes fibronectin and ovomucin: F. M. Burnet revisited
Authors: Finkelstein Richard A. , Boesman-Finkelstein Mary , Holt Peter
Published: May 2006

Cholera vibrios produce a single polymeric protein that (
i
) causes hemagglutination; (
ii
) appears to participate in their attachment to gut epithelium; (
iii
) may mediate their detachment from gut epithelium; and (
iv
) is a protease that hydrolyzes fibronectin and mucin, cleaves lactoferrin, and nicks the A subunit of the choleragen-related heat-labile enterotoxin of
Escherichia coli
.
Microinjection of purified ornithine decarboxylase into Xenopus oocytes selectively stimulates ribosomal RNA synthesis.
Authors: Russell D H
Published: May 2006

This study has utilized stage VI oocytes of Xenopus laevis which have amplified the rDNA gene 1,000-fold to assess whether the microinjection of ornithine decarboxylase (OrnDCase) would stimulate [alpha-32P]guanosine incorporation into 45S and 18S/28S RNA selectively. The injection of purified OrnDCase into individual oocytes resulted in a greater than 2-fold increase in the incorporation of [32P]guanosine into 45S RNA and 18S/28S RNA with no increased incorporation into low molecular weight RNA. Further, an irreversible inhibitor of OrnDCase, alpha-difluoromethylornithine (CHF2-Orn), rapidly inhibited the endogenous activity of OrnDCase when added to the buffered Hepes solution bathing the oocytes and also inhibited the incorporation of [32P]guanosine into rRNA. The inhibitory effect of CHF2-Orn could not be reversed totally by addition of 10 microM putrescine to the oocytes. OrnDCase injected into oocytes in the presence of CHF2-Orn in the media did not stimulate incorporation of [32P]guanosine label into rRNA. However, when CHF2-Orn was removed from the buffered medium at the time of the injection of label and enzyme, a 3-fold increase of 32P incorporation into 18S/28S RNA occurred. Therefore, in an in vivo model in which amplified extrachromosomal rDNA gene copies are present, the microinjection of OrnDCase was capable of specifically stimulating rRNA synthesis. CHF2-Orn, a suicide enzyme inactivator of OrnDCase, was able to inhibit rRNA synthesis and, after washout, there was a more marked stimulation of rRNA synthesis than occurred after only the injection of OrnDCase alone. These data suggest further that OrnDCase is the labile protein that regulates the initiation of RNA synthesis.
Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody.
Authors: Kawamoto T , Sato J D , Le A , Polikoff J , Sato G H , Mendelsohn J
Published: May 2006

Epidermal growth factor (EGF) at 3 nM maximally inhibits the proliferation of A431 epidermoid carcinoma cells. We show that at lower concentrations, in the range of 3-100 pM, EGF has a mitogenic effect on A431 cells. In the presence of 100 nM anti-EGF-receptor monoclonal IgG (designated 528), which inhibits A431 cell proliferation and blocks greater than 95% of EGF binding, EGF becomes mitogenic for A431 cells at concentrations up to 3 nM. These results suggest that a minor population of high-affinity EGF receptors may be involved in stimulation of A431 cell proliferation. Saturation binding assays with 125I-labeled EGF indicate that approximately equal to 0.1-0.2% of receptors for EGF are high-affinity receptors that bind EGF with an estimated Kd of 7 X 10(-11) M. This affinity is nearly 2 orders of magnitude higher than that of the remaining EGF receptors. Although A431 cell proliferation is maximally inhibited by nonsaturating amounts of EGF (3 nM), maximal inhibition by 528 IgG (approximately equal to 70% of maximal inhibition by EGF) requires saturating concentrations of antibody (approximately equal to 15 nM). Unlike EGF, rapid down-regulation is not observed with 528 IgG. These results indicate different mechanisms of growth inhibition of A431 cells by EGF and 528 IgG.
Adaptive significance of differences in the tissue-specific expression of a phosphoglucomutase gene in rainbow trout.
Authors: Allendorf F W , Knudsen K L , Leary R F
Published: May 2006

We have investigated the phenotypic effects of a mutant allele that results in the expression of a phosphoglucomutase locus (Pgm1) in the liver of rainbow trout. Embryos with liver Pgm1 expression hatch earlier than embryos without liver Pgm1 expression. These differences apparently result from increased flux through glycolysis in embryos with liver PGM1 activity while they are dependent on the yolk for energy. Fish with liver PGM1 activity are also more developmentally buffered, as indicated by less fluctuating asymmetry of five bilateral meristic traits. The more rapidly developing individuals begin exogenous feeding earlier and achieve a size advantage that is maintained until sexual maturity. This size advantage produces a significant tendency for earlier age of sexual maturity. These results show that different genotypes at this regulatory gene result in important phenotypic differences that are likely to be of important adaptive significance.
Indirect stimulation of genetic recombination
Authors: Golub Efim I. , Low K. Brooks
Published: May 2006

Recombination between
lac
Z alleles in crosses of λ
lac
Z
1
-
× λ
lac
Z
2
-
and F
-
lac
Z
1
-
× λ
lac
Z
2
-
in
Escherichia coli
(λ) can be stimulated manyfold by UV irradiation of one of the λ
lac
Z phages [Porter, R. D., McLaughlin, T. & Low, B. (1979)
Cold Spring Harbor Symp. Quant. Biol.
43, 1043-1048]. Analogous stimulation has now been observed by coinfection of the cells by UV-irradiated λ phage which carries no
lac
region. This indirect stimulation is not dependent on induction of the SOS system. The bacterial
uvr
system can effectively remove the damages on the λ DNA which cause the indirect stimulation. Among a number of mutations tested, only
ssb-1
was found to cause a drastic decrease in the indirect stimulation. Indirect stimulation was caused only by using phage that had a region of homology with the recombining phage. The homologous region can be separated from the recombining region by an extended nonhomologous region (>7.9 × 10
3
base pairs). This implies that damages to the DNA molecule, which stimulate recombination, can be located very far from the recombining region of the molecule.
Developmental cell interactions in
Myxococcus xanthus
and the
spoC
locus
Authors: Shimkets Lawrence J. , Gill Ronald E. , Kaiser Dale
Published: May 2006

The product(s) of the
Myxococcus xanthus spoC
locus is required for two multicellular activities in fruiting body development, rippling and sporulation. Ripples, which are formed early in development, are spatially separated ridges of cells that move synchronously. Myxospores are heat-resistant resting cells that are formed near the end of the developmental process. To investigate the function of
spoC
, it was cloned in an
Escherichia coli
plasmid, then transferred to
M. xanthus
by specialized transduction with coliphage P1. The plasmid, which cannot replicate in
M. xanthus
, integrated into the
M. xanthus
chromosome, producing two copies of the
spoC
locus in tandem. Cells containing one copy of a mutant allele and one copy of the wild-type allele displayed the wild-type phenotype. Cells containing two different mutant alleles failed to ripple or sporulate, implying that all four independent
spoC
mutations are in the same gene or unit of transcription. Homozygous mutant duplications arose from constructions in which DNA from a
spo
+
donor was transduced into a
spoC
recipient, or vice versa, at an average frequency of 14%, indicating that gene conversion was a frequent event.
Biology of Langerhans cells: Selective migration of Langerhans cells into allogeneic and xenogeneic grafts on
nude
mice
Authors: Krueger Gerald G. , Daynes Raymond A. , Emam Mansoor
Published: May 2006

A major question challenging immunobiologists relates to those mechanisms that control the selective movement of cells involved in immune and inflammatory processes at various tissue sites such as the skin. Little is known about those influences that control the selective migration of macrophage-like Langerhans cells (LC) to normal epidermis, where it is uniformly distributed. Mechanistically, this includes the interaction of blood-borne LC precursors with the vascular endothelium of the skin and those factors that control the migration of the LC into the avascular epidermal component of the skin. By using (
i
) monoclonal antibodies specific for I-region associated Ia antigens found on LC from various inbred strains of animals and (
ii
) the congenitally athymic (
nude
) mouse as an immunologically compromised recipient of allografts and selected xenografts, we developed a model system to study the factors that restrict LC migration into the epidermis. Using this model, which excludes the need to lethally x-irradiate graft recipients, we established that: (
i
) the ingress of LC does not show major histocompatibility complex restriction [LC of the
nude
host are capable of migrating into the epidermis of allogeneic and certain xenogeneic (rat) skin grafts]; (
ii
) host LC are incapable of migrating into the epidermis of guinea pig or human skin grafts; (
iii
) the ingress of host LC into the epidermis of the graft is not accompanied by an overgrowth of the graft by host epidermis; and (
iv
) LC or LC precursors are capable of dividing in the skin or, alternatively, represent an extremely long-lived cell population. The specificity of this model system provides a powerful tool to help understand many aspects of LC biology. Grafting human skin to the
nude
mouse not only provides a biologic support system for the graft but also is, by design, a system that is devoid of contaminating circulating precursor cell types. Manipulation of the experimental conditions is quite easy and provides a highly specific means to investigate many parameters of LC function.
In vitro synthesis of thymosin beta 4 encoded by rat spleen mRNA.
Authors: Filipowicz A W , Horecker B L
Published: May 2006

Thymosin beta 4, containing 43 amino acids and acetylated at the NH2 terminus, is synthesized in vitro in a rabbit reticulocyte lysate or in a yeast protein-synthesis system in the presence of mRNA from rat spleen. The product formed was identified as beta 4 by immunoprecipitation by a specific anti-beta 4 antiserum, comigration with authentic beta 4 in NaDodSO4/polyacrylamide gel electrophoresis and in HPLC, and identity of peptide fragments. The immunoprecipitable product generated in the wheat germ protein-synthesizing system emerged slightly ahead of beta 4 in HPLC and appeared to lack the NH2-terminal acetyl group. There was no evidence for formation of a larger polypeptide precursor of beta 4 in any of the three systems used. In sucrose density gradient centrifugation, the mRNA coding for beta 4 was recovered in the 7-8S mRNA fraction.
Modular structural units, exons, and function in chicken lysozyme.
Authors: Go M
Published: May 2006

By the application of the same algorithm for finding compact structural units encoded by exons as applied previously to hemoglobin, five units, M1-M5, were identified in chicken egg white lysozyme. They consist of residues 1-30, 31-55, 56-84, 85-108, and 109-129, respectively. I call these compact structural units "modules." As in hemoglobin, modules thus identified correspond well to exons--i.e., modules M1, M2 plus M3, M4, and M5 correspond to exons 1, 2, 3, and 4 of the lysozyme gene, respectively. Localization of the catalytic sites glutamic acid-35 and aspartic acid-52 on the module M2 suggests that this module might have worked as a functional unit in a primitive lysozyme. The good correspondence between exons and modules reinforces the idea of "proteins in pieces," which was derived from the fact of "genes in pieces." The evolutionary origin of the introns in globins and lysozyme is discussed.
Switch in the transposition products of Mu DNA mediated by proteins: Cointegrates versus simple insertions.
Authors: Harshey R M
Published: May 2006

Bacteriophage Mu is a self-contained mobile unit encoding functions that mediate its movement. There appear to be two alternate pathways for Mu DNA transposition that differ with respect to the end products they generate. During the lytic cycle of phage Mu growth the end products of transposition are predominantly cointegrates in an experimental system in which the induced Mu prophage is located on pSC101, a low-copy-number plasmid. On the other hand, Mu insertions into the host genome during lysogenization contain Mu DNA as simple insertions. Two Mu functions, encoded by the A and B genes, are required for Mu DNA transposition during its lytic growth. However, during lysogeny the product of gene B is not required for integration of Mu DNA. Evidence is presented here which shows that in the absence of the B gene product the majority of transposition events are simple insertions. This is in striking contrast to the situation in which the majority of the products are cointegrates in the presence of both A and B gene products. Additional evidence also suggests that these simple insertions do not arise through the resolution of cointegrate structures.
Generation of human monoclonal antibodies reactive with cellular antigens.
Authors: Cote R J , Morrissey D M , Houghton A N , Beattie E J , Oettgen H F , Old L J
Published: May 2006

Human lymphocytes from lymph node, peripheral blood, spleen, and tumor specimens have been fused with the LICR-LON-HMy2 (LICR-2) or SKO-007 human cell lines or the NS-1 mouse myeloma line. Over 75 fusions with the three myeloma-lymphoblastoid lines have been performed. Several factors appeared to improve the fusion outcome, including maintenance of the myeloma-lymphoblastoid lines in logarithmic phase growth at greater than or equal to 95% viability, a delay of 24 hr in the introduction of aminopterin to the fused cells, and preselection of the fetal calf serum used in the medium. For a given number of lymphocytes, fusions with NS-1 produced 5-20 times more clones than fusions with LICR-2 or SKO-007, and LICR-2 produced 4 times as many clones as SKO-007. The percentage of clones secreting human immunoglobulin, the range of immunoglobulin production, and the proportion of IgM, IgA, and IgG secretors were comparable for clones derived from the three myeloma-lymphoblastoid lines. Stable Ig-secreting clones were isolated with approximately equal frequency from LICR-2 and NS-1 fusions. A number of stable clones producing human monoclonal antibodies reacting with cell-surface, cytoplasmic, or nuclear antigens have been isolated from tumor-bearing patients and normal individuals. A surface antigenic system present on normal and malignant cells has been defined with a human monoclonal antibody derived from a patient with breast cancer. Techniques for producing human monoclonal antibody now appear to be sufficiently advanced to initiate a serological dissection of the humoral immune response to cancer.
Identification of human T cell leukemia virus in a Japanese patient with adult T cell leukemia and cutaneous lymphomatous vasculitis.
Authors: Haynes B F , Miller S E , Palker T J , Moore J O , Dunn P H , Bolognesi D P , Metzgar R S
Published: May 2006

We have identified a Japanese patient with adult T-cell leukemia (ATL) whose T cells in vitro produced the human T-cell leukemia virus (HTLV). This patient presented with lymphomatous arthritis and leukemia and subsequently developed skin lesions. Skin invasion by malignant T-cells was angiocentric and produced vessel wall destruction, resulting in necrotic cutaneous tumor nodules. Malignant T cells in peripheral blood, skin, and joint prior to culture in vitro did not express p19 HTLV-associated antigen. However, by electron microscopy, intracellular type C viral particles were seen in skin-infiltrating T cells. Peripheral blood malignant cells after 7 days in culture with T-cell growth factor-supplemented media expressed p19 antigen, and type C virus particles were seen by electron microscopy to be budding from malignant T lymphocytes. Mitomycin-C-treated peripheral-blood T cells induced the transformation of cord blood T cells into HTLV-infected p19+ T cells. The demonstration of HTLV in malignant T cells from our patient confirms the association of HTLV with Japanese adult T-cell leukemia. Moreover, HTLV may be associated with a vasculitis-arthritis syndrome.
Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica.
Authors: Coderre J A , Beverley S M , Schimke R T , Santi D V
Published: May 2006

Leishmania tropica promastigotes that are highly resistant to methotrexate, a dihydrofolate reductase inhibitor, have been developed. Organisms resistant to 1 mM methotrexate have a 40-fold increase in dihydrofolate reductase which is associated with thymidylate synthetase, and they contain amplified regions of DNA that may be directly visualized on stained gels of restriction digests. The amplified DNA in these organisms is about 56 kilobases in length, has a copy number about 80-fold higher than that of wild-type organisms, and constitutes about 10% of the nuclear DNA. When the methotrexate-resistant L. tropica are propagated in drug-free medium, the dihydrofolate reductase-thymidylate synthetase protein and the amplified DNA decrease in a parallel fashion until they are indistinguishable from the levels in wild-type organisms. However, when these apparent revertants are again challenged with 1 mM methotrexate, enzyme overproduction and DNA amplification occur rapidly. As in mammalian cells, it appears that drug resistance in parasitic protozoa may be mediated by gene amplification.
Component A of the methyl coenzyme M methylreductase system of Methanobacterium: resolution into four components.
Authors: Nagle D P , Wolfe R S
Published: May 2006

Component A, the oxygen-sensitive protein fraction of the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum, has been stabilized and resolved into three protein fractions and one cofactor that are required to reconstitute component A activity. Component A1 is oxygen-stable and contains hydrogen-dependent deazaflavin (coenzyme F420)-reducing activity. Component A2 is acidic; components A2 and A3 are oxygen sensitive. The specific functions of each component in methyl group reduction are unknown. Resolution of component A revealed a new cofactor requirement of the methylreductase system for FAD. Hydrogen-dependent reduction of methyl coenzyme M to methane and coenzyme M, the terminal step of CO2 reduction by methanogenic bacteria, requires protein components A1, A2, A3, and C in addition to component B, FAD, ATP, and Mg2+.
Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody.
Authors: Bennett J S , Hoxie J A , Leitman S F , Vilaire G , Cines D B
Published: May 2006

Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. To gain further insight into the role of fibrinogen in platelet aggregation and to identify the platelet fibrinogen receptor, we developed a monoclonal anti-platelet antibody that inhibited platelet aggregation. The purified antibody, designated A2A9, inhibited platelet aggregation stimulated by 10 microM ADP, 10 microM epinephrine, and thrombin at 1 unit/ml without inhibiting platelet shape change or platelet secretion. A2A9 was also a competitive inhibitor of fibrinogen binding to ADP-stimulated platelets. Fifty percent inhibition of fibrinogen binding occurred at 65 nM A2A9. Direct binding studies using radiolabeled A2A9 demonstrated 47,000 A2A9 binding sites on unstimulated platelets, with a dissociation constant of 60 nM. Platelets from two individuals with Glanzmann thrombasthenia bound essentially no A2A9. Therefore, these data support the hypothesis that receptor-bound fibrinogen mediates platelet aggregation. In order to identify the platelet fibrinogen receptor, A2A9 immobilized on agarose was used for affinity chromatography. Two platelet polypeptides with Mr = 140,000 and 93,000 were recovered from the immobilized A2A9. After disulfide reduction, these Mr values were altered to 125,000 and 116,000. The smaller polypeptide was also found to contain the PlA1 antigen. These data localize the epitope recognized by A2A9 to the platelet membrane glycoprotein IIb-IIIa complex and suggest that this complex forms the physiologic platelet fibrinogen receptor.
Integrated hepatitis B virus DNA sequences specifying the major viral core polypeptide are methylated in PLC/PRF/5 cells.
Authors: Miller R H , Robinson W S
Published: May 2006

The methylation of various hepatitis B virus (HBV) DNA sequences was examined using the restriction endonucleases Hpa II and Msp I. HBV DNA from virions (Dane particles) and virus-infected liver tissue was digested with Hpa II or Msp I and fractionated by electrophoresis in agarose gels, and the restriction enzyme cleavage pattern was examined by Southern blot analysis. No methylation of the 5' C-C-G-G 3' recognition sequence was detected in either virion DNA or HBV DNA from infected liver tissue. The tissue culture cell line PLC/PRF/5, derived from a human hepatoma, possesses HBV DNA exclusively integrated at several sites. Digestion of PLC/PRF/5 DNA with Hpa II and Msp I revealed that the integrated HBV DNA sequences were methylated. Further analysis using probes specific for various regions of the HBV genome showed that some of the hepatitis B viral DNA sequences, including those specifying the major surface antigen polypeptide, were methylated infrequently or not at all. In contrast, the viral DNA sequences coding for the major core polypeptide were extensively methylated. Because the surface antigen is expressed in these cells while the core antigen is not, our results suggest that DNA methylation could account for the selective expression of HBV genes in this hepatoma cell line.
Renal sulfate transport at the basolateral membrane is mediated by anion exchange.
Authors: Pritchard J B , Renfro J L
Published: May 2006

The transport of sulfate was studied in basolateral membrane (BLM) vesicles isolated from rat kidney cortex by centrifugation on a Percoll self-generating gradient. In contrast to sulfate transport at the luminal membrane, sulfate uptake by BLM vesicles was not sodium dependent. However, imposition of an inside greater than outside bicarbonate gradient stimulated BLM sulfate uptake nearly 10-fold and produced a transient overshoot of about 4-fold. This process appeared to become saturated at high concentrations of either bicarbonate or sulfate. Sulfate itself, thiosulfate, and hydroxyl, but not chloride or thiocyanate, were able to substitute for bicarbonate. None of these anions was as effective as bicarbonate. The sulfate/bicarbonate exchange was unaltered by manipulation of membrane potential, suggesting that it was electroneutral. Both bicarbonate stimulation and overshoot could be prevented by known inhibitors of anion transport, including mercuric chloride, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and phloretin. Bicarbonate-stimulated sulfate uptake also was inhibited by thiosulfate, probenecid, and acetazolamide. Thus, rat kidney BLM vesicles showed carrier-mediated anion exchange. Its properties indicate that this carrier may participate in both reabsorptive and secretory sulfate transport.
Coordinate gene expression during somatic embryogenesis in carrots
Authors: Sung Z. R. , Okimoto R.
Published: May 2006

There are several biochemical differences between the callus and the embryos of carrot culture. Callus tissue produces callus-specific proteins and a conditioning factor that is necessary for the synthesis of callus-specific proteins. By contrast, embryos produce embryo-specific proteins [Sung, Z. R. & Okimoto, R. (1981)
Proc. Natl. Acad. Sci. USA
78, 3683-3687] and develop the capability to inactivate cycloheximide [Sung, Z. R., Lazar, G. J. & Dudits, D. (1981)
Plant Physiol
. 68, 261-264]. A mutant, WCH105, that can inactivate cycloheximide in the callus as well as in the embryos produces the embryo-specific proteins instead of the callus-specific proteins and fails to produce the conditioning factor by the callus tissue. Callus tissues also produce a conditioning factor for callus growth. This factor is not the same as the conditioning factor for the synthesis of the callus-specific proteins, as WCH105 can grow as callus. The existence of WCH105 demonstrates that the callus-specific and embryo-specific traits are coordinately regulated, but in an opposite manner. A common mechanism apparently activates one set and inactivates the other set of functions. WCH105 seems to be impaired in this mechanism.
Characterization of a viable, noninverting herpes simplex virus 1 genome derived by insertion and deletion of sequences at the junction of components L and S.
Authors: Poffenberger K L , Tabares E , Roizman B
Published: May 2006

Earlier studies have shown that the DNA of herpes simplex virus 1 consists of two covalently linked components, L and S, each flanked by inverted repeats. The two components can invert, and viral DNA extracted from infected cells or virions consists of equimolar concentrations of four populations differing solely in the orientation of L and S components relative to each other. This paper describes a recombinant virus (1358) generated by an insertion of a chimeric thymidine kinase gene within the reiterated sequences of the S component and deletions that eliminated most of the internal inverted repeats at the junction between the L and S components. A characteristic of 1358 is that the L and S components are frozen in one (prototype) orientation. Inversion of L and S components is therefore not required for the replication of viral DNA.
In vivo
therapy with monoclonal anti-I-A antibody suppresses immune responses to acetylcholine receptor
Authors: Waldor Matthew K. , Sriram Subramaniam , McDevitt Hugh O. , Steinman Lawrence
Published: May 2006

A monoclonal antibody to
I-A
gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.
Biosynthesis of the subunits of factor VIIIR by bovine aortic endothelial cells.
Authors: Lynch D C , Williams R , Zimmerman T S , Kirby E P , Livingston D M
Published: May 2006

The biosynthesis of the subunit of factor VIIIR was studied in bovine aortic endothelial cells by the techniques of immunoprecipitation and NaDodSO4/polyacrylamide gel electrophoresis. It was determined that the subunit is first produced as a Mr 240,000 glycoprotein precursor, which appears to undergo proteolytic cleavage at or about the time of secretion into the medium with a resultant change in apparent Mr to 225,000, the size of the mature subunit found in plasma. The Mr 240,000 species was detected within 10 min of the start of labeling of cells, but factor VIIIR was not detected in the culture medium until approximately equal to 50 min. Treatment of the cells with tunicamycin resulted in a decrease in the apparent Mr of both species but did not grossly inhibit processing of precursor to product or the secretion of the latter. Thus, much or all N-linked glycosylation of factor VIIIR is not essential for these steps. Accumulation of factor VIIIR in the medium continued over a 24-hr period of cell labeling with [35S]-methionine, without significant net intracellular accumulation of the precursor, suggesting that a large storage pool of factor VIIIR is not present in endothelial cells under these conditions.
Nonenzymatic glucosylation of homologous low density lipoprotein and albumin renders them immunogenic in the guinea pig.
Authors: Witztum J L , Steinbrecher U P , Fisher M , Kesaniemi A
Published: May 2006

Nonenzymatic glucosylation of low density lipoprotein (LDL) and other plasma and structural proteins is enhanced in diabetics. Because conjugated carbohydrates are known to play an important role in the immunogenicity of proteins, we sought to determine if glucosylation of LDL (yielding Glc-LDL) and albumin would make them immunogenic. Therefore, we immunized guinea pigs with homologous glucosylated proteins and measured antibody response by solid-phase radioimmunoassay. Glucosylation of LDL in the presence of cyanoborohydride yields glucitol-lysine as the glucose adduct. Immunization with this Glc-LDL yielded a high-titered antiserum that reacted specifically with guinea pig Glc-LDL but not native LDL. Glucitol-lysine was an effective competitor for binding to the antibody, as were other reductively glucosylated human proteins. Glucosylation of LDL by incubation with glucose in the absence of a reducing agent yields fructosyllysine as the glucose adduct. This product, which has been demonstrated in human plasma, was also immunogenic, though the antiserum produced was of lower titer and affinity. Homologous glucosylated albumin was also immunogenic. These data suggest that nonenzymatic glucosylation of proteins could lead to autoantibody production and the formation of immune complexes in diabetic plasma and tissues.